(Handbook of Sensory Physiology 4 - 1 - Chemical Senses.) J. E. Amoore, M. G. J. Beets, J. T. Davies, T. Engen, J. Garcia, R. C. Gesteland, P. P. C. Graziadei, K.-E. Kaissling, R. A. Koelling, J. LeMa

Handbook
of Sensory Physiology
Volume IV
Chemical Senses Part 1
Editorial Board
H. Autrum R. Jung . W. R. Loewenstein

D. M. MacKay H.1. Teuber
Olfaction
By
J. E. Amoore . M. G. J. Beets' J. T. Davies' T. Engen

J. Garcia' R. C. Gesteland . P. P. C. Graziadei . K.-E. Kaissling
R. A. Koelling' J. LeMagnen . P. MacLeod D. G. Moulton
M. M. Mozell . D. Ottoson' T. S. Parsons' S. F. Takagi
D. Tucker' B. M. Wenzel
Edited by
Lloyd M. Beidler
With 212 Fignres
Springer-Verlag Berlin Heidelberg New York 1971

ISBN-13: 978-3-642-65128-1 e-ISBN-I3: 978-3-642-65126-7
001: 10.1007/978-3-642-65126-7
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Preface
Olfaction is involved intimately in two of the most basic functions of animals:

food intake and reproduction. There are also many other involvements of olfaction
in animal behavior, not the least being communication. The authors of this volume
have collected and evaluated the comparative anatomy, electron microscopy,
electrophysiology, genetics, psychology, chemistry, and biophysics of the olfactory
system and then indicated their roles in animal behavior. The importance of
olfaction in the everyday life of an animal is just being realized fully and recent
years have brought forth a great surge of research in this area. The diverse dis-
ciplines that contribute to our understanding of olfaction make the development
of this volume rewarding for those working in this field.
The olfactory system's very high sensitivity and its great power of molecular
discrimination interests many chemists and physicists. Data from the study of
both vertebrates and insects show that only one molecule of certain odors is
necessary to stimulate a single olfactory receptor! The underlying physicochemical
events are not yet understood. Also, many mammals can discriminate quickly the
difference between two odors of similar structure. Thus the olfactory epithelium
and the more centrally located neural components present the ultimate in chemical
detection and analysis by a biological system. The principles involved are probably
common to those of many other organs.
Electrophysiological techniques have allowed scientists to study the olfactory
system in an objective and quantitative manner. AIl of the authors of this volume
are indebted greatly to Lord Adrian in whose laboratory the first recordings from
a single nerve fiber were realized and who, himself, pioneered in the early researches
of olfaction.
Tallahassee, July 1971
L. M. BEIDLER
Contents
Chapter 1 Anatomy of Nasal Structures from a Comparative

Viewpoint. By T.S.PARSONS. With 14 Figures . . . . 1
Chapter 2 The Olfactory Mucosa of Vertebrates.
By P.P.C.GRAZIADEI. With 24 Figures . . . 27
Chapter 3 The Olfactory Pigment. By D. G. MOULTON.
With 2 Figures. . . . . . . . . . . . . . . 59
Chapter 4 Degeneration and Regeneration of the Olfactory
Epithelium. By S. F. TAKAGI. With 10 Figures 75
Chapter 5 The Electro-Olfactogram. By D. OTTOSON.
With 21 Figures . . . . . . . . . . . . . 95
Chapter 6 Neural Coding in Olfactory Receptor Cells.
By R. C. GESTELAND. With 3 Figures. . . . 132
Chapter 7 Nonolfactory Responses from the Nasal Cavity:
Jacobson's Organ and the Trigeminal System.
By D. TUCKER. With 8 Figures. . . . . . . . 151
Chapter 8 Structure and Function of Higher Olfactory Centers.
By P. MACLEOD. With 5 Figures. . . . . . . . . 182
Chapter 9 Spatial and Temporal Patterning. By M. M. MOZELL.
With 5 Figures. . . . . . . . . . . . . . . . . . 205
Chapter 10 Olfactory Psychophysics. By T. ENGEN. With 12 Figures 216
Chapter 11 Olfactory Genetics and Anosmia. By J. E. AMOORE.
With 1 Figure . . . . . . . . . . . . . . . . . . 245
Chapter 12 Olfactory Response and Molecular Structure.
By M. G. J. BEETS. With 10 Figures . . . . 257
Chapter 13 Olfactory Theories. By J. T. DAVIES. With 10 Figures. 322
Chapter 14 Insect Olfaction. By K.-E. KAISSLING. With 62 Figures 351
Chapter 15 Olfaction in Birds. By B. M. WENZEL. With 7 Figures . 432
Chapter 16 The Use of Ionizing Rays as a Mammalian Olfactory
Stimulus. By J. GARCIA and R. A. KOELLING.
With 7 Figures. . . . . . . . . . . . . . . 449
Chapter 17 Olfaction and Nutrition. By J. LEMAGNEN.
With 11 Figures 465
Author Index 483
Subject Index 500
List of Contributors
AMOORE, John E., Composition Investigations, Vegetable Laboratory, United

States Department of Agriculture, Albany, California 94710, USA
BEETS, M. G. J., International Flavors & Fragrances (Europe), Hilversum,
Netherlands
DAVIES, J. T., Department of Chemical Engineering, The University of Bir-
mingham, Edgbaston, Birmingham, Bl5 2TT, Great Britain
ENGEN, Trygg, Walter S. Hunter Laboratory of Psychology, Brown University,
Providence, Rhode Island 02912, USA
GARCIA, John, Department of Psychology, State University of New York at
Stony Brook, Stony Brook, New York 11790, USA
GESTELAND, Robert C., Department of Biological Sciences, Northwestern Uni-
versity, Evanston, Illinois 60201, USA
GRAZIADEI, P. P. C., Department of Biological Science, The Florida State Uni-
versity, Tallahassee, Florida 32306, USA
KAISSLING, Karl-Ernst, Max-Planck-Institut fUr Verhaltensphysiologie, Abteilung
Schneider, D-8131 Seewiesen, Germany
KOELLING, Robert A., University of California at Los Angeles, Los Angeles,
California 90024, USA
LEMAGNEN, J., College de France, F-75 Paris ve, France
MAC LEOD, P., College de France, F-75 Paris ve, France
MOULTON, David G., Monell Chemical Senses Center, University of Pennsylvania,
Philadelphia, Pennsylvania 19104, USA
MOZELL, Maxwell M., Department of Physiology, College of Medicine, State
University of New York, Syracuse, New York 13210, USA
OTTOSON, David, Kungl. VeterinarhOgskolan, Fysiologiska Institutionen, S-10405
Stockholm 50, Sweden
PARSONS, Thomas S., University of Toronto, Department of Zoology, Ramsay
Wright Zoological Laboratories, Toronto 5, Ontario, Canada
TAKAGI, Sadayuki F., Department of Physiology, School of Medicine, Gunma
University, Maebashi, Gunma, Japan
TUCKER, Don, Department of Biological Science, The Florida State University,
Tallahassee, Florida 32306, USA
WENZEL, Bernice M., Department of Physiology and Brain Research Institute,
UCLA School of Medicine, University of California, Los Angeles, California
90024, USA
Chapter 1
Anatomy of Nasal Structures

from a Comparative Viewpoint
By
THOMAS S. PARSONS, Toronto, Ontario (Canada)
With 14 Figures
Contents
Introduction . . I
Agnatha . . . . 2
Chondrichthyes . 4
Actinopterygii . 5
Sarcopterygii . . . . . . . . . . . . . . . . . . . 8
Tetrapods in General 10
Amphibia 10
Reptilia . 13
Aves 17
Mammalia 18
Evolution of Nasal Structure. 21
References . . . . . . . . . 23
Introduction
This review is concerned almost entirely with the gross anatomy of the nasal
cavities. Such a limitation, while necessary because of the space available, is
unfortunate, since many conclusions reached by different workers depend largely
on the microscopic anatomy and embryology of the nose and its relationship with
other structures, especially the skeleton and nervous system. Most of the references
cited contain information on other aspects and give further references; the review
by MATTHES (1934) is especially important.
Another limitation in any consideration of nasal evolution is the paucity of
information concerning extinct forms. The nasal tissues themselves are, of course,
soft and not preserved in fossils. Unfortunately, in most vertebrates the nasal
capsules are cartilaginous rather than bony, and so are rarely found. With few
exceptions, therefore, all our knowledge of nasal evolution comes from the ana-
tomical study of Recent forms. The exceptions do contribute important informa-
tion, but there is no palaeontological evidence at all on many debatable points.
1 Hh. Sensory Physiology. Vol. IV/l
2 T. S. PARSONS: Anatomy of Nasal Structures from a Comparative Viewpoint
The primary function of the nasal cavities is olfaction. Although in tetrapods

an additional function, serving as the first part of the respiratory passage and con-
ditioning the inspired air, has become important, even overshadowing the sensory
function as in man, most vertebrates still use the nasal cavities mainly, or even
only, as olfactory sense organs. Numerous fishes obtain some of their oxygen from
the air, but few have any connection between the nasal and oral cavities; those
that have such connections do not use them in aerial respiration (ATZ, 1952; PAN-
CHEN, 1967; SCHMALHAUSEN, 1968). Although hagfishes and the teleost Astroscopus
may be exceptions, very few fishes use an oro-nasal connection in aquatic respira-
tion. Thus most of the evolutionary changes in nasal structure, at least in an-
amniotes, should be explicable in terms of increased efficiency in olfaction, and few
of them related to respiration or other functions.
Agnatha
The modern cyclostomes, both lampreys and hagfishes, are unique among living
vertebrates in possessing single median nares and nasal cavities. Those of the
lamprey Petromyzon have recently been described in detail by KLEEREKOPER and
VAN ERKEL (1960), while MATTHES (1934) has reviewed the nasal anatomy of
cyclostomes generally.
Fig. 1. Petromyzon marinu8, the marine lamprey. Sagittal section of the nasal cavity, showing
the single mid dorsal external naris, the large nasal sac with the septum.like median lamella,
and the posteroventral nasopharyngeal pouch. Anterior is to the right
In lampreys (Fig. I) the naris lies on the top of the head, just anterior to the
eyes, and a short nasal tube runs posteroventrally from it. The nasal sac lies im-
mediately anterior to the brain and posterior to the ventral part of the nasal tube
which continues posteroventrally and posteriorly as a large, thin-walled naso-
pharyngeal pouch. A ventrally attached flap arises from the wall of the nasal tube
Agnatha 3
and acts as a valve controlling the passage of fluid between the tube and the nasal
sac. The nasopharyngeal pouch lies between the notochord and the brain. Its
posterior half is expanded into a wide, but low, sinus, while its anterior half is more
duct-like. Although this pouch has commonly been termed hypophyseal or naso-
hypophyseal, it probably lacks any relation to the hypophysis; it functions as an
aspirator, expanding and thus drawing water into the nasal cavity when the
muscles of the gills contract the branchial basket, and contracting and thus expell-
ing water as the branchial basket expands (KLEEREKOPER and VAN ERKEL, 1960).
The nasal sac is a more or less spherical chamber into which projects a series of
olfactory lamellae. The number of lamellae appears to vary, probably depending
on the species studied, and they may not be symmetrically arranged. Some 20 to
25 lamellae are attached peripherally, except anteriorly, and project into the
lumen of the sac; the median one is commonly better developed than the others
and forms a partial septum. Posteriorly the parts of adjacent lamellae may fuse
and thus restrict the space between them to tubular structures. Small accessory
olfactory organs lie at the ends of these tubules. Although these accessory organs
have generally been considered to be glands, HAGELIN and JOHNELS (1955) have
demonstrated that they are lined by sensory olfactory epithelium like that
covering most of the surfaces of the olfactory lamellae.
The nasal cavities of hagfishes differ from those of lampreys in three main ways:
(1) the naris lies at the anterior end of the snout and thus the long nasal tube runs
posteriorly; (2) the nasal sac contains fewer olfactory lamellae; and (3) the
nasopharyngeal pouch forms a duct that perforates the dorsal wall of the pharynx.
This pharyngeal connection allows a respiratory current of water to be drawn in
through the nasal cavity (STRAHAN, 1958).
Since all jawed vertebrates differ from cyclostomes in possessing paired nasal
cavities, the nasal structures of the extinct agnathans (ostracoderms) are of great
interest. Three main groups may be recognized, the Osteostraci, Anaspida, and
Heterostraci, as well as a few poorly known forms which cannot definitely be
placed in these groups. Both the Osteostraci (STENSIO, 1958, 1964, and 1968) and
the Anaspida (SMITH, 1957; HEINTZ, 1958; STENSIO, 1958, 1964, and 1968) have
single median nares piercing the dermal bones of the skull roof in the same position
as the nares of lampreys. Presumably the nasal anatomy of these forms was
basically similar to that of lampreys, but the fossil specimens, especially those of
anaspids, are not well enough ossified to allow the region to be studied in detail
(STENSIO, 1958 and 1964, gives elaborate descriptions and reconstructions).
The Heterostraci do not have a dorsally placed naris like that of other ostra-
coderms. Although most workers believe that the nasal cavities were paired and
lay within a pair of grooves on the ventral surface of the snout just anterior to the
mouth (HEINTZ, 1962), STENSIO (1958, 1964, and 1968) reconstructs these forms
with a single median naris lying just anterodorsal to the mouth. The fossil speci-
mens do not appear to allow any decision on this disagreement, but the paired
grooves are clearly present in most forms and there is no evidence in the bones for
a median naris. If the Heterostraci did have paired nasal cavities, they were the
only diplorhinal agnathans and might well be related to the ancestors of jawed
vertebrates; the cyclostomes, osteostracans, and anaspids would form a monorhinal
assemblage. If, on the other hand, STENSIO is correct, then all agnathans are
1*
monorhinal, with the lampreys, osteostracans, and anaspids having a dorsal naris,
and the hagfishes and heterostracans an anterior one.
Chondrichthyes
The paired nasal cavities of the cartilaginous fishes (Class Chondrichthyes) are
simple, round to oval chambers, each with a single, commonly elongate naris
Fig. 2. Raja erinacea, the skate. Ventral view of the single left external naris, showing its con-
nection, via a labial groove, to the mouth
Fig. 3. Squalus acanthias, the spiny dogfish. Anterior view of the left nasal cavity, showing the
single naris partly subdivided by flaps and the olfactory lamellae
Actinopterygii 5
opening on the ventral surface of the snout (reviews by MATTHES, 1934, and
TESTER, 1963; I know of no recent detailed studies of chondrichthyean nasal
anatomy). The long axes of the nasal cavities and nares are usually anterolateral
to posteromedial.
The naris is largely covered by flaps, usually one medially and one laterally
attached, which overlap at their centers and divide the naris into an anterior
incurrent aperture and a posterior ex current aperture. In some sharks and chimaer-
ans the excurrent aperture lies at the corner of the mouth and may be partly
covered by labial folds (Fig. 2). This arrangement, which superficially parallels the
formation of choanae in tetrapods in that the posterior nasal opening lies nearly or
even actually within the oral cavity, may result in a more effective drawing of an
inhalent current of water into the nasal cavity and over the sensory endings
(PANCHEN, 1967).
A ridge usually runs along the long axis of the nasal cavity opposite the naris.
On either side of this ridge, and at right angles to it, a series of olfactory lamellae
(Schneiderian folds) project into the nasal cavity (Fig. 3). In forms with nearly
circular nasal cavities, the central ridge may be very short, and the lamellae
radially arranged around it. The lamellae, which may bear smaller secondary folds
or lamellae, are covered by sensory olfactory epithelium.
Actinopterygii
The ray-finned or actinopterygian fishes, especially the teleosts, are the most
numerous and varied of vertebrates. Although there appear to be no extensive
reviews of the nasal anatomy of all of them, several studies describe or review
numerous species (BURNE, 1909; DERSCHEID, 1924; LIERMANN, 1933; MATTHES,
1934; REINKE, 1937; TEICHMANN, 1954; SCHMALHAUSEN, 1962; HOLL, 1965) or
consider one species in detail (LAIBACH, 1937; BRANSON, 1963; PFEIFFER, 1963).
The structural variation is so great that only a few of the different patterns can be
reviewed here.
The basic structures are a pair of ovoid cavities, each connected to the outside
by two openings, the anterior and posterior external nares (Fig. 4). A short ridge
or raphe runs along the central part of the long axis of the floor of the nasal cavity;
a series of olfactory lamellae extend out from this raphe, at right angles along its
sides and radially at its ends, and project into the cavity (Fig. 5). In most cases
each lamella bears a small "tongue" or linguiform process on its free margin and
numerous secondary lamellae along its sides (Fig. 6). The sensory epithelium is
concentrated on or restricted to part or all of the surfaces of the olfactory
lamellae.
There are many variants on this basic pattern. The raphe may be reduced so
that a groove appears between the olfactory lamellae. The shape of the raphe and
of the entire nasal cavity may vary, the latter being cssentially circular with the
raphe a central projection or both becoming extremely elongate. In other cases the
olfactory lamellae may be reduced in size or in number or may even be completely
absent. A series of parallel lamellae may occur in the absence of any raphe. Several
workers have tried to define different morphological types based on this variation,
but intermediate cases always occur and no type is sharply distinct. The figures
in the papers cited above demonstrate clearly the nature and extent of this
variation.
The structure and position of the nares also vary. In many forms the anterior
nares, and in some the posterior as well, are at the ends of short projections or
"tentacles". The posterior nares may have variously shaped valves and in some
Fig. 4. Aplodinotus grunniens, the freshwater drum. Lateral view of the two (anterior and
posterior) right external nares. Anterior is to the right
Fig. 5. Aplodinotus grunniens, the freshwater drum. Lateral view of the left nasal cavity,
showing the rosette of olfactory lamellae and entrance to the ethmoid (dorsal) and lachrymal
(ventral) accessory sacs or sinuses. Anterior is to the left
forms the bar of tissue between the two nares forms a vertical flap projecting out
from the surface of the fish. Most of these arrangements are believed to insure that
a stream of water will be drawn into the nasal cavity and passed over the sensory
areas of the olfactory lamellae. Certain fishes, such as sticklebacks (Gasterosteus)
Actinopterygii 7
and viviparous blennies (Zoarces), have only a single naris opening into each nasal
cavity; this opening may, in some cases, represent a conjunction of anterior and
posterior nares, but is usually the anterior, the posterior naris being secondarily
lost (LIERMANN, 1933). In a few eels, the posterior nares open within the oral cavity
(ATZ, 1952).
Fig. 6. Catostomus catostomus, the longnose sucker. Lateral view of the left nasal cavity,
showing the rosette of olfactory lamellae. Anterior is to the left
Many teleosts have accessory nasal sacs or sinuses associated with the main
cavities. There may be a single such sinus dorsal, posterior, or ventral to each nasal
cavity, or there may be both dorsal (ethmoid) and ventral (lachrymal) sinuses
(Fig. 5); the positions and sizes of these sinuses vary greatly in different fish
(BURNE, 1909). The sinuses are lined by non-sensory epithelium and are believed
to function as aspirators, drawing water into the nasal cavities and over the
olfactory lamellae (see PANCHEN, 1967, for references).
A few teleosts show more extreme variation. In Esox and Ctenolabrus (HOLL,
1965), the olfactory lamellae are not simple flaps, but are branched in different
ways to produce a more complex, sometimes irregular rosette in the nasal cavity.
Astroscopus possesses choanae which open from the nasal into the oral cavity.
These openings are not formed by one of the two external nares, which resemble
those of other teleosts, but by a new connection between the accessory sinus and
the oral cavity (ATZ, 1952). Different species of Tetraodon show different aberrant
patterns (WIEDERSHEIM, 1887): some have a projecting cylindrical nose with
anterior and posterior nares opening into it, others have the cylinder terminally
forked and without distinct nares, and still others have the nose reduced to a small
sensory area on the surface of the snout.
The more primitive actinopterygian fishes have been little studied, but
apparently resemble teleosts in their basic nasal anatomy (MATTHES, 1934; J AR-
VIK, 1942; SCHMALHAUSEN, 1962). However, Polypteru8, which is frequently con-
sidered to be in a group (Brachiopterygii) distinct from the Actinopterygii, is quite
different (JARVIK, 1942; PFEIFFER, 1968). Both the anterior, which is at the end of
a "tentacle", and the posterior nares enter a small chamber that also connects
with an anteroventral recess and, posterodorsally, with the main nasal cavity. AU
of these structures except the last are lined by non-sensory epithelium. Longi-
tudinal septa usually divide the ovoid main nasal cavity into five well separated,
radial sectors. These septa bear many olfactory lamellae that project into the nasal
cavity. A sixth, rather isolated sector lies medial to the anterior part of the nasal
cavity. The olfactory nerve runs down the solid core of the nasal cavity, with its
branches extending out into the septa. This complex arrangement does not
resemble closely that of any other vertebrates except the coelacanths.
Sarcopterygii
The fleshy-finned or sarcopterygian fishes include two main groups, the Dipnoi
(lungfish) and the Crossopterygii. Members of these groups are not especially
similar, and the closeness of their relationship is uncertain. The Dipnoi appear to
be a rather homogeneous group, and the living forms may be taken as probably
typical in their nasal anatomy; the Crossopterygii are much more varied, and the
only living genus is of an aberrant subgroup far removed from tetrapod ancestry.
Recent studies of dipnoan nasal anatomy are those of BERTMAR (1965 and
1966b) and THOMSON (1965), while major reviews include those of MATTHES (1934)
and JARVIK (1942). Each nasal cavity has two openings on the ventral surface of
the snout, the anterior just outside of the mouth and the posterior within the oral
cavity. Despite the position of the latter opening, it is almost universally, though
sometimes for different reasons, considered homologous with the posterior external
naris of actinopterygian fishes and not with the choana of tetrapods (JARVIK, 1942;
ATZ, 1952; THOMSON, 1965; BERTMAR, 1966b; PANCHEN, 1967). Lungfish do not
use their nasal cavities in respiration, and the intra-oral position of the posterior
naris is believed to produce a more efficient current of water over the sensory cells
of the nasal cavity (ATZ, 1952). The ventral position of the nares is characteristic
of fossil, as well as Recent, dipnoans (THOMSON, 1965).
The nasal cavities themselves are ovoid chambers with more or less transversely
oriented, dorsal and lateral olfactory lamellae. These lamellae may bear secondary
lamellae and are connected by small, irregular, longitudinal ridges. The pattern is
thus basically similar to that in most other fish and not to that of tetrapods.
Although various recesses and diverticulae have been reported, BERTMAR (1965)
has shown them merely to be the spaces between olfactory lamellae. There is no
evidence for a Jacobson's organ in lungfish (PARSONS, 1967).
The Crossopterygii are divided into three or four groups (JARVIK, 1966). One,
the Coelacanthini (= Actinistia), includes the living Latimeria; a second, the
Struniiformes, is poorly known and will not be discussed here; the last, the
Rhipidistia (= Osteolepiformes plus Porolepiformes), is known only from fossils,
Sarcopterygii 9
but, since this group includes the ancestors of tetrapods, is important in any con-
sideration of nasal evolution.
The best known coelacanth is, of course, Latimeria (MILLOT and ANTHONY, 1954
and 1965), but JARVIK (1942) has demonstrated that several extinct forms were
basically similar in their nasal anatomy. There is no choana, and the anterior and
posterior external nares, the former at the end of a short papilla, resemble those of
actinopterygian fishes. JARVIK reports the branching of the tube leading to the
posterior naris of fossil forms, thus producing two posterior nares on each side; on
the basis of early studies, he believed Latimeria to be similar, but MILLOT and
ANTHONY describe only a single posterior naris in that genus. Both anterior and
posterior nares enter the anterolateral corner of the nasal cavity which forms a
blind sac medial to them. JARVIK also reports the presence, at least in fossil forms,
of a small anteromedial recess of the nasal cavity. He thus considers the gross form
of the nasal cavity to be extremely similar to that of Polypterus.
The main nasal cavity of Latimeria contains five papilla-like olfactory lamellae
which appear to lie three on one side and two on the other of a median raphe much
like that of other fishes (MILLOT and ANTHONY, 1965, Fig. 42). However, medial to
the lamellae, in the deeper parts of the nasal sac, the epithelium forms a spongy
mass divisible, according to MILLOT and ANTHONY, into five lobes. Here again the
structure of Latimeria resembles closely that of Polypterus and is quite different
from that of other vertebrates.
The Rhipidistia have been studied in great detail by JARVIK (1942); his findings
have been challenged by THOMSON (1964) and reaffirmed by JARVIK (1966).
Rhipidistians are the only fish known to possess true choanae like those of tetrapods,
and they may also have both anterior and posterior external nares. The origin of
the choanae and their relation to the nares of other fish are much debated, and
there is still not complete agreement. The anterior external naris of fish becomes
the external naris of tetrapods. Most workers agree that the posterior external
naris of fish becomes the lachrymal duct; JARVIK (1942) has demonstrated the
presence of a fish-like posterior naris in Porolepis and of a tetrapod-like lachrymal
duct in Eusthenopteron among the rhipidistians. Although SCHMALHAUSEN (1968)
and some other workers believe that the choanae also develop from the posterior
external nares (the lachrymal duct developing in part, from the infraorbital sensory
canal), most consider the choanae to be a new development of rhipidistians (see
PANCHEN, 1967, and BERTMAR, 1969, for extensive discussions and references).
The nasal cavities of rhipidistians were relatively large, roughly ovoid cham-
bers. Porolepis is said to have had relatively simple cavities with lateral nasal
sinuses like those of urodeles, while Eusthenopteron had more complex nasal
cavities with principal and inferior cavities, the latter with medial and lateral
recesses like those of anurans (JARVIK, 1942; terminology that of this paper).
THOMSON (1964), who studied Ectostereorchachis, was unable to confirm JARVIK'S
conclusions, but JARVIK (1966) attributes this to the incompleteness of the ethmoid
region in THOMSON'S specimens. The closeness of similarity between the different
groups of rhipidistians and modern amphibians and its significance are currently
debated topics which cannot be treated here. Certainly the rhipidistians had nasal
cavities which resembled those of tetrapods at least as much as they did those of
other fishes. That the shape of their nasal cavities is perfectly reflected in the
surrounding bones appears unlikely, since most modern forms have considerable
connective tissue around the cavities and since olfactory lamellae, for example,
would not be indicated on the bones; nevertheless JARVIK (1942) presents an
admirably detailed study of what can be seen in this region in the fossil material.
Tetrapods in General
Before considering the different groups of tetrapods, it is convenient to define
several terms (PARSONS, 1967 and 1970); this terminology is based on the work
of many investigators. The nasal cavity usually consists of three main parts. First,
the cavum nasi proprium is usually a large, variably shaped space partly lined by
the olfactory (sensory) epithelium. Second, anterior to this, a more or less tubular
vestibulum may connect the external naris and the cavum; some forms lack a well-
marked vestibulum and the naris leads directly into the cavum. Various definitions
of the vestibulum, based on embryological or histological criteria, have been
proposed, but they are generally difficult to use, and the term is here used for any
chamber or tube between the naris and the cavum. Third, posteriorly there may be
another tubular portion, the nasopharyngeal duct, between the cavum and the
choana (= internal naris). Some workers restrict the term to cases in which a true
secondary palate is present, but it is here used in a more general sense for any
tubular structure in that position (FUCHS, 1915; PARSONS, 1959a).
In many amniotes, there are projections of the lateral nasal wall, generally
within the cavum nasi proprium; all such projections are here termed conchae.
GEGENBAUR (1873) and DEBEER (1937) have proposed more restricted (and mutu-
ally exclusive) definitions, but these have not been generally accepted (see PAR-
SONS, 1959a, for discussion).
Two types of sensory epithelium may be recognized within the nasal cavities of
tetrapods. One, the normal olfactory epithelium, is typically dorsally located, con-
tains Bowman's glands, and sends nerve fibers to the main part of the olfactory
bulb; the other, here termed vomeronasal epithelium, is ventrally located, lacks
Bowman's glands, and sends fibers to the accessory olfactory bulbs. Although
SEYDEL (1895 and 1896) and some other workers have designated all regions with
vomeronasal epithelium as Jacobson's organ, I prefer to restrict the latter term
to the vomeronasal epithelium of amniotes (except turtles) which forms in a distinct
ventromedial pocket at a very early embryonic stage and, when well developed,
forms a quite separate organ rather than simply a region of the cavum nasi
proprium (see PARSONS, 1959a and 1967, for discussion). BROMAN (1920) suggested
that the tetrapod vomeronasal epithelium is homologous with the olfactory
epithelium of fish (which lacks Bowman's glands), and that the tetrapod olfactory
epithelium is a new evolutionary development.
Amphibia
The amphibians are now represented by three rather highly specialized orders,
the Anura, Urodela, and Gymnophiona (= Apoda); their relationships to each
other and to other tetrapods are still poorly understood (PARSONS and WILLIAMS,
1963; JARVIK, 1966). The Palaeozoic amphibians, the labyrinthodonts and lepo-
Amphibia II
spondyls, are generally quite similar to rhipidistians and to primitive reptiles, but
difficult to relate to the living amphibians. Almost nothing is known of the nasal
anatomy of the extinct forms since the nasal capsules were but rarely ossified. One
genus, Eryops, had two canals, between the cranial cavity and each nasal cavity,
which have been interpreted as containing the olfactory and vomeronasal nerves;
thus this animal is said to have had a large Jacobson's organ (ROMER and EDINGER,
1942; SCHMALHAUSEN, 1968). Such an interpretation is, however, far from certain,
and the structures of the nasal cavities themselves are unknown.
The most important of the earlier studies of amphibian nasal anatomy is that
by SEYDEL (1895); a more recent study of urodeles is that of SCHUCH (1934) and of
anurans that of HELLING (1938). There are no detailed recent studies of gymno-
phionans, and the basic works remain those of SARASIN and SARASIN (1887-1890)
and WIEDERSHEIM (1879). MATTHES (1934) and PARSONS (1967) have reviewed
amphibian nasal anatomy .
.Fig. 7. Ambystoma maculatum, the spotted salamander. Dorsolateral view of the right nasal
cavity, showing the very simple nasal structure of this form. Anterior is to the right
Most urodeles have very simple nasal cavities lacking separate vestibula and
nasopharyngeal ducts. The cavum nasi proprium is a dorsoventrally flattened
ovoid chamber, with the external naris opening into its anterolateral corner and
the choana from its posterior border (Fig. 7). The lateral margin of the cavum is
extended laterally as a very low lateral nasal sinus (seitliche Nasenrinne); the
lachrymal duct enters the anterior half of this sinus, and vomeronasal epithelium
lines much of its posterior half which is, therefore, often called the Jacobson's
organ. Much of the literature on the nasal cavities of urodeles and other amphibians
is concerned with the formation of the choanae and whether or not they are
homologous in all tetrapods. This topic has recently been treated by BERTMAR
(1966a) who concludes that choanae are indeed homologous throughout the group.
The neotenous urodeles are more varied. Some, like axolotls (Ambystoma), show
the normal urodele pattern. Others, like Proteus, lack a lateral nasal sinus, and
have a longitudinal ridge along the ventral wall of the nasal cavity and a series of
smaller transverse ridges like the olfactory lamellae of most fish. Such transverse
ridges also occur in some fully metamorphosed forms (Ranodon; SCHMALHAUSEN,
1968), but are not typical of them. The nasal cavities of Siren (put into a separate
order, the Trachystomata, by GOIN and GOIN, 1962) are distinctive, with the
lateral nasal sinus ventrally located and shaped like an inverted "T".
Gymnophionans have more complex nasal cavities. A vestibulum is lacking,
and the external naris enters the anterior end of the cavum nasi proprium. Although
there is no well-marked nasopharyngeal duct, the posterior end of the cavum does
form a short tubular connection to the choana. This connection bears a small
anterior diverticulum and, just dorsal to the latter, a tubular structure extending
medially, then anteriorly, and finally laterally ventral to the cavum nasi proprium;
vomeronasal epithelium lines the floor of this tube which is entered laterally by the
lachrymal duct. The cavum itself is a rather large cavity, dorsoventrally flattened
and roughly triangular to oblong in dorsal view. Its ventral side is indented by a
well developed olfactory eminence which parallels the conchae of amniotes in its
general structure.
Fig. 8. Rana catesbeiana, the bullfrog. Lateral view of the right nasal cavity, showing both
internal and external nares and the complex form of the nasal cavity; the medial recess of the
inferior cavity appears here as an isolated opening. Anterior is to the right
Anurans vary considerably in their nasal anatomy, and, although it applies to

most, the following description is not universally valid. There is no nasopharyngeal
duct and little or no development of a vestibulum, but the nose is complex, with
Reptilia 13
the cavum nasi proprium divided into three chambers, the principal, middle, and
inferior cavities (Figs. 8 and 9). Olfactory epithelium is restricted to the first of
these and vomeronasal epithelium to the medial recess of the third; other areas
have non-sensory epithelium.
The principal cavity is a moderately large, nearly spherical chamber lying
dorsomedial to the rest of the nasal cavity. The external naris usually enters it
directly, but there may be a small vestibulum leading to the middle cavity instead.
The floor of the principal cavity is often raised into an olfactory eminence of
variable form, sometimes hemispherical and sometimes thinner and more lamellar.
Ventrolaterally this cavity connects with the middle cavity anteriorly and with the
inferior cavity and choana posteriorly. The smaller middle cavity is dorsoventrally
flattened and connected to both the principal and inferior cavities; it is also entered
by the lachrymal duct. The large inferior cavity is greatly flattened dorsoventrally.
The middle cavity enters it anteriorly, and farther posteriorly, it has a small con-
nection with the principal cavity and forms most of the choana. There is a small
medial and a large lateral recess of the inferior cavity.
Fig. 9. Rana catesbeiana, the bullfrog. Anterior view of the left nasal cavity showing the
superior and inferior cavities; the string passes through the choana
Reptilia
Reptilian nasal anatomy has been reviewed by MATTHES (1934) and PARSONS
(1959a, 1967, and 1970; the last is especially detailed). Other major studies
include the works of SEYDEL (1896) on turtles, HOPPE (1934) on Sphenodon, MALAN
(1946), PRATT (1948), STEBBINS (1948), and BELLAIRS and BOYD (1950) on squa-
mates, and BERTAU (1935) on crocodilians. The living orders differ markedly in
their nasal anatomy and must be described separately.
Most turtles have relatively simple nasal cavities. The vestibulum is a short
tubular structure leading from the anteriorly placed external naris to the cavum
nasi proprium. Posteriorly a nasopharyngeal duct of variable length leads to the
choana. There is often a flap or one or more papillae along the lateral margin of the
choana, and some turtles have a small anterolateral recess off the nasopharyngeal
duct.
The cavum nasi proprium is divided into a posterodorsal olfactory region and
an anteroventral intermediate region. The former is a mediolaterally compressed
hemisphere lined, at least in part, with olfactory epithelium. Tortoises have
relatively large olfactory regions, sometimes with a bulge (the Muschelwulst) in
theIr lateral wall, while sea turtles and soft-shelled turtles have much smaller ones
(Fig. 10). The larger intermediate region is roughly tubular and extends from the
vestibulum to the nasopharyngeal duct. Within this region are areas of vomeronasal
epithelium: in sea turtles in an anteroventral and an anterodorsal pocket, in soft-
shelled turtles in a complex pattern of shallow grooves or sulci, and in most other
turtles in one to four shallow longitudinal sulci. Most of the dorsal side of the inter-
mediate region is widely open into the olfactory region, but lateral and medial
ridges (the Grenzfalten) mark the boundary.
Fig. 10. Chelonia mydas, the green turtle. Medial view of the right nasal cavity, showing the
small posterodorsal olfactory region, large complex intermediate region, long nasopharyngeal
duct, and papillae along the choanal margin. Anterior is to the left
Sphenodon, the only living rhynchocephalian, has a small vestibulum leading

medially from the external naris to the anterior end of the cavum nasi proprium.
The latter is a large chamber with two, simple lamellar, partially fused conchae
projecting in from its lateral wall. Olfactory epithelium lines the dorsal half of the
cavum. The anterior concha runs posteroventrally and theposteriorposterodorsally,
so the two form a Von the wall of the cavum. There is no nasopharyngeal duct,
and the cavum enters the mouth directly along nearly its entire ventral extent. A
Reptilia 15
lateral projection at the base of the nasal septum (the vomerine cushion) and a
more ventral medial projection from the lateral portion of the palate (the choanal
fold) overlap to restrict the oronasal opening. Jacobson's organ is a small tubular
structure lying medial to the anteroventral portion of the cavum into which it
opens; the lachrymal duct enters the anterolateral wall of the cavum.
The Squamata are the most varied of the living reptiles, and this variation is
reflected in their nasal anatomy. In snakes (Fig. 11), amphisbaenians, and many
lizards, the vestibulum is a rather small, simple chamber or tube, leading medially
or posteromedially from the external naris to the anterior end of the cavum nasi
proprium. In some lizards, however, it is more complex and may become S-shaped
or greatly elongated dorsal to the cavum even, in extreme cases, entering the
posterodorsal corner of the latter. Such adaptations occur in desert inhabiting
Fig. 11. Crotalus atrox, the western diamondback rattlesnake. Medial view of the left nasal
cavity, showing the single large concha; Jacobson's organ is a separate structure ventral to the
nasal cavity. Anterior is to the right
iguanids, probably to prevent the entrance of sand into the nasal cavities (STEB-
BINS, 1948), in arboreal and microsmatic chamaeleonids, and in several other
groups.
The cavum nasi proprium is typically a relatively large chamber with a single
concha projecting medially from its lateral wall. The concha may be a large, in-
rolled, lamellar structure or may be smaller and simpler; in some forms, such as
marine snakes and many arboreal lizards, it is greatly reduced or absent. Olfactory
epithelium commonly lines most of the dorsal half of the cavum, but the amount
may be greatly reduced in some forms, generally those in which a concha is lacking.
The shape of the cavum varies considerably depending on the form of the concha,
but there are few basic differences in the structure of different squamates.
There is a short nasopharyngeal duct in snakes and some lizards, but most
lizards have the posterior part of the cavum nasi proprium opening directly into
the oral cavity. The basic structure is almost always the same and like that of
Sphenodon, and the variation, which may be extensive even with a single family,
is in the amount of fusion between the sides of the primitive choanae. There is
always some fusion anteriorly. The lachrymal duct may enter the cavum near its
oral connection, open onto the palate, join the duct of Jacobson's organ, or even
open into two of these places.
Jacobson's organ is usually large, especially in snakes, and forms an organ
entirely separated from the nasal cavities in adults. It is usually a roughly hemi-
spherical structure, with the ventral side indented by the mushroom body which
nearly fills its lumen. Vomeronasal epithelium lines its dorsal walls. Embryolo-
gically the organ forms as part of the nasal cavity, but becomes isolated with the
closure of the anterior end of the choana. Jacobson's organ may be reduced in some
lizards and is virtually absent in chamaeleonids.
Crocodilians have very complex nasal cavities. The vestibulum is short, running
ventrally from the external naris to the anterior end of the long cavum nasi pro-
prium. A true secondary palate is present, and a very well developed, tubular
nasopharyngeal duct leads posteriorly from the floor of the cavum to the choana.
Within the cavum there are three projections of the lateral wall, the pre concha ,
concha, and postconcha. The first two are more or less lamellar projections
developed from the concha of the embryo, and the last is simply a marked con-
vexity projecting into the posterolateral portion of the cavum. The extension of
the cavum behind the pre concha is the preconchal recess and that behind the
concha is the extraconchal recess; the dorsal half of these recesses and of the rest
of the cavum is lined by olfactory epithelium. There are also several accessory
sinuses or recesses, comparable but not homologous to the accessory nasal sinuses
of mammals: a maxillary sinus anteroventrally, a caviconchal recess within and
lateral to the concha, a postconchal cavity within the postconcha, a tubular post-
turbinal sinus connecting the postconchal cavity and extraconchal recess, and a
posterolateral recess ventrolateral to the postconcha. Jacobson's organ, with its
vomeronasal epithelium, is completely lacking in adults.
For only three of the numerous and diverse groups of extinct reptiles is any-
thing known about the nasal anatomy. In Phytosaurus, a large,rather crocodile-like
form, the external naris was high on the head, directly dorsal to the choana;
there was, on the lateral wall of the nasal cavity well anterior to the naris,
a "turbinal" or "lateral ethmoid" which has never been described in detail
(CAMP, 1930). Many hadrosaurid dinosaurs had elaborate crests formed by
the premaxillary and nasal bones and containing part of the nasal cavities which
thus followed a complicated path from the external naris to the choana. Many
diverse theories have been proposed to explain this unusual structure, and OSTROM
(1961) believes the enlarged nasal area to have been used for an increase in the
amount of sensory epithelium. Finally, in advanced mammal-like reptiles, such as
Diademodon and Thrinaxodon, a series of ridges occurred on the inner surfaces of
the nasal, frontal, and prefrontal bones which appear to have been the bases of
unossified ethmoturbinals like those of mammals. These forms also had well
developed secondary palates and hence nasopharyngeal ducts, but there appears
to be no evidence for other mammalian characters such as maxilloturbinals or
accessory nasal sinuses (WATSON, 1913 and 1951: BRINK, 1957).
Aves 17
Aves
Olfaction is not a major sense in most birds, and there have been relatively few
detailed studies of their nasal anatomy. TECHNAU (1936) and KISELEVA (1937)
examined large series of forms while JONES (1937 a, b, c) and BANG (1964 and
1968) have made more detailed studies of selected species. The most important
reviews are those by MATTHES (1934) and PORTMANN (1961).
The external nares lie at the base of the bill in almost all birds, but are at its
tip in the kiwi. They open into a relatively large but poorly defined vestibulum.
The exact shape of the external nares varies greatly; there may be various flaps
which partially close it or other special structures associated with it. The lateral or
dorsolateral vestibular wall commonly bears a simple lamellar structure, the
anterior concha, projecting into its lumen, but this is reduced or absent in some
forms. In most birds the nasal septum is complete, but in some it is perforated and
the vestibula of the two sides are connected (e.g., cathartid vultures; BANG, 1964).
The cavum nasi proprium consists of two rather weakly separated parts, an
anteroventral main chamber and a posterodorsal olfactory chamber (Fig. 12). The
former is continuous with the vestibulum anteriorly and with the olfactory chamber
posteriorly; ventrally it is widely open into the oral cavity through a long, single,
median choana. A relatively large middle concha projects into the main chamber
from its lateral or dorsolateral wall. This concha is normally a rather thin, lamellar
projection which bends ventrally and then laterally, and may form a much coiled
scroll. The lachrymal duct enters the nasal cavity ventral to the middle concha.
Fig. 12. Turdus migratorius, the American robin. Medial view of the left nasal cavity, showing
the well developed anterior and middle conchae; the small posterodorsal posterior concha is
not evident. Anterior is to the right
The pocket-like olfactory chamber lies posterior to the main chamber. It is the
only part of the nasal cavity lined, in part, with sensory olfactory epithelium, and
its size and complexity vary with the development of the olfactory sense in dif-
ferent birds. In most the lateral wall of the olfactory chamber bears a more or less
hemispherical projection, the posterior concha or olfactory tubercle, which is
covered by sensory epithelium. However, in forms in which the sense of smell is
especially poorly developed, the posterior concha may be completely lacking (e.g.,
2 Rh. Sensory Physiology, Vol. IV!l
pigeons; MATTHES, 1934, and TECHNAU, 1936). On the other hand, it may, in
forms with a well developed sense of smell, be a complex, coiled structure much
like the middle concha (e.g., albatrosses and cathartid vultures; JONES, 1937 c; and
BANG, 1964). Finally, in the kiwi (Apteryx), instead of a single posterior concha,
there is a series of five projections resembling the ethmoturbinals of mammals
(MATTHES, 1934).
Both nasal cavities enter the mouth through a common, elongate choana, and
there is no nasopharyngeal duct. Although a rudimentary .Jacobson's organ is
visible in embryos, it is completely lacking in adult birds (GREWE, 1951). A well
developed system of accessory sinuses within the bones of the skull, in the beak and
around the eye, are connected with the nasal cavities. BANG (1964) shows these
orbital sinuses and beak cavities entering the main chamber near the posterodorsal
corner of the middle concha.
Mammalia
The nasal cavities reach their greatest complexity in the Mammalia. This
complexity is related not only to the high development of the olfactory sense
characteristic of most mammals, but also to the need, in rapidly breathing,
homoiothermic animals, to insure that the inspired air is warm, moist, and relatively
clean. However, there is much variation in nasal structure, and some mammals,
most notably cetaceans, chiropterans, and primates, have secondarily simplified
nasal cavities. Mammalian nasal anatomy cannot be discussed here in any detail.
Major surveys, which contain numerous references to more detailed works, include
those by MATTHES (1934), KEILBACH (1954), and NEGUS (1958).
The external naris opens into a small vestibulum that, although histologically
distinct, is not grossly separated from the cavum nasi proprium. The walls of the
vestibulum are commonly simple, but may bear a conchal structure laterally; the
concha may form a separate atrioturbinal or appear as the anterior end of the
maxilloturbinal. In many forms, especially aquatic ones (NEGUS, 1958), specializa-
tions allow the opening and closing of the external nare. These modifications are
most highly developed in the cetaceans which have a complex musculature con-
trolling the narial opening (LAWRENCE and SCHEVILL, 1956). In a few mammals,
most notably proboscideans, the vestibulum is greatly elongated within a motile
trunk.
The cavum nasi proprium is very large, extending posteriorlyuntilit is separated
from the brain only by the cribriform plate of the ethmoid; there is no interorbital
septum in mammals. A series of conchal formations project into the cavum from
its lateral wall (Fig. 13). The largest is usually the maxilloturbinal which extends
along nearly the entire length of the ventral half of the cavum. Dorsal to it lies the
smaller nasoturbinal, and, posterior to the latter, there are generally a number of
ethmoturbinals. The ethmoturbinals and extreme posterior end of the nasoturbinal
may bear olfactory epithelium, while the maxilloturbinal and most of the naso-
turbinal always bear non-sensory respiratory epithelium. The various turbinals
nearly fill the nasal cavity, and the passages ventral to the maxilloturbinal, between
it and the other turbinals, and dorsal to the nasoturbinal and ethmoturbinals are
Mammalia 19
often referred to as ventral (inferior), middle, and dorsal (superior) meatuses

respectively. The lachrymal duct enters the anterolateral part of the ventral
meatus.
The maxilloturbinal functions mainly in conditioning the inspired air. Three
degrees of complexity may be recognized, differing in the amount of surface area
over which the air must pass. The simplest is an unbranched, lamellar projection
which may be coiled like a scroll; such a maxilloturbinal occurs in man and many
Fig. 13. Sus scrota, the domestic pig. Medial view of the left nasal cavity of a late foetus,
showing the anteroventral maxilloturbinal, anterodorsal nasoturbinal, and series of posterior
ethmoturbinals. Anterior is to the right
other primates, most bats, and a few other forms. The second type branches once
and thus resembles a T in section; usually both free ends are coiled, so that this
type of concha is often called a double scroll. Most artiodactyls, many rodents, and
various other mammals have such maxilloturbinals. Finally, in the branched type,
in rabbits, most carnivores, and various other forms, and reaching its greatest
complexity in seals, the turbinal is very highly branched and forms a large laby-
rinthine mass. Some workers recognize a further category, intermediate between
the last two described above.
The smaller nasoturbinal is almost always a simple, lamellar projection which
may be coiled. In some carnivores, such as the cat, it lies partly within the frontal
sinus rather than within the nasal cavity proper (Fig. 14).
The ethmoturbinals, although generally coiled, are usually branched slightly or
not at all. In most mammals a series of large endoturbinalia project nearly to the
medial wall of the nasal cavity; between them lie much shorter ectoturbinalia
which are completely hidden in medial view. Both the size and the number of
ethmoturbinals vary greatly, depending on the olfactory acuity of the form. There
2*
may be 0 to 7 endoturbinalia in each nasal cavity, but most macrosmatic mammals

have 3 to 5. The ethmoturbinals form a rather spongy mass with an enormous
surface area and very restricted air spaces. In some carnivores ethmoturbinals may
extend into the frontal and sphenoidal sinuses.
The Jacobson's organ of mammals is a small tubular structure lying within the
nasal septum beside the anteroventral part of the cavum nasi proprium. It has a
very narrow duct which may enter the nasal cavity directly or may enter the
nasopalatine duct, the latter running between the nasal and oral cavities. In
microsmatic mammals Jacobson's organ is frequently absent.
Fig. 14. Felis domestica, the domestic cat. Medial view of the right nasal cavity, showing the
large and complexly ridged maxilloturbinal, the nasoturbinal which extends into the frontal
sinus dorsal to the nasal cavity, and the ethmoturbinals. Anterior is to the left
In most mammals a series of accessory sinuses enter the nasal cavity. Their
number, shapes, and interconnections vary so greatly that it is impossible to
summarize briefly their structure. Although this classification is unsatisfactory,
paranasal sinuses are frequently divided into a lateral maxillary sinus, a dorsal
frontal sinus, and a posteroventral sphenoidal sinus. The sinuses are normally lined
by non-sensory epithelium, but in some carnivores the nasoturbinal may lie within
the frontal sinus and ethmoturbinals within the frontal and sphenoidal sinuses;
such turbinals bear sensory olfactory epithelium. The size and shape of the sinuses
depends on the shapes of the snout and of the nasal cavities, and has been reviewed
by NEGUS (1958).
Mammals possess a well developed secondary palate and thus have long
nasopharyngeal ducts. These ducts are normally rather simple tubular structures.
They empty into the median nasopharynx, dorsal to the soft palate, which, in
turn, enters the main portion of the pharynx dorsal to the larynx. The nature of
the nasopharyngeal ducts and the nasopharynx has been much debated; CAVE
(1967) has recently reviewed this problem.
Evolution of Nasal Structure 21
The nasal anatomy of various mammals is much modified. There may be a

general simplification, as in man, or more drastic changes, as in cetaceans. Such
modifications are considered in some detail by MATTHES (1934) and NEGUS (1958).
Evolution of Nasal Structure

Due to our lack of knowledge of the nasal anatomy of most extinct forms, it is
impossible to present a detailed and accurate picture of nasal evolution. Some
points, however, can be made and some questions raised.
Most, possibly all, agnathans are monorhinal, with the single nostril usually
dorsally located; all gnathostomes, and probably the extinct Heterostraci among
the agnathans, are diplorhinal, with the paired nostrils usually anterior. This might
suggest that monorhiny is primitive for vertebrates and that diplorhiny developed
in the line leading to gnathostomes. However the monorhinal cyclostomes have
paired olfactory nerves and bulbs which could be considered evidence for the
fusion of paired nasal cavities, that is for primitive diplorhiny. STENSIO (1968) has
recently argued for the originally paired nature of the olfactory sacs in all verte-
brates; he believes the single nostril (and the nasopharyngeal pouch) of agnathans
to represent an "extracephalic" pre nasal sinus. Lower chordates appear to lack
nasal organs completely; Ki:illiker's pit, once thought to be the nasal cavity of
amphioxus, is now considered merely a remnant of the anterior neuropore (DRACH,
1948).
Most gnathostome fish have basically similar nasal cavities with two more or
less well separated external nares and a series of olfactory lamellae, the latter
usually arranged around a central projection or ridge. Two types of modifications
arise independently in different lines, both apparently serving to increase the
efficiency with which water is drawn over the sensory areas (BERTMAR, 1969). One
is to develop various accessory chambers which act as aspirators to draw water in
and out; these occur in many actinopterygians as well as in lampreys. The second
modification is to develop a connection between the nasal and oral cavities so that
the respiratory stream of water draws water through the nasal cavity; such a con-
nection may result from the migration of a posterior external naris to the margin
of (some chondrichthyeans) or into (Dipnoi) the oral cavity or from the develop-
ment of a new opening between the nasal and oral cavities (a few teleosts and the
rhipidistians) .
A few fish do not follow the usual pattern. Polypterus and Latimeria are highly,
and similarly, modified, but the significance of this is unknown. The taxonomic
position of both forms has been debated, but most workers agree on their extremely
isolated evolutionary positions among living fish. The rhipidistian crossopterygians
also appear to have differed from other fish in their nasal anatomy and to have
resembled tetrapods in many ways (JARVIK, 1942), but the available fossils do not
permit a complete reconstruction of their nasal anatomy.
The situation in tetrapods is more complex and not well understood. I have
twice reviewed the nasal evolution of tetrapods (PARSONS, 1959b and 1967), but
still cannot present a detailed or completely convincing account.
There are two major problems concerning amphibians, solutions to which are
necessary for a proper interpretation of their nasal structure. First, the relationships
of the modern Amphibia are not well understood. JARVIK (1942) and others have
postulated that urodeles (and probably gymnophionans) are only remotely related
to other tetrapods; if this is the case, their nasal anatomy cannot be taken to
indicate anything about that of other tetrapods. However, other workers have
disagreed and thought urodeles to be much more closely allied to frogs and other
living tetrapods (PARSONS and WILLIAMS, 1963). The second problem is to deter-
mine, assuming that urodeles are relevant to this story, how much of the simplicity
of their anatomy is primitive and how much secondary.
Urodeles do have simple nasal cavities with virtually no specializations which
would make them structurally unsuitable as ancestors of other tetrapods. Obvi-
ously modern urodeles are not ancestral to other groups, but their nasal anatomy
could reflect the ancestral condition. It is interesting, although of uncertain
significance, that the neural separation of olfactory epithelium (fibers going to the
main olfactory bulb) and vomeronasal epithelium (fibers to the accessory
olfactory bulb) is incomplete, whether primitively or secondarily, in urodeles
(HERRICK, 1921). The other amphibians, frogs and gymnophionans, have more
complex nasal cavities which do not particularly resemble those of any other group
and which must be assumed to show specializations developed within the living
orders or their immediate ancestors.
The homologies of the conchae are important in considerations of amniote nasal
anatomy. Turtles lack them and the Squamata have only one, the one that is first
to develop and largest in members of almost all groups, being homologous with the
posterior concha of Sphenodon, the pre concha and concha of crocodilians, the
middle concha of birds, and the maxilloturbinal of mammals. The crocodilian
post concha and avian posterior concha are homologs, and some workers, for no
convincing reasons (see PARSONS, 1959a), have also included the mammalian
nasoturbinal with these; its homology with the others, although possible, is unlikely
when the phylogeny of the various forms is considered. The other conchal struc-
tures all appear to be independent developments within the groups possessing them.
The nasal cavities of turtles lack conchae and distinct Jacobson's organs and
resemble those of urodeles; it is thus tempting to consider them primitive as well as
simple. In all other amniotes both Jacobson's organ and at least one concha appear
in very early embryonic stages, even in forms in which they are lacking in adults.
From this embryonic form, with a ventromedial pouch representing Jacobson's
organ and one concha, it is easy to derive all non-testudinine amniotes by accepting
ontogeny as a guide to phylogeny: in squamates Jacobson's organ becomes
separated from the nasal cavity; in Sphenodon a second concha develops; in
crocodilians and birds Jacobson's organ is lost and a more posterior concha develops
as does, independently in the two groups, a more anterior one; and in mammals a
series of new conchal formations appears.
The preceding paragraph gives a simple, plausible picture of amniote nasal
evolution. It nowhere conflicts violently with generally accepted ideas on phylogeny,
although the origin of the Testudines, from exceedingly primitive forms according
to this argument, is not well known (OLSON, 1965). However, despite its simplicity
and plausibility, there is no real evidence in favour of this picture of nasal evolu-
tion. Complex structures, such as conchae, could have been present in the an-
cestors of turtles and have been lost completely; structures which appear homo-
References 23
logous could represent parallel developments in separate lines. Only speculation is

possible, and the available evidence does not, to me, appear sufficient to make
speculation beyond that given above and in earlier papers (PARSONS, 1959b and
1967) profitable.
Note Added in Proof. Since the above was written, several major works have appeared, the
most important of which is Herman Kleerekoper's excellent book (H. KLEEREKOPER: Olfaction
in fishes. Bloomington: Univ. Indiana Press 1969) on most aspect of olfaction in fishes, includ-
ing a review of their gross nasal anatomy.
Acknowledgement
I thank Dr. C. S. CHURCHER and Dr. M. C. PARSONS for critically reading this manuscript,
Miss C. R. GRUENWALD, Miss B. M. HALL, Miss A. r. FEDORENKO, and Miss N. M. SCOTT for
their assistance in its preparation. Two of the photographs were taken by Mr. N. HATTON.
Some of the work was supported by Grant A-1724 from the National Research Council of
Canada.
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Chapter 2
The Olfactory Mucosa of Vertebrates

By
p. P. c. GRAZIADEI, Tallahassee, Florida (USA)
With 24 Figures
Contents
The Nasal Cavities . . . . . . . . 29
Macroscopic Observations . . . . 29
Histology of the Olfactory Mucosa 29
Ultrastructure of the Olfactory Mucosa 31
The Olfactory Receptor . 31
The Olfactory Vesicle. 31
The Dendritic Process. 34
Receptor Cell Bodies 34
Cilia . . . . . 37
Basal Bodies . 40
Supporting Cells 42
Basal Cells . . 44
The Vomeronasal Epithelium . 48
Summary 52
References 55
The vertebrate olfactory mucosa is that part of the lining membrane of the
nasal cavities that contains olfactory receptor neurons. It is structurally different
from the surrounding respiratory mucosa in many important respects, such as,
a) the presence of Bowman's glands, b) a characteristic yellow to brown color due
to a pigment, c) and cilia not coordinately beating as opposed to the rhythmically
beating cilia ofthe respiratory regions. The sensory area may be recognized macro-
scopically by the naked eye or with the help of a low power dissecting microscope
because of its properties mentioned in b) and c). Recognition of the chemo-sensory
property of the olfactory mucosa dates back to the macroscopic observations of
early anatomists (see SAPPEY for a survey, 1877). In 1536 MASSA demonstrated the
olfactory nerves and subsequently SCARPA (1789) showed that the fine branches of
the fila olfactoria were actually ending in the olfactory region. The histological
structure of the olfactory mucosa was first described by ECKHARD (1855), ECKER
(1855), SCHULTZE (1856-1862), and KRAUSE (1876). These early studies revealed
28 P. P. C. GRAZIADEI: The Olfactory Mucosa of Vertebrates
that the olfactory mucosa in vertebrates is composed of three cellular elements:

supporting cells, basal cells, and receptor cells; the latter being provided with a
variable number of cilia. The continuity, however, between the intraepithelial
receptors and the terminals of the olfactory nerves was not clear from these
researches. However, SCHULTZE postulated continuity between the nerve fibres
and receptors, the demonstration of this continuity was not given till the discovery
of new nerve staining methods. BABUCHIN (1872), EHRLICH (1886), ARNSTEIN
(1887), GRASSI and CASTRONOVO (1889), VAN GEHUCHTEN (1890), VON BRUNN
(1892), RETZIUS (1892-1894) and CAJAL (1890) provided this evidence. Obser-
vations from these workers clearly showed that the olfactory fibres originated from
the olfactory receptors and that these fibres run directly to the olfactory bulb
without collaterals.
That the olfactory mucosa receives branches ofthe V cranial nerve was established
both by macroscopic (MILNE EDWARDS, 1844; SAPPEY, 1877) and subsequently by
histological methods (GRASSI and CASTRONOVO, 1889; VON BRUNN, 1892; RETZIUS,
1892; VON LENHOSSEK, 1892; MORRILL, 1898; JAGODOWSKI, 1901). The functional
importance of this nervous compound is not completely understood, however
trigeminal electrical activity after odor stimulation has been recorded (TUCKER,
1963).
Due to the obvious topographical relations with the odorous particles, the
surface of the olfactory mucosa with its morphological details attracted the atten-
tion and speculation of the early observers. MILNE EDWARDS (1844) regarded the
mucous layer covering the epithelium as responsible for adsorbing the odor
particles. Cilia were observed by all students after the first observations by
SCHULTZE (1856 -1862) and their reduced motility, as opposed to the very motile
respiratory cilia, was determined under the microscope (SAPPEY, 1877). JAGO-
DOWSKI (1901) observed that the cilia reached the outer surface of the mucous
layer. Their role as receptive elements of the olfactory receptors was postulated by
many and emphasized by PARKER (1922)in his review on thf'. anatomy and physiol-
ogy of the chemical senses. The classic observation of the early students, conducted
on many animal species, stressed the rather uniform structural pattern of the
olfactory mucosa all through the vertebrates. Further histological observations
(HOPKINS, 1926) and comprehensive reviews (ALLISON, 1953; LE GROS CLARK,
1956, 1957; KLEEREKOPER et al., 1960) confirmed the homogeneous plan of the
organ, fundamentally represented by receptors, supporting and basal cells in the
epithelium, and by a lamina propria where a set of specific glands, the Bowman's
glands were contained.
Ultrastructural observations with the electron microscope (EM) substantiate
the previous light microscope (LM) findings complementing them with finer details.
The first observations were performed by BLOOM and ENGSTROM (1952) in humans
and BLOOM (1954) in amphibia. They established the presence of typical 9 + 2 set
of filaments in the ciliary processes of the receptors and the presence of microvilli,
rather than cilia, in the supporting cells. Presumed secretory granules were also
observed in the latter. The constant presence of mitochondria in the receptor
dendrites and a system of membranes later recognized as endoplasmic reticulum
(ER) in their perikaryon were also described. GASSER (1956) measured the diameter
of the olfactory axons giving a mean value of 0.2 fl. Subsequent to these pioneering
Histology of the Olfactory Mucose 29
observations many authors described the olfactory mucosa in many animal species.
Their findings, when pertinent, will be referred to in the following description.
The Nasal Cavities

The olfactory mucosa is a discrete portion of the covering epithelium found in
the nasal cavity where bipolar receptor neurons are found. In terrestrial vertebrates
this area is usually situated posterior to the vestibular and respiratory regions. In
fish no true nasal cavity is observed and the olfactory epithelium lies in a pair of
pits situated in the head. These pits can be modified to canal-like passages with
two openings.
The primary sensory cells of the olfactory mucosa differentiate directly from a
portion of the cranial ectoderm. From the olfactory placode, the centripetal proc-
esses of the receptors constitute the olfactory nerves and secondarily reach the
olfactory bulb. While the eNS neurons do not regenerate or reduplicate in adult
animals, the possibility of regeneration in the olfactory cells has been recently
proved (GRAZIADEI and METCALF, 1971). A survey on this subject is given elsewhere
in this series by TAKAGI (this volume). The area occupied by the olfactory mucosa
in the nasal cavity is not well known for many vertebrate species. Estimates of
2-4 cm2 are usually reported for humans, while rabbit has 9.3 cm 2 (ALLISON, 1953)
and cat has 20.8 cm 2 (NEGUS, 1958). Dimensions of the sensory epithelium are not
a good predictor of the olfactory acuity of the animal, however, as the receptor
density varies not only from one animal species to another but in the same animal
as well from one point to another of the olfactory epithelium (GRAZIADEI, 1971).
Macroscopic Observations
Macroscopic observations of the nasal cavity show that the olfactory area has
a faint yellow to brown color as opposed to the pinkish color of the surrounding
respiratory mucosa. This is due to the presence of a pigment which has been
localized mainly in supporting cells and Bowman's gland cells by some authors
(ALLISON, 1953; BARADI and BOURNE, 1953; MULLER, 1955; ARDOUIN, 1964;
TAKAGI et al., 1965) and in receptor cells as well (GEREBTZOFF et al., 1952). The
importance of this pigment in the olfactory mechanism was stressed by early
observers (OGLE, 1870) and more recently MONCRIEFF, (1951-1967) has discussed
in detail the importance of pigment in olfaction. However, proof has not been so
far collected in favor of a specific role of the pigment in the olfactory mechanism
(see MOULTON and BEIDLER, 1967 and MOULTON, this volume for a review). There
is at present no ultrastructural study on the precise localization of the pigment in
the olfactory mucosa. Its chemical composition is also unclear. KURmARA (1967)
has recently found that in bovines the pigment is principally an insoluble chro-
moprotein.
Histology of the Olfactory Mucosa

The olfactory mucosa of the adult vertebrates is composed of a} a columnar,
pseudo-stratified epithelium, also called neuroepithelium as it contains the
olfactory neurons; b} a basal lamina; c} the lamina propria mucosae which directly
adheres to the underlying bony or cartiligeneous tissue without a discrete sub-
mucosa layer. Tubulo alveolar glands called Bowman's glands with the alveoli
lying in the lamina propria and opening their ducts at the epithelial surface are
distributed over the olfactory area of all vertebrates except fish (BANG and BANG,
1959).
mu
Fig. 1. Histological section of Gopher tortoise's olfactory mucosa stained with iron hematoxylin.
The surface is covered with a layer of mucous (mu) in which cilia (ci) are embedded. Three
nuclear layers are discretely arranged in the epithelium. From the top they are: supporting cells'
nuclei (scn), neurons' nuclei (nn), basal cell's nuclei (bcn). The duct of a Bowman's gland (Bg)
penetrates the epithelium 1
The olfactory epithelium as seen in histological sections is thicker than the

surrounding respiratory epithelium. Its thickness, however, varies from one
animal to the other ranging from 30 [L in moles to 150-200 [L in frogs and turtles.
1 All the light microscope preparations were fixed in Bouin's and stained in iron hematoxylin.
All the material for electron microscope pictures was fixed in 2% OS04 in phosphate buffer
and stained in lead citrate and uranil acetate.
The Olfactory Vesicle 31
The nuclei of the three cellular components (receptor cells, supporting cells
and basal cells) are discretely arranged in separate layers as can be seen in Fig. l.
The epithelial surface is normally covered by a layer of mucus 10 to 40 11- thick
in which receptor cilia and the microvilli of supporting cells are embedded.
A continuous basal lamina underlines the epithelium, i.e. continuous with the
lamina enveloping the Schwann cells of the nerve bundles and the Bowman's
glands. The thickness of this structure is 0.1-0.211- and, while it is usually observed
in EM preparations, histological sections often fail to demonstrate it. The lamina
propria mucosae consists of loose connective tissue in which are found the alveoli
of the Bowman's glands, the fila olfactoria, and blood vessels.
Ultrastructure of the Olfactory Mucosa

The olfactory neuroepithelium is a structural unit where each histological
component is integrated anatomically and functionally with the others. For
descriptive purposes, however, each cellular component will separately be described.
The Olfactory Receptor

The olfactory receptor is a typical primary sensory neuron. It is located in the
epithelium with the olfactory vesicle emerging free at the mucus-cell interface,
and the axon crossing the basal lamina on its way to the olfactory bulb. The
receptor has a flask-like shape, its length is directly proportional to the thickness
of the epithelium, while the diameter of its processes remains more constant. The
olfactory vesicle, ubiquitously provided with cilia, has an approximate diameter of
2-311-, the dendrite of 1-211-, the cell body of 5-8 11-. The axon, is always un-
myelinated and soon after its origin attains a diameter of 0.2-0.3 11- which is con-
stant during its entire course to the olfactory bulb. Olfactory receptors in spite of
a common basic design are rather polymorphic as observed by many students.
DOGIEL (1887), MORRILL (1898), JAGODOWSKI (1901) described in fish cylindrical,
conical and spindle shaped receptors. Recently LE GROS CLARK (1957) described
at LM level many morphological differences in the receptors, and proposed that
this could indicate functionally different units. At an electron microscopic level it
is common to find in the same species receptors varying in shape and content of
organelles, as well as in the general density of the cytoplasm. ANDRES (1969a, b)
has suggested that these differences may represent steps in the maturation of
receptors. Previously ANDRES (1966) described special blastema cells that he con-
sidered to be precursors for regenerating sensory neurons. Recent observations with
autoradiographic techniques have provided positive evidence that olfactory
neurons are continuously replaced in adult vertebrates from staminal elements
(GRAZIADEI and METCALF, 1971) previously described as basal cells. It appears
likely that the differences observed in the receptors' morphology may be at least
partially related to the different stages of maturation and subsequent degeneration
of each receptor.
The Olfactory Vesicle

The olfactory vesicle, the only bare portion of the dendrite, (Fig. 2) is con-
sistently provided with cilia, and it usually but not always protrudes from the
epithelial surface. In many species the olfactory vesicle has cilia and microvilli
(BROWN and BEIDLER, 1966; GRAZIADEI and BANNISTER, 1967; WILSON et al.,
1967; GRAZIADEI, 1968). The shape of the vesicle varies between species and among
species as well (Fig. 3). BANNISTER (1965) has described receptors in fish bearing
Fig. 2. In a section tangential to the surface of the frog's olfactory mucosa only the olfactory
vesicles of the receptors (v) and microvilli (mi) of the supporting cells are observed. Cilia
directed in their first part upward are not shown in this picture
only microvilli but not cilia. GRAZIADEI et al. (1967) have estimated that cilia and
microvilli of duck may increase the receptor bare surface some one thousand or
more times. Protrusion of the olfactory vesicle from the epithelial surface is often
hidden and covered by the protruding distal portion of supporting cells (Fig. 5).
These latter often show vigorous secretory activity and the free epithelial surface of
these cells forms a very irregular outline. That the dendritic portion of the olfactory
receptor may undergo some active movement has been proposed by VINNIKOV and
TITOVA (1957). This phenomenon, called "olfactomotor reaction" may be partially
responsible for the variable prominence of the olfactory vesicles in fixed material.
The olfactory vesicle membrane is continuous with the membrane outlining cilia
and microvilli. Along the border of the olfactory vesicle proper, the surface mem-
brane often shows vesiculated profiles which are the expression of pinocytotic
activity (DELORENZO, 1963; BALBONI, 1965). In the interior of the vesicle sparse
mitochondria and neurotubules 200 A in diameter are constantly observed. A
variable number of basal bodies, seen in turtles, frogs and birds with rootlets, are
The Olfactory Vesicle 33
also a constant organelle in the olfactory vesicle. A constricted zone normally

occurs in the receptor between the vesicle and the more proximal dendrite. This
zone corresponds to the level where the olfactory neurons becomes surrounded by
supporting cells. It is at this level that a junctional complex between the receptor
Fig. 3. Olfactory neurons in box turtles have usually a long cylindrical dendrite with a balllike
olfactory vesicle. In the receptor (R) here represented, a unique cilium (ci) was found, the
olfactory vesicle is not prominent, and the dendrite has a ball-like shape. These morphological
variations are common in most vertebrates. Supporting cells (sc) filled with secretory granules
surround the receptor
and supporting cell surface membranes is observed. This is characterized by the

sequences of zonula occludens, zonula adherens, and macula adherens as it is
described in other vertebrate epithelia (FARQUHAR and PALADE, 1965). The zonula
occludens, as it appears in sections parallel to the epithelial surface, is continuous
around the neck of the receptor and provides a morphological sealing device between
the environment and the epithelial intercellular space. Supporting cells do not
always isolate the receptors. FRISH (1967) has described the occurrence of two
dendritic processes in the same intercellular space. These contacts (Figs. 7, 8, 9)
have been recently observed to occur routinely in some amphibians and reptiles
(GRAZIADEI, 1969, 1971). Their occurrence and frequence as well as their possible
functional role warrant further investigation.
3 Rb. Sensory Physiology, Vol. IV!l
The Dendritic Process

The dendritic process or olfactory rod, proximal to the olfactory vesicle has a
diameter of 1 to 2 microns and a length which varies from one receptor to the other
depending on the location of the perikaryon in the epithelium (Fig. 3). The external
outline is cylindrical or bottle-shaped. Fine, spinelike projections protrude from
Fig. 4. This is a transverse section at the supranuclear level close to the origin of the dendrite
in the olfactory receptor of a frog. A Golgi complex (G) is surrounded by profiles of the rough
ER which show a rather orderly circular arrangement. Shrinkage phenomena artificially induced
in this preparation for the study of intercellular contacts, induce little changes in the overall
satisfactory fixation of the receptor cell
the main shaft and establish direct contacts with neighboring processes of other
receptors (GRAZIADEI 1971). The olfactory rod is consistently filled with mitochon-
dria and neurotubules longitudinally arranged along the major axis. The ground
cytoplasm shows a variable overall density in different receptors of the same
animal. These variations together with a variable number of other organelles give
rise to receptors with generally different appearances.
Receptor Cell Bodies

Receptor cell bodies are located in a rather homogeneous band occupying the
medium third in the height of the epithelium. In the cell body region the receptors
lie in direct membrane to membrane contact over most of the surface, without
intervening supporting cells. A large nucleus 4 to 6 !L in diameter as seen in
Receptor Cell Bodies 35
EM sections occupies most of the cell body area. The cytoplasm then accumulates
at the distal pole of the nucleus while around it only a thin layer 1 to 2 fL thick
remains between the nucleus and membrane. In the supranuclear portion there is
a prominent Golgi apparatus and ER of both the smooth and rough type
Fig. 5. In a number of physiological and experimental conditions the secretory activity of

supporting cells (sc) is dramatically increased, as in this case. Club-like processes of the support-
ing cells (cl) filled with secretory granules (sg) complicate the free surface of the mucosa of
turtle. The number of granules (sg) contained in each supporting cells vary greatly.
Compare SCI and sc 2
(Fig. 4). At times the rough ER is arranged in orderly lamellae similar to the Nissl
bodies of CNS neurons. The extent of these organized portions of the ER is,
however, minimal if compared to the other CNS nerve cells. Free ribosomes
aggregated in rosettes are present in the perikaryon. Mitochondria and tubules are
sparse. A conical projection of the cell body at the proximal pole originates the
axon which, after a short course of a few microns, tapers down to 0.2 fL diameter.
Axons inside the epithelium unite to form small axon bundles running in shallow
invaginations of the supporting or basal cells. Organelles inside the axons are
essentially neurotubules and sparse mitochondria. Inside the epithelium olfactory
axons are commonly aggregated in bundles of tens. In the fila olfactoria, imme-
diately below the basal lamina, where they are enwrapped by the Schwann cell cyto-
S'
P. P. C. GRAZIADEI: The Olfactory Mucosa of Vertebrates
Fig. 6. TEM picture of supporting cells (Be) in the olfactory mucosa of box turtles. The supra-
nuclear portion of the cells is filled with granules of different density (gr) and finely textured
profiles of the ER system (ER)
plasm, they can be often seen running singularly or in a few units in pockets of the
sheet cells. Further in their course they lose their discrete packaging. In the lamina
propria it is frequent to observe large number ofaxons, in the range of several
Cilia 37
hundreds, running in reciprocal close contact in single pockets of the Schwann's

cells. It is not known if the axons, as they continue to the bulb, retain the asso-
ciations initially seen within the pockets.
Fig. 7. In a transverse section through the upper third of the frog's olfactory mucosa dendrites
show repeated contacts (arrows) through processes emerging from the main shaft. These
contacts hold even in preparations like the one shown, where considerable shrinkage has been
induced using hypertonic fixation solution. One dendrite (d 1 ) is partially enwrapped by the
process of a second dendrite (d.)
Cilia
From the early observations of the olfactory mucosa till recent times cilia have
been established as a morphological detail of the olfactory neuron (Fig. 1, 9) and
emphasis has been put on their role, however, not precisely identified, in the per-
ception of odors (SCHULTZE, 1862; PARKER, 1922; OTTOSON, 1963; SEIFERT et al.,
Fig. 8. Repeated interreceptor contacts occur at the dendritic level in the olfactory mucosa.
The two receptors (R) in frog show a large portion of their dendrite in direct contact. Supporting
cells (Be) only partially isolate the upper part of the dendrite
1968). BANNISTER (1965), however, describes in minnow two types of receptors one
of which lacks cilia. TUCKER (1967) was able to experimentally remove cilia from
turtles' olfactory receptors and still record action potentials fro:n olfactory nerve
twigs after odor stimulation. Recent EM observations (BANNISTER, 1968; ALTNER
et al., 1968; GRAZIADEI et al., 1970) have demonstrated that vomeronasal receptors
Cilia 39
Fig. 9. Two olfactory receptors (R), provided with cilia (ci) show intim'1te connections through
a tight junction (ti) and along their dendritic shaft (arrows)
which have functional properties similar to the olfactory receptors proper, lack
cilia. These data invite a more cautious attitude regarding an "essential" role of
cilia. Further studies are needed in order to clarify their role, if any, in addition to
the obvious one of increasing the bare receptor area. The structure of cilia does not
vary from one species to another. All of them have the typical 9 + 2 pattern of
filaments, well known in all cilia throughout the animal kindgom. Variations,
however, have been found in their length and number. OKANO et al. (1967) found
some hundred cilia per receptor in dog while only a few are found in some receptors
of moles (GRAZIADEI, 1966). Usually their number varies from 10 to 15 per receptor.
Variations in the caliber and number of filaments inside the cilia have been described
in frog by REESE (1965), by ANDRES (1969a, b) in rat and by many of the authors
who have made ultrastructural observations of the olfactory mucosa (GRAZIADEI,
1971). These pecularities, however, have not so far been related to any specific
role in the ciliary mechanism.
Basal Bodies
Basal bodies related to each cilium of olfactory neurons are provided with basal
feet. These structures, unique in motile cilia and showing in them a definite orien-
tation, are instead multiple and not oriented in olfactory receptors. Rootlets are
lacking in many species, but have been observed in others such as turtles, birds and
frogs. That olfactory cilia have some motility has been observed by many authors
a long time ago (SAPPEY, 1877). More recent observations seem to confirm an
asynchronous beat of these appendages (BRONSHTEIN, 1954; REESE, 1965;
TUCKER, 1967; GRAZIADEI, personal observations 1968, 1959). It is the author's
Fig. 10. The basal region of the olfactory mucosa is occupied by basal cells (be) and the columnar
processes of the supporting cells (se). It is noticeable that the granular content of the latter
fills the entire cellular profile up to the surface. The cells presumed to be basal cells (be) have
their cytoplasm filled with filaments. A basal membrane is indicated by arrows
opinion that the beating rate of cilia depends in part on the viscosity of the mucous
layer, their length and the temperature of the environment. It seems possible that
shorter cilia which have been broken have been visualized. In SEM observations it
has been observed that olfactory cilia do not have a wavelike arrangement as
respiratory cilia do (Figs. 18, 19).
Basal Bodies 41
Fig. II. The polymorphic appearance of the supporting cells (se) is demonstrated by this figure
as opposed to Figs. 5, 6, 10, 12, 15 from turtle olfactory mucosa. The content of granules (gr)
and the overall density of the cytoplasm varies from one cell to the other. A quantification of
these aspects seem b be necessary before any attempt of classifying the cells could be considered
Supporting Cells
Supporting cells are columnar epithelial cells. They extend vertically from the
epithelial surface, where they show a series of irregular microvilli to the basal
lamina that they reach with branched digitiform processes. Microvilli are long in
cat (2-5 [.L), but can be lacking in other animals. The number and length of
microvilli are quite variable in different animals and no simple statement can be
made in their regard (Fig. 15). In Lamprey BRONSHTEIN et al. (1965) and THORN-
HILL (1967) have described supporting cells provided with cilia. The nucleus of
Supporting Cells 43
these cells forms a discernible band from the nuclei of the receptor cells. The
supporting cell nuclei can also be recognized from the neuronal nuclei due to more
elongated shape and for a denser chromatic reticulum in LM preparations. The
ultrastructural characteristics of supporting cells show relevant variations from
one species to another and in the same species from one animal to another
(Figs. 5,6,11,12,13). Variations are also observed in cells of the same animal. As
these cells are actively secreting, it is suggested that these variations are at least in
part due to the functional stages in which each cell has been fixed in the course of the
preparative procedures. The most prominent organelle observed in supporting cells is

the ER. Both the rough and smooth type are represented and the profiles often
arranged in orderly circular lamellae (Figs. 11, 12, 13, 14). Granules of varying size
and electron density are also obvious and at times they almost completely fill the
distal part of the cells (Fig. 5). Strong odor stimuli as well as perfusion of the nasal
cavity with dilute solutions of detergent substances induce the release of these
granules from the cells so that they may appear, after this procedure, completely
empty of granules. In these instances ER profiles completely fill the cell outline.
In his pioneering observations of olfactory mucosa with EM, BLOOM (1954) men-
tioned granules released from supporting cells into the surface mucous layer.
Mention of granular secretion from supporting cells since then has come from
several authors in many animals (BRONSHTEIN et al., 1965; REESE, 1965; FRISH,
1967). Active secretion has been confirmed by the author in amphibia, birds,
reptiles and mammals. We do not know, however, the specific role of the secretory
product and its chemical composition. Due to the active secretory role of supporting
cells, it is not surprising that they are caught by the fixation procedure in stages of
this process. The description of different "types" has been suggested by many
authors but little agreement so far has been reached as to the number or terminology
of these stages. A more quantitative study of the cells of the olfactory epithelium
seems to be needed before categorizing them as "stages" or "types". According to
LE GROS CLARK (1957) supporting cells have phagocytotic properties. No such
property has so far been described at an ultrastructural level.
Each supporting cell contacts the neighboring ones and the surrounding
receptors by means of a series of specialized structures. Near the epithelial surface
a tight junctional complex describes a belt-like structure around the perimeter of
every cell (Fig. 16). Below this apparatus specialized contacts in the form of
desmosomes are scattered along the surface of supporting cells both where they
contact each other or where they contact the neurons. Tight junctions, however,
have not been reported so far, other than at the junctional complex level.
Basal Cells
Basal cells are prismatic cells located close to the epithelial surface of the basal
lamina and arranged between the basal processes of the supporting cells. They are
morphologically variable from one species to another; their organelle constituents
vary considerably in the elements of the same species (Fig. 10). RODHIN (1963) has
compared them to undifferentiated basal cell of pseudostratified epithelia. They
do show tonofilaments in their cytoplasm. Their function, other than the
possibility of replacement elements, has been so far not clear. Frequent mitosis in
this cell category was observed by SEIFERT et al. (1967). Recently they have been
recognized as stem cells for the replacement of the neurons (GRAZIADEI et al.,
1971). The basal lamina, the extracellular material which is commonly observed at
the base of all epithelia, lines the base ofthe olfactory epithelium of all vertebrates
and, when fila olfactoria and Bowman's glands ducts emerge from it the epithelial
lamina is continuous with the one surrounding these structures. It is usually very
thin in the order of 0.1 fJ. and rests on a loose connective of the lamina propria. This
explains why classic histologists often fail to describe the presence of the basal
lamina in the olfactory epithelium.
Basal Cells 45
Fig. 14. Supporting cells have in the same animal many different morphological aspects. The
cell here illustrated (se) is from olfactory mucosa of box turtle and shows some secretory
granules (sg) and a peculiar but not uncommon system of ER lamellae concentrically arranged.
A second system is partially seen in the upper left corner of the figure. Within its center is
found an electron dense granule. Nucleus (N) and mitochondria are also observed
Olfactory Axons. Olfactory axons run for a short distance inside the epithelium
and leave it in small bundles on the order of a few tens of units; after crossing basal
lamina, these bundles are enwrapped by Schwann cells. The axons in their initial
extraepithelial course run either singularly or in small groups in the Schwann cell
Fig. 15. The secretory process of supporting cells (Se) often modifies the free surface. But very
often entire portions (p) of the cell seem to fall into the mucous layer. The destiny of these
fragments is not well known. They are usually provided with ER profile and/or secretory
granules. Vulture olfactory mucosa
pockets. Very rapidly, however, and certainly before leaving the lamina propria,
they emerge in large fasicles each one contained in a single pocket of the ensheath-
ing cells. These pockets may contain hundreds of small axons each of which have a
mean diameter of 0.2 fL. The fila olfactoria and the roots of the olfactory nerves are
believed to be neurally homogenous having only olfactory axons. No other sensory
fibers or efferent fibers have so far been described in the olfactory fila olfactoria or
Basal Cells 47
nerves. Trigeminal fibers, however, have been described by early histologists to

reach the olfactory mucosa, although their findings have not been followed by more
recent ultrastructural observations. Myelinated fibers are observed in the lamina
Fig. 16. A junctional complex (arrows) unites at the surface two adjoining supporting cells.
This structure is continuous all around the neck of each supporting cell. Large granules are
observed in these frog's supporting cells
propria mucosae and their termination inside the epithelium warrants further
study.
Bowman's Glands. Bowman's glands are tubular alveolar units found only in
the olfactory region of all vertebrates except fish. They are localized in the lamina
propria with their ducts opening at the epithelial surface. While their histological
composition is quite similar among species, the ultrastructural details of the
secretory cells vary from species to species. Furthermore, within a species, cells
with a variable organelle content is observed. In mice, FRISH (1967) described
light and dark cells and intermediate types as well. SEIFERT et al. (1967) in the
same animal described three types of cells. From personal observations of many
species of vertebrates the author suggests that these secreting glandular elements
undergo continuous changes which are incompletely expressed by the attempts to
group them into' 'types". Inasmuch as the number of granules, the extention of the
ER and the number of mitochondria are expressions of the particular secretory

activity of a cell a great many patterns can be expected. Published reports on the
ultrastructure of Bowman's glands, however, are scanty and further information is
needed regarding their comparative morphology and the histochemical charac-
Fig. 17. Bowman's gland in the olfactory mucosa of frog. Secretory cells provided with
granules of high electron density show at their lumen surface a profuse set of microvilli (mi).
One cell not shown in the picture protrudes many ciliary shafts (ei) into the same lumen
teristics of their product. While their secretion is often referred to as serous, histo-
chemical LM observations by GOMPER (1950), SLOTWINSKI (1934), DEAMICIS et al.
(1957) have stated that secretory granules react positively to PAS and other specific
methods for the demonstration of mucopolysaccharides.
Ciliated cells were described by DOGIEL (1887) in frog's Bowman's glands. This
observation has been confirmed at EM level for discrete elements interspersed
among secretory cells (Fig. 17).
The Vomeronasal Epithelium

In addition to the olfactory epithelium proper, which is common to all verte-
brates, the nasal cavities of amphibians, reptiles and mammals (see PARSONS this
volume for details) locate a topographically distinct sensory area called the
Jacobson's organ or vomeronasal (VN) epithelium. The VN epithelium derives
embryologically from the olfactory placode as the olfactory epithelium proper and
contains primary receptor neurons whose axons terminate in the accessory
olfactory bulb. It has been observed that the organ responds to stimuli with
modalities similar to the olfactory organ proper. In spite of many embryological,
The Vomeronasal Epithelium 49
Fig. 18. Respiratory mucosa of Gopher tortoise nasal cavity. The arrangement of the cilia
in ridges repeats the wave-like arrangement of these cilia that in vivo have a metachronal
rhythm
Fig. 19. The olfactory surface from frog's olfactory mucosa shows a pattern of cilia with no
definite orientation. This aspect contrast sharply with the one shown in Fig. 18 of the respi-
ratory region
4 Hb. Sensory Physiology. Vol. IVll
anatomical and functional similarities the VN epithelium has some characteristic

differences when compared to the olfactory epithelium. A description of its ultra-
structural details will be provided below based on the observations of some reptiles.
The organ varies considerably in form from species to species and very little has
been reported so far on the ultrastructure of VN epithelium of any yertebrates.
Fig. 20. Histological frontal section through the nose of Gopher tortoise, showing the transition
zone between the ciliated respiratory epithelium, on the left and the VN epithelium at arrow.
In the latter a layer of supporting cell nuclei (Sen) is discretely arranged from the neuron
nuclei layer (nn). No cilia are observed at the VN epithelium surface
BANNISTER (1968) has studied Anguis fragilis, ALTNER and MULLER (1968) Lacerta
and GRAZIADEI et al. (1968) and GRAZIADEI (1969) have studied many species of
turtles. In light microscopic sections the VN epithelium is distinguishable from the
surrounding respiratory area by a sudden increase in thickness, an abrupt inter-
ruption in the presence of motile cilia and an orderly arrangement of two discrete
layers of nuclei (Fig. 20). The more superficial nuclear layer, characterized by
densely stained ovoid nuclei belongs to the supporting cells. A deeper layer, more
conspicuous, is represented by round finely textured nuclei belonging to the neurons.
At the base of the epithelium sparse nuclei of elongated shape belong to the basal
elements. With the TEM supporting cells show a discontinuous series of microvilli
at their surface (Figs. 23, 24). The cytoplasm is marked by the presence of numerous
The Vomeronasal Epithelium 51
round, electron dense granules. The neurons have a distal portion corresponding
to the olfactory vesicle of the olfactory receptors albeit not so prominent as the
latter and complicated by the presence of branched or somewhat irregularly
shaped microvilli (Figs. 23, 24). Cilia, however, seldom observed, are not present in
the large majority of these receptors. The dendrite is usually longer and more
Fig. 21. Low power SEM picture of thl:' zone of transition between the VM area (VN, lower
right of the picture) and the respiratory area (T). In the latter are observed ridges of cilia
which gradually disappear in the VN region
tortuous than the dendrite of olfactory neurons. Neurotubules are observed in

their dendrites but mitochondria are sparse. The cell body is round, located at
various depths in the epithelium, provided with a large round nucleus leaving a
thin layer of cytoplasm around it. The dendrites have a diameter of 1-2 fL while
the cell body has a diameter of 5-7 fL. From the proximal pole of the perikaryon
the axon arises and joins others in small intraepithelial bundles. The diameter of
these processes is in the order of 0.1-0.4 fL. The present description applies to box
turtle and gopher tortoise, but similar results have been reported by the previously
mentioned authors describing the VN epithelium in Anguis fragilis and Lacerta.
The only substantial differences with the olfactory epithelium proper are seen to
reside in the absence of cilia in VN receptors (Figs. 21, 22), in their long dendrites
which are not provided with the large number of mitochondria observed in
olfactory dendrites and in the absence of Bowman's glands. A study with the
SEM has confirmed the absence of cilia from the VN area as compared with the
classical olfactory area and respiratory area (Figs. 18, 21, 22).
4
Summary
If we compare the architectural complexity of a peripheral auditory apparatus
or of a retina in a vertebrate with an olfactory epithelium the latter seems to be a
relatively simple structure. It is basically composed of a mosaic of receptors
arranged between supporting cells overlying a single layer of basal cells. There are
Fig. 22. The free surface of the VN organ of turtle does not show cilia but microvilli only.
These appendages, belonging primarily to the receptors but complicating the free surface
of supporting cells as well create aspects as the one shown here which are readily differentiated
from the one produced both by respiratory (Fig. 18) or olfactory cilia proper (Fig. 19)
no synapses at the epithelial level, no demonstrated efferent fibres and the neuronal
pattern seems to be of digramatical simplicity. Complex electrical responses,
however, derived from the olfactory nerve after odor stimulation suggest that the
above picture may be greatly oversimplified. A closer ultrastructural analysis of
the epithelium points out that many details of the organ are still unclear as to
their role; and further study is needed. A list of facts and doubts regarding the
olfactory mucosa as observed from an ultrastructural point of view can be pre-
sented.
The receptive pole of the neuron is likely to be confirmed as the bare portion,
the olfactory vesicle with attached cilia. These appendages have been attributed
with an important role in the transduction process from the first studies on the
organ. The hypothesis is not supported by any specific evidence. The fact that VN
receptors, embryologically, anatomically and physiologically are similar to the
classical olfactory receptors but lack cilia is a well supported observation and
Summary 53
should be considered before assessing the role of cilia in olfaction. It is true that
cilia are common in many receptors, but they are ubiquitous elements well
demonstrated in all types of cells from macrophages to neurons. Certainly they
have the function of increasing the receptor surface many thousand times but if
Fig. 23. The free portion of a VN receptor from box turile (R) is complicated by several
microvilli (mi). Many tubules (tu) but only sparse mitochondria fill the dendritic profile
this is their primary function it is not known. The motility of the cilia was observed
by many authors and the pecularity of their irregular beating, as opposed to the
rhythmicity of the respiratory cilia is not clearly understood. A number of authors
has reported fine structural details of olfactory cilia in many animal species.
However, we still lack a comparative study of cilia showing the consistent presence
of any detail throughout many species such as those precisely described by REESE
(1965) and ANDRES (1969a and b). So far the function of cilia in olfaction is
obscure and the many hypothesis provided are not on a firm experimental basis.
We do not even know if they are indispensable organelles in the olfactory mecha-
nism or if they simply facilitate or complement the function of the receptors.
The sustentacular cells, commonly thought of as insulating and/or supportive
elements have more recently acquired a different significance. Inter-receptor con-
tacts have been frequently observed at the perikaryon lcvel where large areas of
the receptor cell bodies are in mutual contact. Further, zones of interreceptor
contact have been demonstrated at the olfactory vesicle, dendrite and axonal level
so that the insulating function seems not to be pertinent (GRAZIADEI, 1971). More
than the function of supportive cells, the sustentacular elements seem to allow a bet-
ter patterning of the receptors. But their possibly most important function is their
secretory activity. This function has been observed by many authors and can be
enhanced by odor stimulation or more general chemical stimuli. This activity renders
the cell extremely variable in appearance due to its manifold functional stages. We
do not known however, the chemical composition of its secretion nor its importance
Fig. 24. Vomeronasal receptors (R) as seen in TEM pictures lack ciliary processes, but are
provided with a complex set of microvilli (mi). Supporting cells (se) in the VN epithelium have
the cytoplasm filled with secretory granules
in the olfactory mechanism. Morphologically the secretory granules appear dif-

ferent from the ones observed in the Bowman's glands. If the two secretions are
dramatically different their role on the olfactory surface could prove to be quite
different. The layer of secretion covering and embedding the cilia is often referred
to as mucous, but further histochemical analysis is needed to establish its true
nature. There are reports by numerous authors attempting to chessify the non-
neural cells of the olfactory epithelium so far included in the substentacular type
in more than one category. These attempts have so far not demonstrated that the
proposed types have any type-associated functional property and if the different
types are observed all through the vertebrates or in some species only. These
researches, however, emphasize the need of "more dynamic anatomical and func-
tional approach" to the study of the olfactory mucosa than has been so far
followed. It has been recently demonstrated that both olfactory newrans and sup-
porting cells are labile elements replaced all through the life of adult vertebrates
(GRAZIADEI and METCALF, 1970, 1971). Autoradiographic techniques both at LM
and EM level have shown that tritiated thymidine is incorporated into the nuclei
of olfactory neurons during the adult life of frogs and rats. The replacement is
References 55
operated by a continuous duplication and maturation of stem cells recognized as

the so-called basal cells. This phenomenon could be partly responsible for the dif-
ferent morphological patterns observed both in nervous and supporting cells. These
data are, moreover interesting from a clinical point of view, as replacement of
neurons in high vertebrates has so far not been commonly described.
The study of the trigeminal component described by earlier classic neuro-
histologists (see page 28) is commonly neglected in the more recent ultrastructural
studies of the olfactory mucosa. TUCKER (1963) was able to record, however, from
trigeminal fibers in the olfactory mucosa. Myelinated fibres are observed in the
lamina propria but their final destiny is not clear. The study of these terminals is
important as their modalities of distribution and their relation with other tissue
components could clarify the non-olfactory innervation (or possibly efferent
innervation) of the olfactory mucosa.
Acknowledgement
This work was supported in part by Florida State University and the National Institutes
of Health grant number 5 R 01 NS 08943-03.
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Chapter 3
The Olfactory Pigment

By
D. G. MOULTON, Philadelphia, Penn. (USA)
With 2 Figures
Contents
Introduction 59
Distribution and Location 60
Properties 61
a) Carotenoids and Vitamin A 62
b) NonCarotenoid Pigments . 63
Pigment and Olfactory Sensitivity 64
a) Albinism, Olfactory Pigment Density and Sensitivity to Odors 64
b) Vitamin A, Carotenoids and Sensitivity to Odors . . . . . . 66
Theories Implicating Pigment in the Olfactory Transduction Process 67
Summary . 71
References 71
Introduction
The ultimate action of odorant molecules on an odor receptor is to produce
electrical changes within the cell. These may appear as the firing of a nerve impulse
or in an alteration in the rate of impulse initiation. But before the odorant energy
can elicit these effects it must be absorbed by the appropriate receptor molecules.
Provision must also be made for the rapid deactivation or removal of the odorant
after it has achieved its effect, and for the restoration of the system to the resting
condition.
In vision, the comparable cycle of events depends on the presence of a caro-
tenoid-protein (rhodopsin or porphyropsin in rods; iodopsin or cyanopsin in cones).
Carotenoid pigments have also been implicated in the mechanisms of photo-
synthesis and bacterial respiration. More generally, various pigmented compounds
participate in vital physiological processes, and are precursors of substances which
fulfill essential functions of the organism. Consequently, it is reasonable to ask
whether pigments may also be implicated in the primary events of olfactory
transduction process.
60 D. G. MOULTON: The Olfactory Pigment
In fact, the olfactory epithelium can usually be distinguished from the red of
the surrounding mucosa by its yellow or brownish color - a contrast reflected in
the older name for this region: the lowsluteus. As early as 1870, OGLE - noting the
parallel between the presence of visual purple in the retina and the yellow of the
olfactory organ - suggested that pigment, if not essential to olfaction, at any rate
"conduces much to its keenness and perfection". The parallel became more striking
with the demonstration that there are significant concentrations of carotenoids in
the olfactory area of steers (MILAS et al., 1939), and that odor perception can be
restored to some anosmic patients by administration of vitamin A (DUNCAN and
BRIGGS, 1962). There are other - less direct - lines of evidence (for example, the
reports that albinism is associated with reduced olfactory sensitivity), and for
some the conclusion has seemed incontrovertible: pigment is essential to olfaction.
On closer inspection, however, the problem grows more complex: the mere
presence of pigment is no indication of functional importance. There are carotenoids
in the ear wax of cattle (PALMER, 1922), pigments in the dura mater of various
species of mammals (SOKOLOV, 1963), and in some species of vultures - but not
others - there are heavy concentrations of pigment around the collecting tubules
of the nasal glands (BANG, 1964). Yet pigments at these and other sites throughout
the body serve no obvious function and in some cases, at least, may well be
excretory products. Furthermore, there is not one, but several olfactory pigments
and it is important to examine the location, distribution and properties of each
component before considering evidence concerning their possible role in olfaction.
Distribution and Location

Olfactory pigment appears in all vertebrate classes from cyclostomes to
mammals. The color, on gross inspection, ranges from a pale yellow - as in man
and the frog, through yellowish-brown - as in many mammals, to a dark black-
as in certain fish. At the outset, however, it is important to recognize two classes
of pigments: those contained in specialized pigment cells and those arising in
Bowman's or analogous glands, and in (or between) the cells of the olfactory
epithelium.
The pigment cells are probably restricted to the submucosa of cyclostomes,
amphibia, fish and reptiles, and generally form discrete bands or spots (as opposed
to the more uniformly dispersed pigments of the second type). In the fish Ohanna
argus, for example, black and gold regions ring the pinkish mucosa of the ol-
factory lamellae, while in certain carp and mudfish the white olfactory epithelium
is scattered with black dots (SHIBUYA, 1960). The pigment is not present in the
epithelial layers of the sea lamprey (Petromyzon spp.) or slow worm (Anguis
tragilis) but is distributed around nerve bundles in the submucosa (NEGUS, 1955).
In the case of the Xenopus toad, NEGUS (1955) has shown that the black pig-
mentation of the olfactory region is due to the presence of specialized melanin-
containing cells, or melanophores, in the submucosa. Similar cells are found
scattered throughout the dermis of many lower vertebrates, where their ability to
expand and contract contributes to the color changes seen in these species.
Removal of the pituitary glands leads to a clumping of both the olfactory and
dermal melanophores, while anterior corticotrophic hormone, containing inter-
Properties 61
medin, causes expansion. The olfactory melanophores do differ in one respect,

however; they fail to respond on exposure to light (NEGus, 1958).
No pigment migration occurs following stimulation of the olfactory epithelium
with aniseed (NEGus, 1955). In some species the dermal pigment cells are yellow,
and apparently contain carotene. But whatever the pigment involved it seems
unlikely that such chromatophores have any significant function in olfaction and
are probably fortuitous dermal inclusions. At any rate we will not consider them
further.
The second class of pigments are responsible for the pale yellow-to-dark brown
appearance typically associated with the olfactory epithelium of mammals and
birds, but also found among certain lower vertebrates including the lamprey
(KLEEREKOPER and VAN ERKEL, 1960). For example, in the turkey vulture
(Cathartes aura), the Trinidad oilbird (Steatornis caripensis), and three species of
albatross, the well-developed olfactory organ is conspicuously yellow (BANG, 1960).
Factors reported to influence the degree of color saturation include rhinitic
infection, age, individual breed and species differences, and length of time the
epithelium has been exposed by dissection (JACKSON, 1960; KOLMER, 1929;
GEREBTZOFF and PHILIPPOT, 1957). For example, in 2 month old albino rats the
olfactory epithelium is a very pale yellowish-brown. In 12 month old rats, the
color is noticeably darker. However, there has been no systematic exploration of
the action of these variables, and there is no evidence to suggest that the pigment
is decomposed by odorants.
Most histologists agree that within the olfactory epithelium, pigment granules
are present predominantly in the supporting cells and in the Bowman's glands
(which are found only in the terrestrial vertebrates). There appears to be only one
report that the basal cells contain pigment (BARADI and BOURNE, 1953). In the
critical case of the receptor cells, however, the evidence is less clear. Generally, it is
either stated that no pigment can be detected in the receptors or no mention is
made of its presence at this site (ALLISON, 1953; ARDOUIN and MAILLET, 1964;
BARADI and BOURNE, 1953; KLEEREKOPER and VAN ERKEL, 1960; MULLER, 1955).
However, there are certain exceptions. Thus HERBERHOLD (1968), suggests that
pigment may be present in fish receptors although he could find none in mammalian
receptors. VINNIKOV and TITOW (1949) were impressed with the similarity of the
olfactory receptors to the rods and cones of the retina and, on the basis of histologi-
cal studies, claim that the pigment is present in both the receptors and supporting
cells, but the evidence is not convincing. GEREBTZOFF and SHKAPENKO (1952),
using frozen sections, also identified pigment in the peripheral processes of the
olfactory receptors. However, localization was mainly based on Feulgen's plasmal
reaction and on Sudan black, scarlet red and Nile blue sulfate staining which
might also stain non-pigmented neutral fats, fatty acids and phospholipids. They
further assumed that the pigment had the same location as the lipids (GEREBTZOFF
and PHILIPPOT, 1957).
Properties
From a functional standpoint it is appropriate to consider the olfactory pig-
ments as comprising vitamin A and carotenoids and non-carotenoids.
a) Carotenoids and Vitamin A

Vitamin A is the lower isoprenologue of monocyclic carotenoid pigments having
20 carbon atoms. The term is a non-specific one which includes retinol (vitamin AI) ;
retinal (retinene, whose ll-cis isomer is the chromophore of visual pigments);
retinoic acid (vitamin A acid); 3-dehydroretinol (vitamin A 2 ); and biologically
active esters of vitamin A. Retinal can be derived in vivo by central cleavage of the
carotene molecule (GOODMAN and HUANG, 1965; Fig. 1).
{i-Carotene
/H
~C=O ~CH20H
Retinal Retinol
(Retinene) (Vitamin AI)
Fig. 1. Structure of members of the vitamin A series and p-carotene
Strong indications of vitamin A and carotenoids have been found in the ol-
factory epithelium of cattle. Vitamin A occurs as free vitamin A (BRIGGS and
DUNCAN, 1961) or more specifically as retinol (MILAs et al., 1939; MOULTON, 1958).
There is also a suggestion that 3-dehydro-retinol may be present (MILAS et al.,
1939).
MILAS et al. (1939) also noted 4 adsorption bands which they concluded were
due to carotenoids, and later workers have confirmed the presence of {3 carotene
(MOULTON, 1958, 1962; KURIHARA, 1967). It is important to note that, although
BRIGGS and DUNCAN (1961) and DUNCAN and BRIGGS (1962) found evidence for
protein-bound carotenoids in bovine olfactory epithelia, KURIHARA (1967) was
unable to confirm this.
The occurence of these compounds in cattle epithelium may not be too surpris-
ing since vitamin A is essential for maintaining the integrity of many cell systems,
and cattle - as opposed to many other species - preferentially store carotenoids
throughout the fatty tissues of their body (GOODWIN, 1954). It is therefore im-
portant to know whether carotenoids also occur in the epithelia of species known
not to be primary carotene accumulators. DUNCAN and BRIGGS (1962) found, in
addition to retinol and vitamin A esters, traces of other carotenoids in the
olfactory epithelium of a dog. However, there is some question concerning the
status of the dog as a carotene-accumulator since carotenemia has been reported
(DUHIG, 1931). Rats and pigs seem to fall more clearly into the category of non-
carotene accumulators. In these species, MOULTON (1962) could find no epithelial
carotenoids although the same technique gave indications of high concentrations
of carotene in the olfactory epithelium of a cow. There is also a report that WRIGHT
Properties 63
(1962) could find no carotenoids in the olfactory epithelia of salmon. Thus, it has
yet to be clearly established that carotenoids are generally present in the olfactory
epithelia of vertebrates.
b) Non-Carotenoid Pigments
Even in those species in which carotenoids are lacking, or present only in trace
quantities, the olfactory epithelium is still in most cases a yellowish-brown in gross
appearance. Biochemical analyses suggest that several pigments contribute to this
color.
A series of studies by GEREBTZOFF and his associates at first suggested that the
non-carotenoid pigment derived from pooled cat, dog and rabbit epithelial extracts
were composed essentially of fatty acids - mainly acetal (HEUSGHEM and GEREBT-
ZOFF, 1953). However, in later studies on sheep they concluded that the pigment
itself was merely bound rather weakly to phospholipids (predominantly lecithins)
and possibly in part to protein. Acidic and basic pigment fractions were derived
but no characteristic absorption bands were seen. None of the tests for melanin
or other commonly occurring body pigments gave positive results (PHILIPPOT and
GEREBTZOFF, 1956, 1958; GEREBTZOFF and PHILIPPOT, 1957).
JACKSON (1960) concluded from an analysis of pigment extracts from the
olfactory epithelia of cats, pigs, and rabbits that the color was due in part to
phospholipids - probably lecithins - but primarily to the auto-oxidation products
of these phospholipids. He did not consider the possibility that the pigment was
only bound to phospholipids.
Fractionation of saponifiable extract from bovine olfactory epithelia by paper
chromatography gave a further indication that several non-carotenoid pigments
occur (MOULTON, 1958). Three yellow or yellowish-brown fractions separated on
alumina-impregnated paper in a water-saturated butanol solvent system. A fourth
band remained close to the origin. The first fraction showed absorption maxima at
about 248, 260, and 270 mfL; the second showed no distinctive peaks, and the
third had a peak at about 250 mfL with a weak maximum at about 305 mfL.
Using more adequate purification and extraction procedures than in these
earlier studies, KURIHARA (1967) found that the main bulk of the bovine olfactory
pigment was tightly bound, not to lipids but to proteins, in both a water soluble
fraction and in pigment granules. (Pigment from the granules showed an absorp-
tion peak at 260 mfL and a single 4.0-8 boundary). After liberation from the
protein, the pigment was separable into an acetone-soluble and -insoluble fractions.
With chromatography on a silica gel column the acetone-soluble fraction was
further divisible into a yellow pigment with peaks at 242, 317, and 365 mfL, and a
red pigment. The remaining pigment, extracted from the acetone-insoluble frac-
tion was light yellow and showed a peak at 260 mfL.
In a fraction containing water-soluble pigments KURIHARA (1967) also separated
a further pigment with an absorption maximum at 293 mfL, and chromoproteins
with spectra similar to those isolated from pigment granules. Olfactory chromo-
protein isolated from dog and horse epithelia was spectroscopically identical to
those isolated from cattle epithelia.
KURIHARA estimated that the ratio of the contributions of chromoprotein in
pigment granules, the water soluble pigments, and pigments soluble in organic
solvents, to the color of the olfactory epithelium is roughly 100: 15: 1.2. But he
used epithelial shavings that did not include Bowman's glands.
In summary, it appears that the olfactory pigment is primarily composed of at
least four non-carotenoid compounds present in granules within the olfactory
epithelium as well as in Bowman's glands. There are indications that much of the
pigment may be bound to proteins. In no case, however, is the chemical constitution
of these pigments known.
Pigment and Olfactory Sensitivity

a) Albinism, Olfactory Pigment Density and Sensitivity to Odors
The view that some relation exists between pigment and olfaction draws
heavily on reports that albinism is associated with anosmia - or, at least with
reduced olfactory sensitivity - and that conversely, the darker the pigmentation
of the skin or olfactory epithelium, the more sensitive the sense of smell.
Most of the accounts linking albinism with anosmia can be traced to DARWIN
(1875). He was impressed with the apparent immunity of dark-skinned animals to
certain vegetable poisons and gave some examples, two of which follows: In parts
of Florida, all the pigs except the black ones suffer severely from eating the roots of
Lachnanthes tinctoria (The Paint Root); while in Sicily, Hypericum crispum (St.
John's Wort) is poisonous to white sheep, but not to black. Accordingly, the
inhabitants of both places raise only black individuals.
Such examples are frequently cited as evidence that albinos are anosmic, the
implication being that albinos alone eat the poisonous plants because they cannot
smell them. But this is not DARWIN'S point. On the contrary, he suggests that all
the animals eat the plants, but that the black ones are immune to the effects of the
poison. He was probably correct. The effects described are characteristic of photo-
dynamic sensitisation. If cattle eat the Hypericum plant unpigmented skin becomes
sensitised to light. Exposure of these areas to the sun can then lead to a rash,
oedematous swelling, and in some cases, to convulsions and death: pigmented areas
are generally unaffected. A fluorescent pigment, hypericin, can be extracted from
the Hypericum plant. When this is injected into white rats, it produces typical
photodynamic lesions when the animals are exposed to light (HORSELY, 1934;
BLUM, 1941).
But a basic weakness in the arguments linking albinism with anosmia is the
underlying assumption that albinos lack olfactory pigment. Even if some olfactory
impairment did exist in albinos, it has still to be shown that this is related to lack
of pigment. [Deafness in white cats is associated not only with coat color but also
with the lack of structures essential for hearing: the hair cells of the Organ of Corti
(WOLFF, 1942).] In fact, a comparison of the gross appearance of the olfactory
epithelium of pigmented and albino rats shows no evident difference in the degree
of color saturation. Furthermore, when extracts of olfactory epithelia from these
two groups are fractionated by paper chromatographic techniques no differences
between them can be seen in either the number or appearance of the pigmented
fractions (MOULTON, 1958).
It might still be argued, however, that some component of the pigment com-
plex, absent from albinos but involved in olfaction, had been overlooked. Con-
Pigment and Olfactory Sensitivity 65
sequently, some impairment of olfactory powers might still occur in albinos. Indeed,
KEELER (1942), concluded that the albino gene has a profound dulling effect on
the sense of smell in rats. Thus in one of his tests, the responses of black, gray and
albino rats to a piece of garlic held 2 mm from their noses was observed. 71 per cent
of the albinos backed away after sniffing the garlic for an average of 9.9 seconds
while 66 per cent of the pigmented rats backed away after an average of 4.9 seconds.
The differences are statistically significant. When this test was repeated using
10 Hatai black hooded and albino rats, the black rats backed away after 7 seconds,
whereas the albinos never backed away. However, it is well known that many
behavioral traits segregate with coat color (WINSTON et al., 1967), - a fact which
could easily account for these findings. Thus KEELER'S conclusion seems un-
warranted.
Clearly, a more adequate evaluation of KEELER'S hypothesis requires a more
sensitive technique providing quantitative data on concentration-response rela-
tions. This was attempted by MOULTON (1960) who investigated the detectability
of n-hexanol by black and albino male Norway rats in an odor discrimination
apparatus. In the first 24 trials of training, black rats made significantly higher
scores than did albinos. In fully-trained animals, however, the position was
reversed. There was evidence to suggest that the initial differences were deter-
mined by differences in behavioral traits rather than in olfactory capacities. In any
case, it is evident that the albinos were not anosmic and were at least as sensitive
to the test odorant as were their pigmented littermates. Similar conclusions have
been reached concerning the relation of coat color to saline preferences in mice
(WOLF and LAWRENCE, 1963).
The converse of the view that albinism reduces olfactory sensitivity can be seen
in suggestions that olfactory sensitivity is more acute in dark-skinned races than
in light (OGLE, 1870; MONCRIEFF, 1967). The conclusions of most comparative
studies conflict with this view (MCCARTNEY, 1968).
But in any case, these various lines of evidence neither prove nor disprove that
pigment is directly implicated in olfaction, since in no case was any observed dif-
ference in sensitivity correlated with differences in the composition or concen-
tration of the olfactory pigment. However, such a correlation is frequently claimed
in relation to the olfactory powers of various species of mammals, despite the fact
that no direct quantitative comparisons between behavioral response and depth
of pigmentation have been made. On the other hand, ADRIAN (1956) notes that a
more deeply pigmented organ "is more sensitive and shows far less evidence of
exhaustion with continued stimulation" than one with little color. ONAGAWA
(1957 a, 1957b, 1957 c) also concludes that depth of color and degree ofresponse,
as recorded electrophysiologically, are related. While such may be the case it need
not imply a specific function for pigment in olfaction. In a given species a number
of features may control the apparent depth of pigmentation, including the thick-
ness of the epithelium, the ratio of supporting to receptor cells, and the density of
Bowman's glands. But these features may themselves control olfactory sensitivity.
We have now evaluated the evidence for three - not necessarily related -
propositions:
(a) Albinism is associated with anosmia or reduced olfactory sensitivity.
5 Rb. Sensory Physiology. Vol. IV/l
(b) Albinism is associated with the lack or reduction in intensity of olfactory

pigment.
(c) Olfactory sensitivity is related to the degree of color saturation of the
olfactory pigment.
The first proposition is untenable, and the evidence in the other cases is
inconclusive. The persistence of beliefs to the contrary is an interesting example of
how an accummulation of uncritically selected data can lead to an apparently
compelling argument.
b) Vitamin A, Carotenoids and Sensitivity to Odors

Experimental evidence concerning a specific function for vitamin A or
carotenoids in olfaction is of two kinds: anosmia or loss of sensitivity to odors fol-
lowing vitamin A deprivation, and restoration of a response to odors in anosmic
patients following vitamin A administration.
WOLBACH and HOWE (1925) noted that loss of smell was late but constant
symptom of vitamin A deficiency in rats. However, this seems to be based on the
observation that the rats would eat only if food was placed in their mouths. In
more extensive experiments, LE MAGNEN and RAPAPORT (1951) gave 20 male
albino rats continuous access to two bottles, one of which contained a solution of
bitter quinine sulfate and had eucalyptus smeared on the nozzle, while the other
contained pure water and had benzaldehyde smeared on the nozzle. The relative
positions of the bottles were changed twice daily. When a preference became
established, both bottles were filled with pure water. The discrimination remained.
The rats were then fed a diet deficient in vitamin A. In general, the discrimination
(as judged by water intake) broke down only in the few days before death or in a
very advanced state of the deficiency. The authors assumed that anosmia had
developed, but do not appear to have investigated its cause, its degree of rever-
sibility, or its effects on olfactory epithelium or pigment.
In a study of 56 patients with anosmia (believed to be uncomplicated by nasal
pathology) DUNCAN and BRIGGS (1962) found that treatment with vitamin A
alcohol preparations was followed by a full or partial restoration of sensitivity to
odors in 50 cases. No controls were used. However, two stages of recovery were
noted: the appearance of an unpleasant lingering olfactory sense in 4 to 6 weeks,
and a later return of pleasant olfactory sensations in a smaller number of patients.
The results of both these experiments are only significant in the present context
if it can be demonstrated that they do not depend on some non-specific function of
vitamin A. But this compound has widespread effects. Thus in the early stages of
vitamin A deficiency, keratinizing metaplasia leads to atrophy of secretory
epithelia, including the olfactory epithelium, and Bowman's glands (WOLBACH,
1954; WOLBACH and BESSEY, 1941; WOLBACH and HOWE, 1925). There is also a
blocking of the synthesis of mucopolysaccharides - compounds reported to be
present in the secretions of Bowman's glands in some species at least (NEGUS, 1958).
It is this effect which is thought to be responsible for decline in taste sensitivity
found during vitamin A deprivation in rats (BERNARD and HALPERN, 1968). [In
this context it is significant that the first biochemical investigation of olfactory
epithelia arose not from any theory implicating carotenoids in olfaction but from
the knowledge that vitamin A is needed to prevent drying of mucous membranes

and should therefore occur at this site (MILAS et al., 1939)].
A further non-specific effect of vitamin A deficiency is the appearance of
nervous lesions including the nerves of the special senses. This is thought to result
from a disproportionate growth of the central nervous system in relation to its
bony casing (WOLBACH and BESSEY, 1941).
Clearly, anyone or more of these abnormalities might account for the findings
of LE MAGNEN and RAPAPORT. Similarly DUNCAN and BRIGGS do not seem to have
eliminated the possibility that the anosmia they studied arose from a non-specific
effect such as an alteration of mucous membrane function. These membranes are
particularly sensitive to increases, as well as decreases, in vitamin A concentrations
of the body (WOLBACH and BESSEY, 1941; MOORE, 1957; BANG and BANG, 1963),
and furthermore it is known that changes in nasal mucous membrane function are
associated with changes in olfactory sensitivity (SCHNEIDER and WOLF, 1960).
Indeed, the very existence of anosmia argues a non-specific action since it implies
the loss of all ability to detect odors including that of the highly chemosensitive
"free" endings of trigeminal nerve in the nasal mucosa. There is no evidence that
any characteristic pigment is associated with these endings. The initial return of
"unpleasant olfactory sensations" following the administration of vitamin A might
thus be due to the more rapid restoration of function to the non-olfactory areas of
the nasal mucosa containing the trigeminal endings, while the later return of more
pleasant olfactory sensations would reflect the return of true olfactory sensitivity.
These experiments allow no final conclusion concerning the function of vita-
min A or carotenoids in olfaction.
Theories Implicating Pigment

in the Olfactory Transduction Process
The color of the olfactory epithelium - in mammals, at least - seems to be due
mainly to the presence of non-carotenoid pigments. Nevertheless it is the existence
of carotenoids in bovine olfactory epithelia that has generally seemed most
significant to those assigning a primary role to pigment in olfaction. In fact
carotenoids do possess several features which might well equip them for such a
function. In particular, they show a diversity of possible geometrical isomers,
unusual among substances of low molecular weight. It is, for example, the iso-
merism of ll-cis retinal by light which determines the critical role of that com-
pound in vision.
A further significant feature of carotenoids - shared with compounds of the
vitamin A series - is their high electron mobility, a property conferred by their
conjugated chain structure of alternating single and double bonds. This may allow
them to function as electron mediators in photosynthesis, bacterial respiration,
and in superconduction and semiconduction effects. For example, on the basis of
an earlier hypothesis of JAHN, DINGLE and Lucy (1965) have suggested that
carotenoids or vitamin A compounds may be implicated in the production of
membrane potentials by facilitating the movement of electrons from one side of a
biomolecular phospholipid leaflet to the other.
5*
But while there have been several suggestions that carotenoids are implicated
in the primary events of olfactory transduction (for example, CAMPBELL, 1956;
BRIGGS and DUNCAN, 1961) the only attempt to provide a detailed theory supported
by experimental observations, has been that of ROSENBERG et al. (1968). The
essential observation is that when various odorous gases are adsorbed on all-trans-
p-carotene powder, the semiconduction current increases by factors up to 107
(Fig. 2). This is accounted for by a decrease in activation energy for semiconduc-
tion. The current increase depends on the amount and nature of the adsorbed gas,
and the process is reversed by desorption of the gas.
10- 6
10-7
10- 8
0-
E
0
C 10- 9
~
:;
v
""(;
0
10- 11
60 100 I~O lBO 220 250

Time Imin)
Fig. 2. Time course of the current rise and decay in a p-carotene cell when nitrogen gas is
bubbled through various alcohol liquids kept at 15 C and passed through the chamber.
Arrows indicate when pure nitrogen is flushed through the chamber (from ROSENBERG et al.,
1968)
In the case of oxygen, the low binding energy (0.17 eV) found, indicates a
weakly-bound complex. There was also an increase in the dielectric constant of
p-carotene and they concluded that they were probably dealing with a donor-
acceptor complex. The effect of this would be to produce an increase in the
equilibrium number of charge carriers which, in turn, causes the observed increase
in conductivity (MISRA et al., 1968).
These investigations were carried out in a purely physical system. To evaluate
their relevance to the olfactory mechanism ROSENBERG and his associates ranked
test odors - by a technique they did not describe - into three categories: no (or
weak) odor; moderate odor; and strong odor. The current increases due to the
adsorption of these gases on p-carotene were also measured. Strong odors gave
increases of a factor of 105 -1 07 ; moderate odors gave increases of a factor of
102 -103 , while weak odors were included among those that gave increases of less
than a factor of lO.
However, the low current increases attributable to the esters are not consistent
with their strong odors and effectiveness in eliciting responses in the primary
olfactory neurones. ROSENBERG et al. (1968) have therefore investigated the effect
of adsorbed methyl acetate on the semiconductive properties of another organic
semiconductor, vitamin A alcohol crystals. With this compound, esters elicited a
large current increase. The authors therefore propose that different pigments
represent different receptor types and that odor quality might therefore depend
on the formation of different strength of donor-acceptor complexes between
odorous molecules and different pigments.
Further investigations are needed to clarify other anomalies. For example,
carbon dioxide - with little or no detectable odor - gives a current increase larger
than that of the longer chain-length alcohols which have strong odors, according
to other investigations. On the other hand, the short chain-length alcohols - with
relatively weak odor intensities - give larger current increases than the longer-
chain alcohols. It might also be predicted from their data that the odor properties
of a gas diluted in a nitrogen stream would differ from those of the same odorant
diluted in oxygen. There is no evidence to suggest that this is the case.
When ammonia gas was passed over p-carotene crystals another phenomenon
was observed. Following a rapid initial increase of five orders of magnitude the
current declined more slowly by three orders of magnitude. With successive flushes
of the ammonia the current increment became progressively lower. Sensitivity to
ammonia returned after flushing with nitrogen gas for a few hours. The authors
suggest that this phenomenon may explain both olfactory fatigue and adaptation.
But olfactory adaptation may be largely a central phenomenon (BENNETT,
1968). Little signs of it appear in electrophysiological recordings from the primary
neurones or olfactory bulb of a variety of species: prolonged depressions of response
seldom follow repeated receptor exposures to a variety of odorants. In any event,
when adaptation does occur, the subsequent recovery is on the scale of seconds
rather than hours.
ROSENBERG'S theory has the merit of drawing on one known constituent of
bovine olfactory epithelia to account for both odor quality and olfactory trans-
duction. But it also has several weaknesses: it is not yet clear that carotenoids
occur in the olfactory epithelia of all vertebrate groups, and that their charac-
teristics - when incorporated in a biological system - are comparable to those
they exhibit when in crystalline form. A comparison of observed current increases
with data derived from objective assessments of receptor response is also needed.
Some support for the idea that semiconduction effects can account for certain
neural phenomena comes from the analysis of CHALAZONITIS' data on photo-con-
ductance in thc pigmented A plysia neuron before, during and after exposurc to
light. According to solid-state theory a biological reaction which is rate-limited by
charge conduction across an activation energy barrier at the surface of a solid,
should be described by the Elovich equation. COPE (1968) has shown that CHALA-
ZONITIS' data conform to this prediction. The pigments of the Aplysia neuron are a
heme-protein and carotene-proteins (ARVANITAKI and CHALAZONITIS, 1960).
While ROSENBERG'S theory may, in essence, be applicable to non-carotenoid

olfactory pigments, it is primarily concerned with carotenoids. An earlier theory
of WRIGHT et al. (1956) makes no assumptions about the chemical constitution of
the pigment. They proposed that olfactory pigments are normally in an electro-
nically excited state. Resonance between the vibrations of the odorous molecule
and the pigment, trigger a return to the ground state. The resulting release of
energy initiates the nerve impulse. However, WRIGHT (1964) does not now
emphasize pigment as an essential ingredient of this hypothesis, and the theory has
in any case met with severe criticisms by THOMPSON (1957) and others.
But, if non-carotenoid chromoproteins form the bulk of the olfactory pigment,
it becomes relevant to consider the mode of action of other non-carotenoid chromo-
proteins with biologically significant effects. Phytochrome - for example - is
essential to a diverse range of physiological functions in plants (BONNER and
VARNER, 1965). In this case, the chromphoric group is a bilitriene.
If the olfactory pigment were implicated in olfaction in a comparable manner,
it would be reasonable to look for it primarily in the receptor membrane, in as-
sociation with a receptor site. It could then be argued that weak-binding of a
odorant to the receptor site induces a change in the shape of the non-protein part
of the chromoprotein molecule leading to a conformational change in the protein
moiety. This, in turn, leads to a transient alteration in the permeability of the
proteinaceous membrane of the receptor site. The resulting interchange of ions, as
they move down their concentration gradients, brings about a redistribution of
charge across the membrane. When of sufficient magnitude, this initiates the events
which terminate in the firing of a nerve impulse.
One difficulty with this and other hypotheses which emphasize events occurring
at the receptor membrane is that the olfactory pigment is apparently concentrated
within the supporting cells and in Bowman's glands. Now both these elements
contribute their secretions to the aqueous sheet overlying the receptors. Thus if
pigment has a function, it may focus on events occurring in this sheet. However,
the possibility that pigment is, in fact, present in the receptor cell, or that the
primary site of transduction occurs in the supporting cell (which may be electri-
cally coupled to the receptor cell through tight junctions) cannot be excluded.
But whatever theory is proposed to account for olfactory transduction it is
presumably intended to apply to all vertebrate groups. GEREBTZOFF and PHILIPPOT
(1957), however, suggest that olfactory pigment is involved in olfaction only in air-
breathing vertebrates, since they found no pigment in certain urodeles and fish, or
in the vomeronasal organ (which normally lies in a fluid filled chamber). Thus they
seem to equate the absence of color (as determined by gross inspection) with the
absence of all components of the olfactory pigment complex. Conceivably, however,
pigment may still occur, even when its density is too low to impart any charac-
teristic color to the epithelium. But if, in fact, all olfactory pigment is absent from
species in which olfactory sensitivity is well-developed, or from the high chemo-
sensitive vomeronasal organ, it may seriously be doubted whether pigment plays
any unique role in olfaction. Indeed, the absence of Bowman's, or analogous
glands, from non-terrestrial vertebrates demonstrates that pigment is dispensible
at this site at least, although when present, it may have a specific facilitatory, but
non-essential role. But the presence of non-functional pigment in the olfactory
References 71
epithelium would not be surprising. Beginning at the age of 7 -8, a proportion of

the ribonucleic acid in many neurones of the human body is said to be replaced by
a yellow pigment (HYDEN, 1950). This is assumed to be a waste product. Black,
dark brown, or yellow pigments are common in such neural structures as the
substantia nigra, spinal and sympathetic ganglia, and in unmyelinated neurons
(D'ANGELO et al., 1956; MALHORTA, 1957). It should also be recognized, however,
that pigments without function and with different functions may coexist in the
same epithelium.
Despite the accumulation of evidence, reflecting a wide diversity of approaches,
it is clear that we lack reliable information concerning all but a few of the charac-
teristics of the constituents of the olfactory pigment complex. We do not yet know
their identity, whether they are present in the receptor cell (and if so at what site)
and the extent to which they are represented in the olfactory epithelia of different
vertebrate groups. These and other deficiencies seriously impede the formulation
and testing of adequate hypotheses.
Summary
Pigments present in the olfactory chromatophores of lower vertebrates are
probably accidental dermal inclusions without functional significance. The remain-
ing olfactory pigments are concentrated in Bowman's glands of terrestrial vertebra-
tes and in the supporting cells. It is not clearly established that the receptor cell con-
tains pigment. Compounds identified in olfactory epithelia include retinol and
tJ-carotene in cattle, and retinol and vitamin A esters in the dog. The presence of
these compounds in the olfactory epithelia of other species has yet to be established.
Four non-carotenoid pigments, which are primarily responsible for the color of
the olfactory epithelium, have been isolated from, but not identified in bovine and
dog material. They are probably bound to protein and concentrated in pigment
granules.
When albino rats were compared with pigmented littermates no significant
differences were found in either the sensitivity to odors or the depth of pigmen-
tation of the olfactory mucosa. Loss of responsiveness to odors during vitamin A
deficiency in rats is probably due to the non-specific actions of the vitamin.
It has been proposed that the known ability of tJ-carotene crystals to show
semiconduction effects in the presence of adsorbed gases is the basis of the olfactory
transduction process. Other theories implicating pigment are considered. Olfactory
pigment is reported to be lacking in certain fish and from the highly chemosensitive
vomeronasal organ.
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Chapter 4
Degeneration and Regeneration

of the Olfactory Epithelium
By
SADAYUKI F. TAKAGI, Maebashi (Japan)
With 10 Figures
Contents
A. Experiments which Showed no Degeneration 76
B. Degeneration not Accompanied by Regeneration. . . . . . . 76
1. Sectioning of Olfactory Nerve or Ablation of Olfactory Bulb 76
2. Closing of the Nasal Opening 79
3. Effects of Chemicals . . 79
4. Effects of Inflammation. . . 81
5. Summary . . . . . . . . . 81
C. Regeneration of the Olfactory Epithelium after Degeneration 81
1. Removal of the Nasal Organ . . 81
2. Sectioning of the Olfactory Nerve . . 83
3. Effects of Zinc Sulfate . . . . . . . 85
4. A New Cell in the Normal Epithelium 87
5. Summary . . . . . . . . . . 87
D. Electrophysiological Approach. . 89
1. Changes in the Negative EOG's 89
2. Histological Changes. . . . . 91
3. Relation between the Electrical and Histological Changes. 91
4. Positive EOG's in the Degenerated Olfactory Epithelium . 92
5. Summary . . . . . . . . . 92
E. Final Summary and Conclusion. 92
References . . . . . . . . . . . 93
The olfactory epithelium is generally made up of olfactory, sustentacular and

basal cells. The olfactory cells, which are sensory in function and from whose cell
bodies originate the olfactory nerve fibers, are crowded several cells deep between
a superficial layer of columnar sustentacular cells, and a deep layer of irregularly
shaped basal cells. Each of the olfactory cells has a dendritic process, which passes
to the surface of the epithelium between the sustentacular cells. On the surface it
ends in a small vesicle from which arise the long, delicate olfactory cilia. The
76 S. F. TAKAGI: Degeneration and Regeneration of the Olfactory Epithelium
axonal process of each cell, the olfactory nerve fiber, passes between the basal cells
to enter the lamina propria on its way to the brain. Within the lamina propria are
located numerous tubular glands, called Bowman's glands which secrete serous
mucus to the surface of the olfactory epithelium. The sustentacular cells contain
many granules which are also secreted to the surface.
The olfactory cell is a sensory neuron. It has generally been held that nerve
cells which have been destroyed are never regenerated and that nerve cells do not
increase in number by mitosis in the postembryonal stage (cf. FRUHWALD, 1935).
Against this view, however, evidence has been presented in many experiments
that the olfactory cell may regenerate.
The aim of this chapter is to review the research works on the olfactory
epithelium which are concerned with this problem. In the research treated here
various methods were used to induce degeneration of the olfactory cell or
epithelium: the olfactory nerve was sectioned; the olfactory bulb was ablated; the
nasal air passage was obstructed, or some kind of drug was applicd. Although the
results obtained have been conflicting, they can be divided into the following three
categories: (A) olfactory cells do not degenerate, (B) they degenerate but do not
regenerate, and (C) they regenerate after degeneration. The investigations will be
presented here according to this classification, and finally the problem will be
studied from a different viewpoint by comparing electrogenesis in normal and
degenerating olfactory epithelia.
A. Experiments which Showed no Degeneration

BIFFINI (cf. NAGAHARA, 1940) is said to be a pioneer in this kind of research
work. SCHIFF (1859) sectioned the olfactory tracts of dogs but did not find any
degeneration in the peripheral parts of the nerves. Movement of the ciliary cells
was observed in the olfactory region even after the death of the dogs. COLASANTI
(1875) sectioned the olfactory nerve of the frog unilaterally and examined the
olfactory epithelia after one to ninty days. There was not found any difference
between the olfactory epithelia of the normal and thc operated sides. Excepting
these three experiments performed in the early period, all the other experiments
showed degeneration of the olfactory cells after the same manipulation.
B. Degeneration not Accompanied by Regeneration

1. Sectioning of Olfactory Nerve or Ablation of Olfactory Bulb
HOFFMANN (1867) sectioned the olfactory nerves of the frog and found fatty
degeneration of both the olfactory cells and the epithelial cells of the Schultze type.
EXNER (1877) also found fatty degeneration of these two kinds of cells in the frog
after the extirpation of the olfactory tract and bulb. The olfactory epithelia
characterized by the olfactory cilia changed into the cilia-free columnar epithelia
in two months, and all degeneration ended in two months. He attempted the same
experiments on rabbits without success. NAKAMURA (1916) sectioned the olfactory
nerves of the frog. Since degeneration of the olfactory nerve procecded towards the
periphery during the seven days after nerve section, he assumed that degeneration
Degeneration not Accompanied by Regeneration 77
would occur in the olfactory cells if he could observe it for more days. His assump-
tion was later proven in the bullfrog by TAKAGI and YAJIMA (1964, 1965). They
further showed that retrograde degeneration proceeds faster in the summer than
in the winter, the QI0 being about 2. This will be stated in detail later.
Similar experiments were performed in mammals. HOFFMANN (1867) in the
experiment stated above obtained the same results in the rabbit as in the frog.
BAGINSKY (1894) examined the degenerating olfactory epithelia of young rabbits
from 13 to 68 days after the nerve section. The results obtained were that atrophy
occurred in the whole olfactory mucous membrane, not only in the epithelia but
also in the submucosa; that the atrophy was not similar in different parts of the
epithelia in spite of seemingly the same experimental procedures, and that the
atrophy was more advanced the longer the operated animals were kept alive. It
is especially worthy of note that the degeneration of the olfactory nerve was not
parallel with that of the olfactory epithelium. From this finding, he believed it very
likely that the olfactory epithelium alone atrophied when only the olfactory nerve
was sectioned, but that it was accompanied by atrophy of the other parts when the
nerve sectioning was combined with an interference of blood supply. LUSTIG (1924)
destroyed the olfactory lobes uni- or bilaterally in 12 rabbits. After 45 to 65 days,
it was found that not only the olfactory cells, but also the epithelial cells of the
Schultze-type and the basal cells degenerated or disappeared. He added that
these degenerative processes were not completed even 65 days after the manip-
ulation.
TAKATA (1929) sectioned the olfactory nerves of white rats with minimal
injury of the olfactory bulb. Three to 119 days later, he found that sectioning of a
large number of the olfactory fibers in the immediate neighborhood of the cribri-
form plate had led to degeneration of the olfactory cells, and furthermore that in
regions of severe degeneration, the olfactory epithelium was replaced by the
ciliated epithelium which is characteristic in the respiratory region of the nasal
cavity. NAGAHARA (1940), however, attributed such replacement to the inflam-
matory changes due to chronic rhinitis, and CLARK and WARWICK (1946) supported
NAGAHARA'S criticism. It is interesting that TAKATA attributed a trophic function
to the ethmoidal branches of the trigeminal nerve, because he found that the
olfactory epithelia were well preserved in cases where the olfactory nerve was
sectioned but the trigeminal nerve was left intact, whereas the epithelia showed
remarkable degeneration in cases where the trigeminal nerve was injured but the
olfactory nerve was barely damaged. Moreover, he showed that destruction of the
olfactory bulb was not followed by changes in the olfactory region so long as the
vascular supply to the nasal cavity was not interfered with. Against this finding,
however, the following investigators showed that degeneration could occur in the
olfactory region if the olfactory bulb could be removed completely.
CLARK and WARWICK (1946) performcd complete resection of the olfactory
bulb unilaterally and examined the degeneration of the septal mucosa in rabbits.
No change was observed in the epithelium of the affected side during the first
12 hours, and very little before the lapse of 24 hours. But after 24 hours very
striking degeneration became evident, and after 48 hours the olfactory epithelium
decreased in thickness to 2/3 of the normal epithelium. In 2 or 3 weeks, degeneration
was complete and the epithelium decreased in thickness to half its normal size
(Fig. 1). In the meantime, the olfactory cells underwent an actual necrosis, the
Fig. 1. Sections of the olfactory epithelium of the normal side in the left and the operated side
in the right, 6 weeks after removal of one olfactory bulb. Note the reduction in thickness of the
epithelium and the residual receptors on the operated side (Protargol x 540) (CLARK, 1957)
Fig. 2. Sections of the olfactory epithelium covering the first endoturbinal on the operated side
(in the left) and the normal side (in the right). (x 120.) On the operated side the arrow marks
the abrupt transition between apparently normal epithelium and severely degenerated
epithelium (CLARK, 1951)
Degeneration not Accompanied by Regeneration 79
rods fragmenting and the nuclei breaking down into pycnotic granules strewn
throughout the epithelium, and the supporting cells played a significant part in the
phagocytosis of the products of degeneration of the sensory cells. Eventually there
survived only the supporting and basal cells. Up to eight weeks after the ablation of
the olfactory bulb, no evidence of regeneration was apparent. Later, CLARK (1951)
studied the same problem in the same animals, this time examining the degenerative
changes in the whole olfactory epithelia. The extent of the retrograde degeneration
of the olfactory cells was determined indirectly by the degree of reduction in the
total depth of the epithelial layer which, as already noted above, accompanies the
atrophy and disintegration of the olfactory sensory cells. Although this criterion of
the extent of the retrograde degeneration of the receptors only provides data of an
approximate kind, he could show that the reduction in depth of the entire olfac-
tory epithelium following the total resection of one olfactory bulb is 40 % on the
average. Moreover, following partial ablation of the olfactory bulb, he could show
the abrupt transition from the apparently normal to the severely degenerated
epithelia (Fig. 2). In 1957, he further studied this problem. Considering a defective
silver impregnation of the epithelium of the operated side, he reached a different
conclusion, namely that not all the receptors are affected by the removal of the
olfactory bulb. He found that about half (something between 40 and 60 %) under-
goes total degeneration and disappears almost completely in 3 days, but that the
remainder of the receptor cells persists for as long as 6 months after the operation.
This finding is comparable to EAYRS'S finding (1952), that after optic nerve section
in the rat rather less than half the ganglion cells whose axons comprise the fibers of
the optic nerve persists in the retina. In these three experiments, however, CLARK
could not find any sign of recovery in the olfactory epithelia up to a period of
6 months in the rabbit. SEN GUPTA (1967) repeated CLARK'S experiments in
15 young albino rats. After the first 24 hours the olfactory cells began to show a
violent dissolution, while the disrupted olfactory epithelia appeared to be repaired
by the tall sustentacular cells with cilia-like growth at the free margins. By the
fifth day, the olfactory receptor cells became totally absent and then never reap-
peared in the degenerated olfactory epithelia. The total disappearance of the
olfactory cells is a finding which is contradictory to the conclusion of CLARK (1957).
2. Closing of the Nasal Opening

FRUHWALD (1935) studied the effect on the olfactory epithelium of the blocking
of air passage through the nasal cavity. He sewed together the nasal openings uni-
laterally in young rabbits. Three to 12 days later, he undid the seams and found that
the olfactory cells had decreased in number, and that once the cells decreased they
never recovered at all. Consequently, he asserted that the olfactory epithelium is
completely built when the animal is born and that the olfactory sensory cells
never increase in the postembryonal stage, although mitoses are often found in the
supporting cells.
3. Effects of Chemicals
Zinc sulfate solution was once used as a prophylactic agent in poliomyelitis
epidemics. SMITH (1938) tried to clarify the effect of this solution upon the olfac-
tory epithelium of rats (Fig. 3). It was made clear that the olfactory epithelium is
completely destroyed by 1 % of this solution and that the destruction may go on
to sloughing, or the necrotic olfactory cells may become enmeshed in regenerating
cells derived from the non-sensory elements of the epithelium and then undergo
Fig. 3. Effects of ZnS0 4 1 (x 300). Normal olfactory mucosa as a control. 2 (x 300). The
olfactory mucosa two days after treatment with 1% zinc sulfate. 3 (x 300). The olfactory
mucosa after complete disappearance of the olfactory cells, showing early regeneration.
4 (x 300). The olfactory mucosa 12 days after treatment showing the differentiation of the
regenerated non-sensory elements to form ciliated columnar epithelium (SMITH, 1938)
gradual removal. This removal is completed within seven to ten days after treat-
ment, and a well developed ciliated columnar epithelium is present by the twelfth
day. It is interesting, however, that under the same experimental procedures he
found later that the olfactory cells can regenerate in the frog. This will be stated
later.
It is well known that colchicine, a plant alkaloid, in very small doses blocks
the mitotic process in cells at metaphase. JACKSON and LEE (1965) examined the
drug's effect on the olfactory bulb by means of the microinfusion technique (SIN-
GER, 1954). The disappearance of the olfactory cells and the reduction in thickness
Regeneration of the Olfactory Epithelium after Degeneration 81
were found in most regions of the olfactory epithelium. Intraperitoneal injection

of the drug also produced similar degeneration of the receptor cells. In either case,
although 20 to 80 % of the olfactory cells were lost due to degeneration, some
olfactory cells survived.
4. Effects of Inflammation
KOLMER (1927) showed in man that patches of the olfactory epithelium are
replaced commonly by ciliated columnar epithelium after they are destroyed by
inflammatory processes.
5. Summary
In summary, so far the results were stated which showed degeneration not
accompanied by regeneration of the olfactory cells or epithelia. HOFFMANN (1867),
EXNER (1877) and NAKAMURA (1916) sectioned the olfactory nerve in the frog and
QUASTLER and WEINGARTEN (1930) in the fish. HOFFMANN (1867), EXNER (1877),
BAGINSKY (1894), LUSTIG (1924), FRUHWALD (1935), CLARK and WARWICK (1946)
and CLARK (1951 and 1957) removed the olfactory bulb totally or partially in the
rabbit. TAKATA (1929) and SEN GUPTA (1967) did the same experiments in the rat.
Degeneration of all the receptor cells was observed in most experiments, but in a
few experiments varying amounts of receptor cells were found to have survived. In
this regard, it might be recalled that retrograde degeneration of the neuron is
generally considered to occur more severely the closer the axon is sectioned to the
cell body, and that the neuron is destroyed completely when the sectioning is
performed in the immediate vicinity of the cell body. Since the animals and the
experimental procedures are different in these experiments, it is no wonder that
the results do not coincide perfectly.
It is worthy of note that replacement of the degenerated epithelium by non-
sensory epithelium was observed in four cases. EXNER (1877) in the frog and SMITH
(1938) in the rat, found a nonciliary columnar epithelium in place of the olfactory
epithelium. TAKATA (1929) found a ciliated respiratory epithelium in place of the
olfactory epithelium in the rat. In the same animal, SEN GUPTA (1967) found that
the degenerated epithelium is repaired by a concomitant hypertrophy and cilia-
like growth at the free margins of the sustentacular cells. In this connection, it is
essential to demonstrate whether or not such changes occurred solely due to the
experimental procedures. Mammals are frequently affected by acute or chronic
rhinitis and replacement or repair of the affected area by non-sensory epithelium
can be found in many cases.
C. Regeneration of the Olfactory Epithelium after Degeneration

1. Removal of the Nasal Organ
BELL (1907) studied the development and regeneration of the nasal organ in
frog embryos. It was found that the nasal anlage, represented by a pigmented
thickening of the epithelium, can be readily regenerated if it is removed, and that
the nasal anlage very probably develops from a predetermined area of ectoderm
independently of the brain and buccal epithelium with which it normally connects.
6 Hb. Sensory Physiology, Vol. IV!I
82 S, F, TAKAGI: Degeneration and Regeneration of the Olfactory Epithelium
II
"
"
Fig,4
SZUTS (1914) performed the following two experiments in tritons: in the first
group he removed the nasal cavity and the olfactory epithelium, and in the second
group he removed the olfactory nerves and lobes together with the whole upper
jaw. The noses of the tritons in the first group completely recovered 3 weeks later.
In the tritons of the second group, not even an indication of regeneration was found
on the surface of the wound. He concluded, therefore, that the olfactory lobes are
needed for the regeneration of the nose, and that the olfactory nerves regenerate
from the forebrain.
2. Sectioning of the Olfactory Nerve

NAGAHARA (1940) examined the histology of the normal olfactory epithelium
in the mouse and divided the receptor cells into two types: type I cell is the "func-
tioning or active cell" which has a small, round and chromatin rich nucleus and is
mostly situated in the superficial part of the olfactory epithelium, and type II cell
is the "resting cell" which has a relatively large, oval and honey-combed nucleus,
lacks dendritic and axonal processes and is generally situated in the basal area.
Soon after sectioning the olfactory nerve, he found extensive degeneration in the
olfactory epithelium. Most of the functioning cells degenerated and disappeared in
3 days. On the other hand, the resting cells, although they often showed mitotic
division even in the normal olfactory epithelium, began to show very active
mitosis after nerve section: the resulting daughter cells extended their proximal
processes at first and their peripheral processes later. In this way the whole
epithelium was completely regenerated within 90 days after the operation (Fig. 4).
In these observations he could not find any degeneration in the sustentacular and
basal cells. Consequently, he believed that the olfactory cells increase by mitosis
even in the postembryonal stage, and that the olfactory epithelium has a dif-
ferentiation zone (Wachstumszone) in the basal area just as in other epithelia.
Although CLARK (1946, 1951, 1957) argued against NAGAHARA and attributed
his findings either to the incomplete destruction of the olfactory bulb, or to the
after-effects of rhinitis, NAGAHARA'S findings are supported by the recent findings
of ANDRES (1965, 1966), as will be stated later.
Fig. 4. Degeneration and regeneration of the olfactory epithelium. a Normal olfactory

epithelium. M figures of mitotic division of the olfactory cell nuclei. F "functio-
ning or active olfactory cell." R "resting olfactory cell." b Olfactory epithelium
a day after the section of olfactory nerve. The thickness of the epithelium has
not changed yet, but the nuclei of the olfactory cells (mostly functioning cells)
show as degenerative phenomena, pycnose, hyperchromatose of the nucleus wall, and
karyorrhexis. c Olfactory epithelium 3 days after nerve section. The epithelium decreased in
thickness to about half of the normal one. The nuclei of the resting cells show the figures of
division in various stages. The lumen of the Bowman's gland is clearly broadened, and the
gland cells are mostly swollen. d Normal olfactory epithelium (by axis cylinder staining).
e Olfactory epithelium, 3 days after nerve section. The sensory dendritic process of separated
resting olfactory cells is winding and thickened and has the appearance of a string of pearls here
and there. f Olfactory epithelium in the regenerative stage, 7 days after nerve section. Regener-
ative sprouts of the olfactory cells a.nd an increase of functioning cells are observed. The
thickness of the epithelium has slightly increased (NAGAHARA, 1940)
6"
Fig. 6
3. Effects of Zinc Sulfate

SMITH (1951) repeated the same experiment in the frog as in the rat (1938). He
found that 1 % zinc sulfate solution caused the olfactory epithelium to degenerate
and slough within two days (Fig. 5). By the time this sloughing was completed, the
lamina propria was already covered by a newly formed layer of flattened cells.
These new cells increased in number and by the 28th day the epithelium acquired
almost its full complement of cells. At this stage the cells looked much alike, had
large round nuclei and little cytoplasm, and were irregularly arranged and closely
crowded. Then in the second 4 weeks of regeneration, the regenerated cells progres-
sively assumed their characteristic structure and position. The sustentacular cells
acquired a columnar superficial portion and formed a palisade-like layer at the
surface. The sensory olfactory cells developed superficially directed processes which
ended at the surface as olfactory vesicles. The deepest cells of the epithelium, which
remained as a residue of undifferentiated cells, formed a now recognizable basal
layer. During the next two weeks maturation of the newly-formed epithelium con-
tinued and by the 70th day the regenerated epithelium was found to be identical
in structure with the normal epithelium.
In a second series of experiments, a small disc of the olfactory epithelium with
its supporting lamina propria was punched out with a needle and regeneration of
the lost tissue was examined. The lost tissue was at first replaced with blood clot.
Then the cells of the epithelium bordering the punched out area lost the specialized
characteristics that identify them as sustentacular, sensory or basal cells, and
mitotic figures appeared among these cells. Migration of these cells to close the gap
in the epithelium occurred, and at the 31 st day the hole was covered by round,
closely crowded, undifferentiated cells. Finally, at the 64th day the epithelium
became very similar with the normal epithelium. Thus, contrary to his own finding
in the rat (1938) he observed regeneration of the olfactory epithelium in the frog.
SCHULTZ (1941) showed in monkeys the restoration of the olfactory cells which
had been destroyed by 1 % zinc sulfate solution. He further studied (1960) the
effect of this drug upon the olfactory epithelium of monkeys (Fig. 6). Separation of
the degenerated olfactory epithelium from the lamina propria and generation of a
single layer of new flat cells covering the lamina occurred in nearly the same way
as SMITH (1951) observed in the frog. The cells making up this new epithelium
were derived largely from the lamina propria, partly from the duct cells of the
glands of Bowman, and some possibly from the sheath cells of Schwann. In its
early development, the new epithelium resembled an atypical, pseudostratified
epithelium. Soon after the tenth day, normal-appearing olfactory cells with
Fig. 5. Regeneration of the olfactory epithelium after degeneration. 1 Normal olfactory

epithelium. Control x 310. 2,3,4, and 5 Olfactory epithelium 3,14,28 and 56 days respectively,
after applying 1% zinc sulfate. x 310 (SMITH, 1951)
Fig. 6. Separation of necrotic olfactory epithelium. a Normal olfactory epithelium. x -135.

b En masse separation of necrotic olfactory epithelium from the lamina propria; near the nasal
vault; 28 hours after treatment. x 135. c En masse separation of necrotic epithelium 2 days
after treatment. A layer of new epithelial cells has begun to cover the denuded lamina propria.
Hematoxylin and eosin stain. x 135 (SCHULTZ, 1960)
Fig. 7. The olfactory epithelium of an adult cat. Edge of the olfactory epithelium (os) with
ciliary olfactory vesicles (sk) and microvilli (only suggested). In the outer cytoplasm zone of
the supporting cell (stz) lie cross- and oblique-sections of the shafts (d) of the sensory dendrites;
mitochondria (mi); nucleus of thc sustentacular cell (n). In the darkly stained cytoplasm of the
olfactory cell light Golgi-apparatus (gz) can be seen. A similar arrangement is distinctly seen in
the lighter blastema cell (bl); basal cell (bz); basal membrane (bm). In the center, mitosis of a
blastema cell is seen. A light-microscopic photograph (x 2,200) (ANDRES, 1966)
dendritic processes began to appear; by the 20th day they became numerous in
most areas, and by 6 months to a year after the treatment the general structure of
the epithelium was found to be essentially normal. These histological findings are
coincident with the observation that the resistance afforded by an intranasal
irrigation with zinc sulfate solution a day or two prior to intranasal inoculation
with a given neuronotropic strain of poliomyelitis virus (MV strain) was usually
followed in 3 or 4 months by resusceptibility to intranasal inoculation. From the

acquisition of resusceptibility, the restoration of the destroyed olfactory epithelium
had been presumed even before these studies.
4. ANew Cell in the Normal Epithelium

By means of blood vessel-perfusion-fixation with glutaraldehyde, ANDRES
(1965, 1966) found in the dog, cat and rat a new type of cell, a "blastema cell,"
besides the normally found sensory, supporting and basal cells (Fig. 7). The new
cell was usually found below the basal cells. It resembled the olfactory cell in the
structure of the nucleus and cytoplasm, but could be differentiated from the
sensory cell by the following features: (a) the new cell had a lighter and bigger cell
body; (b) lacked the characteristic stratified ergasto-plasmatic reticulum; and (c) its
tonofibrils composed a system connected by desmosomes. The blastema cells were
easily found in dogs and cats up to two months after birth.
Since it appeared that the sensory cells develop from the blastema cells even in
the olfactory epithelium of the adult animals, he assumed that the blastema cells
are a reservoir for the differentiation and regeneration of the sensory cells, and that
the olfactory epithelium, including the sensory cells, undergoes a constant regener-
ation (Fig. 8). The latter assumption corresponds to the findings of BEIDLER and
SMALLMAN (1967) in the rat, that the epithelial cells surrounding the taste bud
divide and that some of the daughter cells enter the taste bud and slowly move
toward the center, suggesting that the cells within the taste bud undergo con-
siderable change with time.
Although ANDRES did not study the changes of the olfactory epithelium which
may occur when the olfactory nerve is sectioned or when the olfactory bulb is
ablated, it may be presumed that regeneration of the olfactory cells after degener-
ation occurs in the same way as above. ANDRES' description of the location, struc-
ture and function of the blastema cell reminds the readers of the "resting cell"
discovered by NAGAHARA (1940). It is very probable that the "blastema cell" is
equivalent to the "resting cell," although it is not known whether the structure of
these two kinds of cells is entirely the same in detail or not.
5. Summary
In summary, all the above works indicated that the olfactory epithelium or the
olfactory cell can regenerate after degeneration. Regeneration of the olfactory cell
or discovery of the "resting cell" (NAGAHARA, 1940) or the "blastema cell"
(ANDRES, 1965, 1966) is generally regarded as very exceptional in the nervous tissue
at the postembryonal stage. However, a possibility that such regeneration may
occur in the olfactory epithelium is suggested by its very structure, in which the
olfactory cell bodies retain their primitive location in the surface epithelium, being
situated for the most part in the mid-zone of the fairly thick, pseudostratified
columnar epithelium of the olfactory mucosa. Moreover, KOLMER (1927) found
large bi-nucleated olfactory cells with two dendritic processes in the olfactory
epithelium of man. Considering these unusual features of the olfactory epithelium
as well as the very probable regeneration of the gustatory cell, a sensory receptor in
Fig. 8. A half-schematic representation of the olfactory epithelium of macrosmatic mammals.

The olfactory cell (rz) and blastema cell (b1) are imbedded in a connnected system of the susten-
tacular (stz) and basal cells (bz) by desmosoms and tonofibrils (tt). Basal spurs of the susten-
tacular cells and the basal cells are rich in lysosomes (ls). The caliber of the olfactory fila (I)
which are surrounded by Schwann's cells (sz) under the basal membrane (bm) are in contrast
to the size of the olfactory cells. The position of the Golgi-zone (gz) is identical in both the
blastema and the olfactory cells. The ergastoplasm in the laminated layer (er) is absent in the
blastema cell. One peripheral sensory dendrite is still not completely differentiated. Increase of
centrosomes is shown in the olfactory vesicles. The olfactory vesicle (sk) is a constituent part of
the olfactory edge (as). The ends (es) of the olfactory cilia lie in the outer mucus film (1m) or
between the microvilli (mz) (ANDRES, 1966)
a closely related sense organ (BEIDLER and SMALLMAN, 1967), it should not be
surprising that the olfactory epithelium has the ability to regenerate after destruc-
tion.
Electrophysiological Approach 89
D. Electrophysiological Approach
Since the pioneer works of HOSOYA and YOSHIDA (1937), YOSHIDA (1950) and
OTTOSON (1956), it has been well known that the olfactory epithelium elicits a slow
potential when an odour is applied. It was called the "Electro-olfactogram" or
"EOG" by OTTOSON (1956). So far five types of slow potentials have been found
in the frog, toad, fish, newt, guinea pig and rabbit. They are "electronegative-on,"
"electronegative-off," "electropositive-on," "electropositive-off" and "electro-
positive-after-potentials" (TAKAGI, 1969). Since the olfactory epithelium is a rather
simple tissue composed of three kinds of cells - olfactory, sustentacular and basal
cells; the origin of these slow potentials has been the subject of research for many
years.
TAKAGI and YAJIMA (1964, 1965) attempted to clarify the changes of these
EOG's in the bullfrog after sectioning the olfactory nerve unilaterally. The changes
of the electrical activity were compared with the histological changes in the degener-
ating and normal olfactory epithelia.
1. Changes in the Negative EOG's

In the first group of bullfrogs, the nerve section was performed in mid-August.
Recording of the electrical responses to odours including amyl acetate, a typical
negative EOG-generating odorant, showed that the negative EOG's gradually
decreased in amplitude and disappeared in eight days after the section (Fig. 9).
EOG Days after Olfactory cell

section Supporting cell
-A- 5 5.2 - 1.3
2 ~ 6 4.7 - 2.5
4
------ 7
8
2.9 - 1.7
1.9-0.14
5. 9 0.7 - 0.2
6 10 2.4 - 1.6
Fig. 9. Electrical responses and ratios of the number of the olfactory cells to that of the
supporting cells in the degenerating olfactory epithelia. In the left, decrease in the amplitude
of the negative EOG's in the bullfrog is shown from the top down in the order of days elapsed
after nerve section (shown in the middle). In the right, ratios of the number of the olfactory
cells to that of the supporting cells are shown in the same order. Further explanation in the
text (TAKAGI and YAJIMA, 1965)
Fig.IOa-d
Electrophysiological Approach 91
The same experiments were repeated successively in three groups of frogs between
September and December. The negative EOG's in these three groups disappeared
in 8 days, 11 days and 16 days respectively. The average temperatures in each of
the four successive experiments were 26.3 C, 22 C, 17.9 C and 10.9 C.
Now, when these four groups are compared, it is clear that as temperature falls
there is an increase in the number of days that elapse before the disappearance of
the negative EOG's. When the second group is compared with the fourth one, the
number of days becomes double in the latter group, while the average temperature
falls by 11C. If the disappearance of the negative EOG's is presumed to be due
to the retrograde degeneration of the olfactory cells, the QI0 of the velocity of nerve
degeneration can be said to be approximately 2.
Electrical activity of the olfactory epithelium continued to be examined, but
after their disappearance the negative EOG's could never be re-elicited by amyl
acetate in bullfrogs. These were kept alive for 103 days, a much longer period
than the two months which EXNER (1877) stated to be enough for all degeneration.
2. Histological Changes
When the normal olfactory epithelium stained by gallocyanin was examined
under a microscope, numerous nuclei could be seen. In many preparations, they
could be easily divided into the nuclei of the supporting, olfactory, and basal
cells, of the Bowman's glands and of phagocytes.
When the degenerating olfactory epithelium was examined under a microscope,
a decrease in the number of nuclei was noted (Fig. 10). Several days after nerve
section, nuclei were found only in the upper and the lower part of the olfactory
epithelium, and in the middle part a nucleus-free space became manifest. This
became more and more striking as time elapsed. The nuclei in the upper part were
identified as those of the supporting cells, and the nuclei in the lower part as those
of the basal cells, surviving olfactory cells, and phagocytes. About 10 days after
nerve section, the number of the olfactory cells was found to be considerably
smaller than in the normal epithelium.
Recovery of the olfactory cell was histologically examined in these degenerating
olfactory epithelia, but not the slightest indication of regeneration of the cell was
obtained up to 42 days after nerve section.
3. Relation Between the Electrical and Histological Changes

The supporting cell is heptagonal or octagonal in the horizontal section (ALLI-
SON, 1953), and each supporting cell has two apexes in common with a neighboring
Fig. lOa-d. Sections of the normal and degenerating olfactory epithelia. a) A section of normal
olfactory epithelium as a control. Slender nuclei of the supporting cells are found in the upper
part and round nuclei of the olfactory cells are found below them. b-d) Sections of degenerat-
ing epithelia. 6 days in b) 9 days in c) and 16 days in d) after the nerve section, respectively.
Slender nuclei are seen in the upper part and round ones in the lower part. Between them a
spacial gap is noted. It becomes more and more striking as days elapse. d) The number of the
supporting cells is far larger than that of the nuclei in the lower part. The olfactory cell has
degenerated considerably and the number is very small in this stage. Most of the nuclei found
in the lower part and the round nuclei in the uppermost part are presumed to be of different
origin. Further explanation in the text (TAKAGI and YUIMA, 1965)
supporting cell. Since the olfactory cells do not always lie in all but only in some of
the apexes of a supporting cell (RETZIUS, 1900), the ratio of the number of the
olfactory cells to that of the supporting cells is less than 5 or 6 to 1.
When the ratio was examined in the degenerating epithelia, the maximal ratio
was found to decrease day by day after the section of the olfactory nerve. Then,
when this ratio was compared with the change in the magnitude of the negative
EOG's, it was found that the negative EOG's disappeared when the ratio went
below 2 to 1 (Fig. 9). From this finding, it was concluded that the negative EOG's
originate in the olfactory receptor cells (TAKAGI and YAJIMA, 1965).
4. Positive EOG's in the Degenerated Olfactory Epithelium

Although the negative EOG's disappeared in the degenerated olfactory epithelium,
the positive EOG's continued to appear (TAKAGI, AOKI, IINO, and YAJIMA, 1969).
Since degeneration of the olfactory cells was confirmed in this epithelium, the
origin of the surviving positive EOG's should be sought in the other elements of
the epithelium. By means of electronmicroscope, OKANO and TAKAGI (1971) ex-
amined the degenerating and degenerated olfactory epithelia and found that
granules were secreted from the supporting cell much more vigorously when
chloroform vapour, a typical positive EOG-generating odorant, is applied than
when amyl acetate vapour is applied. Consequently, it was thought very probable
that at least most of the positive EOG's are generated by the secretory activity of
the supporting cells. It seems that the sustentacular cells retain their secretory
function even after the degeneration of the olfactory cells (cf. CLARK, 1946).
5. Summary
In summary, from the results of the experiments on the electrogenesis of the
olfactory epithelium, it may be said that regeneration of the degenerated olfactory
cells in the bullfrog does not occur for as long as 103 days after nerve section.
However, this does not negate a possibility that the olfactory cell may regenerate
under different experimental conditions, and after a longer period of time.
E. Final Summary and Conclusion

Degeneration and regeneration of the olfactory cell or the olfactory epithelium
have been studied by many investigators after sectioning the olfactory nerve or
ablating the olfactory bulb. The problem has also been studied by a few research
workers after applying zinc sulfate or colchicine solution and by one investigator
after closing the nasal opening. The animals used in these experiments were
various, from tritons up to monkeys.
In many histological studies regeneration of the olfactory cell or epithelium
has not been found. This is in harmony with the generally held hypothesis that the
neurons do not increase in number by mitosis in the postembryonal stage. It was
also supported by one electrophysiological study on electrogenesis or EOG in the
degenerating olfactory epithelium.
On the other hand, however, there has been accumulated much histological
data which indicates regeneration after degeneration. It is very possible that after
References 93
incomplete degeneration the olfactory cells revive, extend new axons and sensory
dendrites and recover completely; for example, when the olfactory nerves have been
sectioned at a distance from the cell bodies or when the drug effects have been
insufficient. This is very probable in the light of the well known cases of the sec-
tioning of motoneuronal axons. In the research works reviewed here, however,
there has not been found a single study which suggested such a result. All the works
indicated a generation of entirely new olfactory cells or epithelium after the
destruction of the old olfactory cells or epithelium.
Since the olfactory sensory neurons retain their primitive location in the super-
ficial epithelium, the olfactory epithelium is presumed to be a poorly developed
receptor organ. Consequently, it is highly conceivable that some undifferentiated
cells increase in number by mitosis, develop and become entirely new sensory
neurons under certain favourable circumstances. In this connection, the finding of
the "resting olfactory cell" by NAGAHARA (1940), the "blastema cell" by ANDRES
(1965, 1966), and regeneration of a new olfactory epithelium from the underlying
lamina propria by SMITH (1951) and SCHULTZ (1960) cannot be overestimated,
and should stimulate research work in the other nervous tissues toward the
solution of a very basic problem, regeneration of the neuron.
References
195-244 (1953).
ANDRES, K. H.: DifIerenzierung und Regeneration von Sinneszellen in der Regio olfactoria.
Natmwissenschaften 17, 500 (1965).
- Der Feinbau der Regio olfactoria von Makrosmatikern. Z. Zellforsch. 89, 140-154 (1966).
BAGINSKY, B.: tJber das Verhalten von Nervenendorganen nach Durchschneidung der zu-
gehOrigen Nerven. Virchows Arch. path. Anat. 137, 389-404 (1894).
BEIDLER, L. M., SMALLMAN, R. L.: Renewal of cells within taste buds. J. Cell BioI. 27, 263-272
(1967).
BELL, E. T.: Some experiments on the development and regeneration of the eye, and the nasal
organs in the frog-embryos. Arch. Entwickl.-Mech. Org. 23,457-478 (1907).
CLARK, W. E. LE GROS: The projection of the olfactory epithelium on the olfactory bulb in the
rabbit. J. Neurol. Neurosurg. Psychiat. 14, 1-10 (1951).
299-319 (1957).
- WARWICK, R. T. T.: The pattern of olfactory innervation. J. Neurol. Neurosurg. Psychiat.
9, 101-111 (1946).
COLOSANTI, G.: Untersuchungen iiber die Durchschneidung des Nervus olfactorius bei Fro-
schen. Arch. f. Anat. u. Physiol. V. Reichert u. d. Bois Reymond, 469 (1875). Cited by
Nagahara Y. (194O).
EAYRS, J. T.: The relationship between the ganglion cell layer of the retina and the optic nerve
in the rat. Brit. J. Ophthal. 38,453-459 (1952).
EXNER, S.: Fortgesetzte Studien iiber die Endigungsweise des Geruchsnerven. S.-B. Wien.
Akad. 78, Abt. 3 (1877).
FRURWALD, V.: Die Folgen des einseitigen N asenverschl usses auf die Riechschleimhaut und auf
den Bulbus und Tractus olfactorius. Arch. Ohren-, Nasen- u. Kehlkopfheilk. 139, 153-173
(1935).
HOFFMANN, C. K.: tJber die Membrana olfactoria. Inaug.-Diss. Amsterdam 1867.
HOSOYA, Y., YosmDA, H.: tJber die bioelektrische Erscheinung an der Riechschleimhaut. Jap.
J. Med. Sci. III, Biophysics. 5, 22-23 (1937).
JACKSON, R. T., LEE, CR.-CR.: Degeneration of olfactory receptors induced by colchicine. Exp.
Neurol. 11,483--492 (1965).
KOLMER, W.: Geruchsorgan. In: VON MaLLENDoRFF, W., Handbuch der Mikroskopischen
Anatomie des Menschen, Bd. III, S. 192-249. Berlin: Springer 1927.
LUSTIG, A.: Die Degeneration des Epithels der Riechschleimhaut des Kaninchens nach Zer-
starung der Riechlappen desselben. S.-B.Wien. Akad. 89, Abt. 2 (1924). Cited by NAGAHARA
(1940).
NAGAHARA, Y.: Experimentelle Studien tiber die histologischen Veranderungen des Geruchs-
organs nach der Olfactoriusdurchschneidung. Beitrage zur Kenntnis des feineren Baus des
Geruchsorgans. Jap. J. Med. Sci. V, Pathoi. 5, 165-199 (1940).
NAKAMURA, Y.: Uber die Veranderung des Geruchsorgans nach der Olfactoriusdurchschnei-
dung bei Froschen. Dainihon Jiji Ih. 22, (1916). Cited by NAGAHARA, Y. (1940).
OKANO, M., TAKAGI, S. F.: In preparation 1971.
OTTOSON, D.: Analysis of the electrical activity of the olfactory epithelium. Acta physioi.
scand. 35, Suppi. 122, 1-83 (1956).
QUASTLER, H., WEINGARTEN, H.: Kiinnen Fische die Riechschleimhaut regenerieren? Arch.
Entwickl.-Mech. Org. 122, 763-769 (1930).
RETZIUS, G.: Cited in RAUBER-KoPSCH'S Lehrbuch der Anatomie des Menschen. Abt. VI.
S.88 (1900). Leipzig: Georg Thieme 1912.
SCHIFF, M.: Der erste Hirnnerv ist der Geruchsnerv. Moleschotts Untersuch. 6, 254--424
(1859). Cited by NAGAHARA, Y. (1940).
SCHULTZ, E. W.: Regeneration of olfactory cells. Proc. Soc. expo BioI. 46, 41--43 (1941).
- Repair of the olfactory mucosa. Amer. J. Path. 37, 1-19 (1960).
SEN GUPTA, P.: Olfactory receptor reaction to the lesion of the olfactory bulb. Olfaction and
Taste II, ed. T. HAYASm, pp. 193-201. New York-London: Pergamon Press 1967.
SINGER, M.: Apparatus for continuous infusion of microvolumes of solution into organs and
tissues. Proc. Soc. expo BioI. 86, 378-380 (1954).
SMiTH, C. G.: Changes in the olfactory mucosa and the olfactory nerves following intranasal
treatment with one per cent zinc sulfate. Canad. Med. J. 39, 138-140 (1938).
- Regeneration of sensory olfactory epithelium and nerves in adult frogs. Anat. Rec. 109,
661-671 (1951).
SZUTS, A. v.: Beitrage zur Kenntnis der Abhangigkeit der Regeneration vom Zentralnerven-
system. Arch. EntwickI.-Mech. Org. 38, 540-546 (1914).
TAKAGI, S. F.: The EOG problems. Olfaction and Taste III, ed. C. PFAFFMANN, pp. 71-91.
New York-London: Rockefeller University Press 1969.
- AOKI, K., IINO, M., YAJIMA, T.: The electropositive potential in the normal and degenerat-
ing olfactory epithelium. Olfaction and Taste III, ed. C. PFAFFMANN, pp. 92-108.
New York-London: Rockefeller University Press 1969.
- YAJIMA, T.: Electrical responses to odours of degenerating olfactory epithelium. Nature
(Lond.) 202, 1220 (1964).
- - Electrical activity and histological change in the degenerating olfactory epithelium. J.
gen. Physioi. 48, 559-569 (1965).
TAKATA, N.: Riechnerv und Geruchsorgan. Arch. Ohren-, Nasen- u. Kehlkopfheilk. 121,43-78
(1929).
YOSHIDA, H.: Bioelectrical phenomena in the olfactory epithelium. Hokkaido J. Med. 2;;,
454-458 (1950). (In Japanese.)
Chapter 5
The Electro-Olfactogram
A Review of Studies on the Receptor Potential of the Olfactory Organ
By
DAVID OTTOSON, Stockholm (Sweden)
With 21 Figures
Contents
I. The Concept of Generator Potentials. . . . . . 96
A. Generator Potentials in Different Types of Receptors . 96
B. Early Observations on Slow Olfactory Potentials . 98
II. Characteristics of the EOG. . . . . . . . . . . . 99
A. Stimulus Intensity-Response Amplitude Relation 99
B. Stimulus Quantity-Response Amplitude Relation. 100
C. Time Course of Activation. 101
1. The Latent Period. 101
2. The Rising Phase . . . 102
3. The Falling Phase. . . 103
D. The Response to Sustained Stimulation. 103
E. Recovery. . . . . . . 104
F. EOG in Different Species 106
G. Oscillations . . . . . 108
H. On- and Off-Responses . 109
J. Origin of EOG III
III. Relation to Stimulus Characteristics. 116
A. Relation to the Dynamic Characteristics of the Stimulating Air Current. 116
B. Responses to Different Types of Odorous Substances . . 116
C. Relation to the Properties of the Stimulating Substances 117
D. Quantitative Analysis of Odor Mixtures. . . . . . . . 119
IV. EOG and Olfactory Input to the Bulb. . . . . . . . . . 120
A. EOG and Impulse Activity of the Olfactory Nerve Fibres . 120
B. EOG and Olfactory Bulb Activity . . . . 121
1. Slow Bulb Potential and Induced Waves 121
2. Unitary Activity . . . . . . . . . . 122
V. EOG and Psychophysical Measurements . . . . 124
Comparative Studies of the Frog's EOG and Subjective Odor Intensity in Human 124
VI. Concluding Remarks 126
References . . . . . . 128
96 D. OTTOSON: The Electro-Olfactogram
I. The Concept of Generator Potentials

A. Generator Potentials in Different Types of Receptors
It is generally agreed today that the impulse discharge in primary afferent
fibres of sense organs is triggered by a local depolarization produced by the receptor
cells. BECK (1899) was probably the first to record a pure receptor potential. In
,
\
\. .
"
\
.... ............... _,
.~ . . . . -._._.f!.....___._._
--
Fig. 1. Graphical reconstruction of the receptor potential of the eye of Eledone. (From BECK,
1899)
studies on the electrical reaction of the eye of Eledone he found that the receptor
layer became more negative during illumination. BECK had no photographic
equipment for recording the response. In order to bring out the time course of the
potential change he therefore made graphical reconstructions from the readings of
the galvanometer. Fig. 1 shows one of these reconstructed responses. BECK
described the response as follows:
"Beim Kommen des Lichtes erfolgt ein starker Anschlag, der rapid das Maxi-
mum erreicht. Sofort nach Erreichung des Maximums tritt ein allmahliches, aber
doch anfangs bedeutend rascheres Sinken als in den Kurven des ersten Typus ein.
Dauert die Belichtung lange, so erfolgt nach dem raschen ein langsames Sinken,
bis wieder eine Grenze erreicht wird, auf welcher die Ablenkung stehen bleibt oder
sich sogar hebt. Bei Eintritt der Verdunkelung berner ken wir wieder ein rascheres
Sinken der Schwankung, besonders rasch und jah nach dauernder Belichtung,
langsamer nach lang dauernder Belichtung. "
This account gives a concise description of all the essential features of a pure
receptor potential as confirmed by later studies in different types of slowlyadapt-
ing sensory end organs_ The response is characterized by an initial rapidly rising
phase followed by a decline to a static level during maintained stimulation and a
fall to zero after cessation of the stimulus.
The eye of Eledone consists of a single layer of visual cells and it was natural for
BECK to assume that the response was produced by these cells. Further evidence
supporting this view was provided by FROHLICH (1914) who confirmed and
extended BECK'S finding. FROHLICH found that the response could be obtained
from the isolated receptor layer which had been separated by dissection from
other structures of the eye. FROHLICH, who was the first to analyze closely the
properties of the pure receptor potential (Fig. 2), showed that the amplitude of
Generator Potentials in Different Types of Receptors 97
the response was a logarithmic function of the brightness of the illuminating light.
He also studied the oscillations superimposed upon the slow sustained potential
and showed that they increased in frequency with increasing intensity of the light.
Responses closely similar to that of the eye of Eledone were later recorded from the
eyes of arthropods by HARTLINE (1928). In some species such as Limulu8 the
response had the same simple shape as in Eledone while in other species the poten-
tials recorded were more complex and often consisted of an "on" -effect in response
to the onset of illumination and an "off" -effect in response to cessation of illumi-
nation.
Fig. 2. The electrical response of the eye of Eledone to illumination. Time marks: 1/5 sec.
(From FROHLICH, 1914)
An important contribution to the understanding of the development of these

polyphasic responses was made in 1942 by BERNHARD in recording from the eye of
the water beetle (Dytiscus). The eye of the water beetle is more complex than that
of Eledone in being closely connected with the optic ganglion. The response to
illumination recorded from the optic ganglion consists of a slow potential with
superimposed oscillatory waves. Cessation of illumination is followed by a pro-
nounced "off"-effect. BERNHARD found that the "off"-effect and the waves
disappeared after the optic ganglion had been crushed but the slow monophasic
response remained unaffected. This observation suggested that the slow response
was produced within the receptor layer. Definite evidence showing the retinal
origin of the slow potential was obtained by treating the preparation with cocaine
to block the conducted impulse activity (Fig. 3). The remaining response was a
smooth monophasic potential which represented the activity of the receptors. This
finding showed that the "on" and "off"-effects recorded from the untreated pre-
paration had to be attributed to the activity within the optic ganglion. BERN-
HARD'S observations clearly pointed to the functional role of the receptor potential
in the generation of the conducted activity.
The concept of the generator potential was formulated by BERNHARD, GRANIT,
and SKOGLUND (1942) in another paper which appeared in the same issue of the
Journal of Neurophysiology as the report on the Dytiscus eye. On the basis of con-
siderations of possible mechanisms underlying the production of potentials in
sense organs and of accommodation properties of nerve fibres, BERNHARD et al.
suggested that "i) the sensory cells themselves develop only generator potential
but no impulses, ii) the generator potential is carried down electrotonically to the
7 Rb. Sensory Physiology, Vol. IV/I
optic ganglion, where both a slow stationary potential difference as well as an

impulse discharge is initiated, iii) in some cases inhibition turns up instead of
excitation, and iv) these phenomena all can be imitated by polarizing currents".
The idea that the receptors are generators of local current which triggers the
nerve impulses has gained support from studies in different types of end organs
and is at present generally accepted as explanation for the initiation of impulses in
sense organs. Since the receptor potentials recorded from simple vertebrate eyes
~------------------------
br--_____________
"---
Fig. 3. Isolation of the receptor potential of the eye of DytisGUS with cocaine. Recordings
from optic ganglion before (a) and after (b) treatment of the preparation with 4% cocaine.
(From BERNHARD, 1942)
like Eledone or Limulus represent the activity of a great number of sensory cells
no definite conclusion can be drawn from the characteristics of these potentials as
to the response of the individual elements. The behaviour of the single receptor unit
remained unknown until 1950. Then, in studies of responses recorded from a single
frog muscle spindle, KATZ (1950) was able to isolate a graded potential after block-
ing of the conducted activity with cocaine. The characteristic behaviour of this
potential and its relation to the impulse discharge during controlled linearly rising
stretches has later been studied in detail by OTTOSON and SHEPHERD (1965). The
response is a purely monophasic negative-going potential with an initial dynamic
peak followed by a decline to a static level during maintained stretch. At termina-
tion of stretch the potential gradually declines to baseline. The receptor potential
of the muscle spindle shares its typical features with the responses recorded from
other sensory organs like the insect chemoreceptor (BOECKH, PRIESNER, and
SCHNEIDER, 1963), the crustacean stretch receptor (EYZAGUIRRE and KUFFLER,
1955), the visual cell of the Limulus eye (cf. FUORTES, 1958) and the response of
the decapsulated Pacinian corpuscle (LOEWENSTEIN and MENDELSON, 1965;
OZEKI and SATO, 1965).
B. Early Observations on Slow Olfactory Potentials

In the course of studies of the patterns of discharge of units within the olfactory
bulb of the rabbit it was noticed by the present author (OTTOSON, 1954) that
olfactory stimulation gave rise to a slow sustained potential. The general properties
Stimulus Intensity - Response Amplitude Relation 99
of the response suggested that it was homologous with the dendritic potentials
recorded from other areas of the cerebral cortex. However, in view of the specific
structural features of the bulb it could not be excluded that the potential repre-
sented the summated impulse activity of the afferent fibres. There was also the
possibility that the response was produced by the receptors in the olfactory mem-
brane and electrotonically conducted to the bulb. The latter view was favored by
the observation that a slow potential closely similar to that obtained from the bulb
could be recorded from the olfactory fibres at their entrance into the cranial
cavity. This response remained almost unaffected after the nasal mucosa had been
treated with cocaine at a concentration sufficiently strong to block the conducted
activity, suggesting that the response recorded from the nerves represented the
receptor potential of the olfactory organ. The close similarity between this potential
and that recorded from the bulb indicated that the two potentials had the same
origin. However, later studies provided definite evidence that the bulb potential
actually was produced by structures within the bulb. Hence, there were two
sustained potentials, one produced by the sensory cells and another developed by
structures in the bulb. Later it was pointed out by TAKAGI and SHIBUYA (1959)
that the mucosa potential had been observed in 1937 by HOSOYA and YOSHIDA.
In the rabbit as in most higher vertebrates the olfactory sensory cells are
spread out over extensive areas of the nasal mucosa covering an elaborate system
of folds of turbinal processes. The complex structure of the peripheral organ
together with the relative inaccessibility of the nerve fibres in the rabbit made it
difficult to analyze the potentials which had been found. Quantitative studies on
the properties of the response of the olfactory membrane were therefore carried
out on the frog's olfactory organ which was found to offer an almost ideal pre-
paration for such a study. In the frog the receptor sheet is spread out over a
relatively flat portion of the nasal mucosa which can be easily exposed together
with the bulb and the olfactory nerve. This makes it possible to apply the stimulus
under well controlled conditions and to make simultaneous recordings from the
mucosa and the nerve fibres or the bulb. The studies on the frog provided evidence
of a strikingly similar behaviour of the response obtained from the olfactory
membrane and the retinogram in the simple invertebrate eye. Because of this and
because of the structural similarity between the sensory olfactory epithelium and
the retina of the simple eye it was suggested that the olfactory potential should be
called the electro-olfactogram, the EOG (OTTOSON, 1956). This denomination was
introduced to designate the monophasic negative potential evoked by odors in the
sensory region of the nasal mucosa and is in the following used in this strict sense.
II. Characteristics of the EOG

A. Stimulus Intensity - Response Amplitude Relation
The electrical response of the frog's olfactory epithelium to a short puff of
odorous air is a slow negative monophasic potential with a steep rising phase
followed by an exponential fall towards the baseline (Fig. 4). The response is only
obtained from regions of the mucosa lined with sensory cells. These regions are
7
easily identified since they have a slightly yellow colour while those with respira-
tory epithelium are pink. Pure air elicits no response or an insignificantly small
response which most likely arises due to traces of odorous material in the air
current. It can therefore be concluded that the response obtained with odorized
air is not produced by mechanical stimulation of the receptors. The absence of
Fig. 4. The electro-olfactogram. Superimposed records of responses of frog's olfactory organ to

brief stimulations with butanol-vapor of different strengths. (From OTTOSON, 1956)
response to pure air also is a crucial control that there is no contribution of non-
biological effects such as may easily arise if inappropriate electrodes are used (cf.
MOZELL, 1961). The fact that the response increases in amplitude with increasing
concentration of odorous material in the stimulating air current provides further
evidence that the potential reflects the activity of the olfactory receptor cells.
Analysis of the relationship between stimulus strength and magnitude of
response shows that the amplitude of the potential is a power function of the
stimulus concentration. This is of interest in relation to the findings in psycho-
physical studies as will be discussed in the following.
B. Stimulus Quantity - Response Amplitude Relation

For a given concentration of the odorous stimulus the response increases up to
a given maximum with increasing volume of the stimulating air current. This
effect may partly be due to the fact that with increasing amounts of odorized air
greater areas of the olfactory membrane are activated. It is also likely that the
olfactory receptors are differentially sensitive and that receptors with higher
threshold are recruited when the amount of odorized air is increased. The fact that
Time Course of Activation 101
the activity is a function of the concentration as well as the amount of odorous

substance carried to the olfactory membrane reveals an important functional
property of the olfactory organ. It implies that for a short-lasting stimulation the
olfactory membrane measures the absolute quantity of the odorous material
impinging upon the mucosa. The behaviour of the olfactory organ is in this respect
closely similar to that of the eye. As shown by HARTLINE (1928) the response of the
photoreceptors of the eye of Limulus follows the Bunsen-Roscow law according to
which a constant amount of energy is required for the production of a constant photo-
chemical effect. When the olfactory mucosa is stimulated with a constant amount of
odorous material distributed in different volumes of air the responses are of equal
height. Apparently the reactions which elicit the EOG in the frog are dependent on
the amount of stimulating material of the odorous stimulus in a manner similar to
that in which the activity of the optic nerve of Limulus is a function of the energy
of the illuminating light. In the eye the reciprocity between time and intensity of
illumination only applies for brief periods of stimulation. The same holds true for
volume and concentration of odorous stimulation of the olfactory membrane.
C. Time Course of Activation

1. The Latent Period
It is generally agreed today that in order to produce excitation the odorous
substance has to be brought in direct contact with the receptors. However, it is
not long ago that various radiation theories still were flourishing. According to
these theories no contact between the stimulating substance and the receptors was
required since the odorous particles were assumed to emit some sort of radiation
which stimulated the receptors in a way similar to that in which the visual cells are
stimulated by light. According to one theory (MILES and BECK, 1949) the olfactory
receptors emit infra-red radiation which was supposed to be absorbed by the
odorous particles. The loss of energy was postulated to be the trigger mechanism.
It has been difficult to obtain definite and unequivocal evidence against or for
the notion that actual contact between the stimulating molecules and the receptors
is necessary for excitation. To prove that contact is required, the odorous particles
should be allowed to come close to the receptors but not to enter into direct con-
tact with them. This has been possible to do in the frog by covering the mucosa
with a very thin film of plastic material and using the EOG as an index of the
effects evoked by the stimulus (OTTOSON, 1956). These experiments provide
definite evidence that the olfactory receptors are not excited unless the odorant
particles are brought into direct contact with the mucosa.
Before the contact between the odorous material and the receptor elements is
established the particles have to pass through the layer of watery mucus covering
the sensory cells. The time required for this passage depends on a number of factors
among which the solubility properties of the substance and the thickness of the
mucus layer may be the most important ones. The thickness of the mucus layer in
the frog varies from one region of the mucosa to another and also with the activity
of the secreting cells. On the average the thickness is 25-30 fL (REESE, 1965). The
hairs protruding from the sensory cells extend to the outer surface of the mucus
102 D. OTTOSON: The ElectroOlfactogram
layer where they form a membrane which is separated from the air in the nasal
cavity by a thin film of mucus. The first stage in the excitatory process can be
assumed to involve the absorption of the molecules of the stimulating substances
on the chemosensitive membrane. This stage may be followed by several inter-
mediate processes which finally lead to a depolarization of the sensory cells. At the
present time there appears to be no means by which we can study the events
taking place during the silent period from that moment when the odorous particles
impinge upon the mucosa until the electrical change appears. However, the dura-
tion of this period can be measured and from this we can draw some conclusions.
The latent period of the frog EOG as measured from the moment when the
front of the stimulating air current reaches the surface of the epithelium and the
onset of the response varies from 200 to 400 msec when butanol is used as stimulat-
ing agent. The true latent period must be shorter since the measured values include
the time taken for the particles to pass through the mucus to the receptors. Another
factor which has to be taken into account in this context is that the ambient air is
pushed ahead of the stimulating air current. The arrival of the air front can there-
fore be assumed to occur somewhat earlier than the actual arrival of the stimulating
particles. If it is assumed that it takes 100 msec before the odorized particles reach
the receptors the true latency for the response to butanol (0.1 M) would be
100 msec or less. It is interesting to note that in the invertebrate eye the latent
period may be of the same value for moderately strong flashes of light (cf. FROH-
LICH, 1914). The fact that the response with strong stimulation appears within
such a relatively short latency has interesting implications as to the origin of the
receptor potential. There are two sites at which the electrical response might arise.
One site is represented by the sheet formed by the cilia close to the surface of the
mucus layer, the other being the olfactory vesicles at the ends of the dendrites of
the receptor cells. In the latter case the particles would have to pass by passive
diffusion through a layer of mucus which is at least 25 !L thick but may be up to
100 !L thick or more. Quite besides the likelihood that most of the particles would
be trapped by the cilia, such a passage would take a considerably longer time than
the measured latent period. It therefore appears most likely that the responses
arise in the cilia. This view is also supported by the observation that the response
is greatest at the outer layer of the mucus layer.
2. The Rising Phase

The time course of the response is of interest since it reflects the temporal
characteristics of the excitatory process in the receptor elements. With a given
substance the potential rises faster with increasing stimulus strength. A closer
study of the rise reveals that the peak of the response is reached at approximately
the same time for different strengths. This is of particular interest since other
receptor potentials like that of the Pacinian corpuscle (GRAY and SATO, 1953) or of
the muscle spindle (OTTOSON and SHEPHERD, 1965) exhibits similar properties. It
would appear therefore that the constant rise time of the response to a given
stimulus reflects a common property of receptor potentials. Since the response
recorded from the olfactory membrane represents the summated activity of all
excited receptors it gives no direct information concerning the electrical behaviour
of the individual receptors. It is of interest in this context to note that in the eye
The Response to Sustained Stimulation 103
of Limulu8 the response of the individual receptor cell is closely similar in shape to
that of the whole eye (of. HARTLINE, WAGNER, and MACNICHOL, 1952; FUORTES,
1958). This similarity suggests that the characteristic features of the response of the
individual units are reflected in the response obtained in recording from the whole
eye. It would appear by analogy with the invertebrate eye that the response of the
individual olfactory receptor elements would have the same general appearance as
the summated olfactory response. Whether this assumption is correct or not cannot
be settled, however, until intracellular recordings have been made from single
units in the epithelium.
3. The Falling Phase

It is generally assumed that excitation of the olfactory cells occurs by absorp-
tion of molecules of the stimulating material on the receptor membrane. The
further fate of the odorous material is at present unknown. There is indirect
evidence that it is rapidly inactivated but how this occurs is not clear. It can be
presumed that the time course of the elimination of the stimulating particles is
reflected by the return of the EOG towards baseline. It is therefore of interest to
examine more closely the characteristics of the falling phase of the EOG.
The response elicited by a brief puff of odorized air is characterized by a
gradual return to the resting level. Following weak stimulation the potential
returns to zero within 1-2 sec while the fall after a strong stimulus may take
4- 6 sec or more. The fall of the potential is exponential and has a time constant
of 0.9-1.4 sec and the curves for different concentrations of a given substance are
approximately parallel. This implies that the decay is a logarithmic function of the
concentration of the stimulating substance. It seems likely that this relationship
reflects the time course of the processes underlying the elimination of the odorous
material. As will be further discussed below it is possible that inactivation is
governed by enzymatic processes within the sustentacular cells.
Until the particles of the odorous substances are wiped out of the olfactory
epithelium the receptors are subjected to a continuous stimulation. With an
increase of the stimulus intensity a greater amount of odorous material impinges
upon the mucosa and consequently the removal of the substance will take a longer
time. An increase of the stimulus strength therefore implies a stimulation not only
of greater intensity but also of increased duration. This would explain why not
only the decay time but also the peak phase of the response is prolonged when the
odour intensity of the stimulating air is increased. It is well known that the per-
sistence of an odorous sensation differs greatly from one substance to another.
Some substances produce a long-lasting sensation while others give rise to sen-
sations which rapidly fade away. In view of these differences it is of particular
interest to note that the time course of fall of the EOG is different for different
substances, as will be further discussed below.
D. The Response to Sustained Stimulation

It was shown in the early studies on the invertebrate eye (BECK, 1899; FROH-
LICH, 1914) that the response to illumination is composed of an initial phase at the
onset of light and a later steady potential of lower amplitude during steady
illumination. Later studies have demonstrated that the response of single photo-
receptors and mechanoreceptors exhibits similar characteristics. These end
organs are able to maintain a constant discharge during sustained stimulation and
are therefore classified as slowly adapting receptors. Other end organs like the
rapidly adapting crustacean stretch receptor and the Pacinian corpuscle, the
receptor potentials of which are characterized by a more or less rapid decay to zero
during maintained stimulation, are accordingly designated as rapidly adapting.
Olfactory receptors have generally been classified as rapidly adapting receptors.
This idea seems to derive from the common experience that the sensation of an
odour often weakens rapidly and may entirely disappear. This disappearance of
the sensation of smell is generally attributed to a presumed inability of the receptors
to respond to prolonged stimulation. This view is contradicted by the observation
by ADRIAN (1950) that the mitral cells in the olfactory bulb of the rabbit discharge
at each inspiration without any appreciable decline for more than one hour.
Recordings of the response of the sensory mucosa to continuous stimulation provide
direct information about the adaptive properties of the olfactory receptors. The
response of the frog olfactory membrane to a constant flow of odorized air is a
potential which declines from its initial peak to a steady level during maintained
stimulation. It would thus appear that the olfactory receptors adapt relatively
slowly to the stimulus.
This view gains further support from observations on the effect of repeated
stimulations. When the olfactory membrane is stimulated with brief puffs of
odorized air at short time intervals (10 sec) the response becomes successively
lower for the first 4-5 stimulations but then remains constant in amplitude for the
ensuing stimulations. This is a direct corroboration at the receptor level of ADRIAN'S
observation that regular discharges were obtained from bulbar neurons over pro-
longed periods. This implies that the olfactory organ when subjected to regularly
repeated stimulations is able to maintain its activity over long periods and supply
the olfactory bulb with information of the chemical composition of the inhaled air.
Olfactory adaptation may be largely due to the intrinsic activity of the bulb and
the effect of centrifugal influences, so that transmission of the incoming signals to
higher olfactory centers is suppressed.
E. Recovery
In most sense organs the termination of stimulation implies that the stimulus
is withdrawn. In the olfactory system the situation is quite different as termination
of stimulation only signifies that no more material is deposited onto the mucosa.
As long as the stimulating molecules remain in the mucus the receptors are subjected
to the action of the stimulus. If the odorous material was not eliminated it would
thus produce persistent excitation and fatigue. For the function of the olfactory
system it is therefore of importance that the stimulating material is rapidly
removed. At present there is little information as to how this occurs. Part of the
material may be carried away by the air current passing over the mucosa. It would
appear likely that only a relative small amount of the material is removed in this
way and that the major portion of the excitatory substance is taken care of within
the mucosa. It is possible that the sustentacular cells play an active role in this
Recovery 105
process. Each sustentacular cell carries a large number of microvilli (Fig. 5) which
protrude into the mucus between the cilia of the receptor cell. There is evidence
that the sustentacular cells have a high metabolic activity. When the receptors
degenerate after ablation of the olfactory bulb the debris is rapidly cleared away by
the sustentacular cells. It is conceivable that these cells also take part in the
removal of the odorous material. It is also interesting to note that there is a wide
Fig. 5. Electron micrograph of a section cut parallel to surface of olfactory epithelium, showing
olfactory vesicles of four receptors cells containing basal bodies from which cilia arise. Sur-
rounding the vesicles are microvilli from supporting cells cut in cross-section. Magnification
X 25,000. (From REESE, 1965)
variety of enzymes in high concentrations in the olfactory mucosa (for ref. see
OTTOSON, 1963). The functions of these enzymes are at present unknown. It is
tempting to assume that at least part of their functions is to eliminate the odorous
material. These considerations lead to the conclusion that the elimination of the
stimulus is an active process, the time course of which is partly reflected by the
falling phase of the EOG. It might also be expected that the time course of removal
would be related to the physicochemical properties of the stimulating substance.
The return of the potential to the baseline does not signal that the receptors
have regained their resting excitability. This can easily be demonstrated by apply-
ing a second stimulus in the aftermath of a preceding one. Following a single brief
stimulus the excitability of the olfactory membrane is initially reduced to about half
of its original value in terms of the amplitude of the response to the test stimulus
(Fig. 6). Recovery takes place gradually but still after one minute the responsiveness
of the receptors is not completely restituted. The reduced sensitivity of the receptors
in the aftermath of a preceding stimulation cannot be attributed to the effect of
remaining stimulating particles in the mucus since the potential has returned to
baseline. It is most likely that the excitatory process involves an ionic flow through
the membrane of the receptors (C. TAKAGI, WYSE, KITAMURA, and ITO, 1968) and
that the reduced sensitivity reflects metabolic processes associated with the
restitution of the internal ionic concentrations.
Fig. 6. Recovery. Records of responses to two successive stimulations of equal intensity, the
second being applied at increasing intervals after the first. A-C: records of responses to first
and second stimulation. Stimulus interval: A, 2 sec; B, 4 sec; C, 6 sec. The records D-G show
the gradual increment of the response to the second stimulation at further increase of stimulus
interval. D, 10 sec; E, 16 sec; F, 38 sec; G, 58 sec. (The response to the first stimulation in each
case being identical with the first potential seen in C). Stimulus: 0.01 M butanol. Volume of
stimulating air: 0.5 cc. Vertical line in G: 1 mV. Time bar: 2 sec. (From OTTOSON, 1956)
F. EOG in Different Species

The histological structure of the olfactory mucosa is essentially the same in all
vertebrates. It would appear therefore that the response of the olfactory membrane
would be alike in different species within the vertebrate phylum. Most of the
studies have been carried out on the frog because of the obvious advantages which
this preparation offers for quantitative studies. Recording of the EOG in verte-
brates can be made either directly from the exposed mucosa or by inserting an
electrode through the small holes of the lamina cribrosa (OTTOSON, 1959a). The
latter method has the advantage that recordings can be made without opening
the nasal cavity and the olfactory mucosa is left untouched. The upper record in
Fig. 7 illustrates the typical response obtained in this way in the rabbit to stimula-
tion with butanol. To bring out the adaptive properties of the response stimulation
was accomplished with a constant stream of air current lasting for about 3 sec. The
response shows that in all essentials the electrical reaction of the rabbit's olfactory
membrane behaves as the EOG in the frog.
An elegant technique for recording the EOG has recently been developed by
MAC LEOD, PERRIN, and LEVETEAU (1966). They implanted electrodes in the
olfactory mucosa of rabbits and recorded the response in the non-narcotized
animal. The preparation remained stable for weeks and allowed for tests under well-
controlled conditions. This technique has been applied in studies on the peripheral
EOG in Different Species 107
interaction of different odorous components (MAC LEOD, 1968). It has thus been
demonstrated that the interaction may be characterized either by a positive synergy
or inhibition depending on the concentration and nature of the constituents of the
odour mixture. In certain cases the ingredients do not interact. The mechanisms
governing the various modes of interaction remain to be explored. Responses with
similar properties as the frog's EOG have been recorded in fish (TUCKER and
SHIBUYA, 1965), tortoise and guinea pig (MACLEOD, 1959).
Rabbit
Fig. 7. EOG of the rabbit (upper record) and the receptor potential of the male cockroach
antenna (lower record). Response from rabbit obtained with a constant flow of odorized air
(oil of cloves). Duration of stimulation: 3 sec. (From OTTOSON, 1956). Response of cockroach
antenna evoked with isolated sex attractant of female. Stimulus duration: 2.5 sec. (From
BOECKH et al., 1963)
It is interesting to note the similarity of the generator potentials recorded in

vertebrates with those in insects (cf. SCHNEIDER, 1957; BOECKH, 1962). The
recordings in insects are made with a microelectrode inserted into the antenna at
the base of a sensory hair. The general properties of the response obtained in this
way and its close correlation with the impulse discharge suggest that the potential
represents the generator potential of the olfactory receptor cells. The lower record
in Fig. 7 show a typical example of the receptor potential obtained by BOECKH
from the antenna of a cockroach to stimulation with a current of air containing the
sex attractant substance of the female. As can be seen the response has the same
general configuration as the EOG recorded from the vertebrate olfactory organ.
Recording of the EOG in man is technically difficult since the olfactory area is
not easily reached with the exploring electrode. This may explain why no studies
on the human EOG have been reported until quite recently. The first successful
recordings were made in 1969 by OSTERHAMMEL, TERKILDSEN, and ZILSTORFF.
They were able to place an electrode in contact with the olfactory region and to
retain it there for a sufficient length of time to obtain reliable records. The response
obtained to a brief puff of coffee-saturated air is shown by the records in Fig. 8. It
is a purely monophasic response which closely resembles the EOG of the frog as
shown in Fig. 4. It may be noted that there is no off-response. The fact that only
an insignificantly small response was recorded with air alone shows that the
response is not artifactual. Direct evidence that the potential is of olfactory origin
was obtained by recording the response to different amounts of coffee-saturated
air.
Coffee
25ml
Fig. 8. The human EOG. Response of the olfactory epithelium in man to brief stimulations
with coffee-saturated air. (From OSTERHAMMEL et al., 1969)
The report of OSTERHAMMEL et al. is of considerable interest. It is the first time

recording has been made of a pure receptor response in man. It is unfortunate that
the technique for recording is too difficult to be used as a routine procedure. Such
a method would provide a valuable tool for correlative studies of the electrical
reaction of the receptors with data from corresponding psychophysical measure-
ments, as is suggested by the preliminary results obtained by FRANZEN, OSTER-
HAMMEL, TERKILDSEN, and ZILSTORFF (1970).
G. Oscillations
Rhythmic oscillatory potentials are well known phenomena within the central
nervous system and are generally regarded as due to synchronous activity of
neurons which are interconnected. However, potential oscillations may also be
obtained from synchronously acting groups without any nervous interconnections.
In 1955, it was reported by ADRIAN that potential oscillations could be obtained
from the olfactory mucosa in rabbit. In the frog oscillations sometimes appear
superimposed upon the slow potential. These oscillations, which have a frequency
of 15-25 per sec, are usually obtained with relatively strong stimuli. They are
usually seen superimposed on the peak of the EOG (Fig. 9). When the mucosa is
stimulated with a continuous stream of odorized air the oscillations sometimes
appear as waxing and waning waves superimposed upon the sustained phase of the
EOG. With repeated stimulations the waves decrease in amplitude and vanish.
The waves may arise as a result of an intermittent synchronous activity in groups
On- and Off-Responses 109
of receptors. However, the possibility also exists that they reflect synchronous
activity of groups of nerve fibres. As shown in electron microscopical studies by
GASSER (1956) the olfactory nerves are densely packed together in bundles sur-
rounded by a common sheath. This structural arrangement provides possibilities
for an interaction between neighbouring fibres within a fascicle and for synchro-
nization of the discharge.
Fig. 9. Rhythmic waves superimposed upon the slow potential. Stimulus: amyl acetate.
Volume of stimulating air: 0.25 cc. Vertical line: 5 m V. Time bar: 1 sec. (From OTTOSON, 1956)
H. On- and Off-Responses

The response obtained with olfactory stimuli from the frog's nasal mucosa,
which consists of a single layer of receptor cells without synaptic interconnections,
is generally a monophasic negative potential and there is no off-effect at cessation
of stimulation. In 1959, TAKAGI and SHIBUYA reported on and off-responses in the
olfactory epithelium of the frog. They stated: "Thus, two slow action potentials
appeared at the onset and the cessation of stimulation. These phenomena are
well known in the retina, in the optic nerve and in the auditory nerve. They are
called' on' - and' off' -responses respectively. It is interesting that similar phenomena
were found in the olfactory epithelium as well." It was found "that the shapes and
magnitudes of the potentials varied from time to time". To explain these responses
it was concluded that there are "two or three types of olfactory cells, corresponding
to the on-and off-, or on-, off-, and on-off-responses". However, because of the varia-
bility of the responses it was suggested that "the response types of the receptive
elements in the olfactory epithelium are not stable" (TAKAGI and SHIBUYA, 1960c).
Later the list of responses was extended to include also a positive potential which
was attributed to hyperpolarization of the receptor cells and it was suggested that
there were two receptive processes in the olfactory cell. "One is an ordinary excita-
tory process which produces an electronegative slow potential in response to general
odors. The other is a process of a different kind which is activated only by the vapor
of an organic solvent of high concentration ... " (HIGASHINO and TAKAGI, 1964). In
general the various responses could only be obtained with certain stimuli such as
saturated vapors of ether or chloroform. The reported potentials have later been
referred to in a series of papers by TAKAGI and his co-workers (cf. HIGASHINO and
TAKAGI, 1964; TAKAGI, WYSE, and YAJIMA, 1966; TAKAGI, WYSE, KITAMURA, and
no D. OTTOSON: The Electro-Olfactogram
ITO, 1968) as being components of the physiological response of the olfactory

membrane. Before this view can be accepted it appears necessary to examine
briefly the experimental conditions under which the reported effects were obtained
and also to consider alternative interpretations for their development.
For the assessment of the observations reported by TAKAGI and his coworkers,
it is essential to keep in mind that under physiological conditions the olfactory
receptors are usually subjected to odorous stimuli in extremely low concentrations.
In studies of the functions of the olfactory organ it is therefore generally agreed
that stimuli of low concentrations should be employed and furthermore that the
substances used should not damage the receptors or block their activity. As
mentioned above, to obtain the reported potentials TAKAGI and coworkers generally
used saturated vapors of ether or chloroform. These substances can for obvious
reasons not be regarded as suitable for olfactory studies and particularly not when
used in concentrated form. It would appear obvious that results obtained with
such stimuli would have little or no relation to the physiological reactions of the
receptors to odorous stimuli. This is also suggested by the fact that the responses
varied not only in configuration and size from time to time to the same stimulus
but also from one region to another. This is in sharp contrast to the behaviour of
the response to an odorous stimulus of physiological strength. Not only is the
response stable for hours at the same site but has the same general features at all
sites of the sensory membrane. It seems clear therefore that the observed responses
can not be taken as signs of a normally functioning olfactory membrane. It is
obvious also that the conclusion by TAKAGI and SHIBUYA (1959) that there are
"off" -or "on-off" receptors is untenable.
The structural and functional similarity between the olfactory sense organ and
the simple invertebrate eye has already been emphasized. To interpret the findings
reported by TAKAGI and co-workers it might be useful to turn back again to some
old observations on the invertebrate eye. In studies of the response of the eye of
Eledone, FROHLICH (1914) found that a polyphasic response could be obtained
when the eye was exposed to strong and/or uneven illumination. In the diagram in
Fig. 10 the records in the left column show some examples of the polyphasic
potentials of the eye; the records in the right column are recordings by TAKAGI and
SHIBUYA (1959) from the olfactory epithelium. FROHLICH undertook a careful
analysis of the development of the polyphasic responses of the eye and showed
that they arose as a result of an interaction between the activity in different
regions of the retina. It would appear likely that the same explanation would
apply to the polyphasic potentials obtained from the olfactory sheet. The obser-
vation by TAKAGI and co-workers that the responses varied greatly from one region
of the mucosa to another provides direct evidence in favor of this interpretation.
TAKAGI and his co-workers suggested that the regional differences arose because the
relative number of the on-and off-elements, or the on-, off- and on-off-elements
differed in different parts of the epithelium (TAKAGI, SHIBUYA, HIGASHINO, and
ARAI, 1960). This explanation which in itself seems highly questionable is con-
tradicted by their own finding that the response to a given substance varies with
time at one and the same region. A more likely explanation would be that the
polyphasic responses appeared as a result of regional blocking of the receptor
activity in the olfactory mucosa. It is also likely that in the course of an experiment
Origin of EOG III
with repeated stimulations differences in time course of recovery from one region
to another may have contributed to the production of polyphasic responses.
The suggestion that the complicated potential patterns obtained by TAKAGI
and co-workers are due to an uneven field of excitation in the olfactory membrane
does not exclude other possibilities. Thus it is conceivable that variations in the
steady potential existing across the mucosal membrane may be responsible for
some of the observed effects particularly when saturated vapors were used for
( a l~ ___, _
(bl - ______
(cl..-/'~' ,~
I
(dl _ _ _J\~
( e ) _________. - 1 -
Fig. 10. Comparison of polyphasic responses. Left column: from the eye of Eledone (from
FROHLICH, 1914); right column: from the frog's olfactory organ. (From TAKAGI and SHIBUYA,
1959)
stimulation. Eddy currents of the stimulating air may also have contributed. It
would appear premature therefore to presume the existence of specific "on" and
"off" receptors on basis of results obtained only under strongly unphysiological
conditions.
J. Origin of EOG
In order that an electrical response recorded from a receptor organ should be
designated as the generator potential of the sensory cells it must fulfil certain
criteria. It should be produced by the receptor ; it should be of graded nature and
remain after the conducted activity is blocked; it should exhibit a characteristic
and regular relationship to the parameters of the adequate stimulus; there should
be a similar close relation between the potential recorded from the receptor and the
discharge of the afferent nerve.
In the frog the sensory region can easily be recognized because of its yellow
colour. As described above the EOG is only obtained from these regions while
adjacent regions with respiratory epithelium produce no response. This obser-
vation strongly suggests that the response is of olfactory nature. Definite evidence
showing that the EOG derives from the receptor cells has recently been obtained
by TAKAGI and YAJIMA (1964, 1965). They sectioned the olfactory nerves in bull-
frogs and recorded the EOG at various stages of retrograde degeneration of the
olfactory epithelium. It was found that the EOG decreased in parallel with the
reduction in number of the sensory cells and disappeared 8-16 days after nerve
section.
There has been a great"deal of discussion in the literature on olfaction as to the

actual site of receptor activation. Since the first contact between the odorous par-
ticles and the sensory cells undoubtly takes place at the surface of the olfactory hairs
it would be natural to assume that these structures represent the receptor elements.
As mentioned above it is also possible that the olfactory vesicle may carry the
odour sensitive sites. The different possibilities underlying the receptor activation
have been considered in detail by OTTOSON and SHEPHERD (1967). On the basis of
observations by REESE (1965) of the dimensions and ultrastructure of the olfactory
Fig. 11. The EOG recorded at different depths of the frog's olfactory mucosa. (From OTTOSON,
1956)
hairs it was calculated that the length constant of a single cilium with a diameter
of 0.1 [L would be about 100 [L. If excitation is assumed to take place along the total
length of the cilium exposed to the surface of the mucus the activity of the cilia
would be transmitted with relatively little attenuation. Since it can be assumed
that the molecules also diffuse into the mucus more proximal sites of the cilia would
be excited and activation would be correspondingly more efficient. If the potential
is produced in the vesicle the electrical situation for spread of excitation would be
equally or even more effective. However, if activation takes place at the olfactory
vesicle only those particles which have escaped from being absorbed on the mem-
branes of the cilia would excite. As discussed above such a process would be bound
to take place with a considerable delay. It would also hardly be compatible with
the extreme sensitivity of the receptors if the greater portion of the stimulating
agent was eliminated before reaching the receptor site. In recording with a micro-
electrode at different depths (Fig. 11) it has been found that the response became
successively smaller as the electrode was advanced into the epithelium (OTTOSON,
1956). This suggests that the potential originates in structures located close to the
surface of the epithelium. Byzov and FLEROVA (1964) confirmed this finding but
Origin of EOG 113
found that in some cases the potential had its greatest amplitude at a depth of
75 fL and therefore concluded that the potential was produced by the cell body.
Experiments by SHIBUYA (1964) strongly suggest that the EOG is produced by the
cilia. He applied a piece of absorbant paper on the mucosa and found that the
EOG disappeared after this treatment. SHIBUYA concluded, for reasons which are
not clear, that the response disappeared because of removal of the mucus. It is
difficult to understand how this conclusion was arrived at since half of the mucus
Fig. 12. Electron micrograph showing dense mat of cilia in mucus layer of frog olfactory
epithelium. Practically all cilia are cut in cross-section; small distal ciliary segments in outer
layer and larger proximal segments in inner layer; olfactory vesicles and microvilli in bottom
layer. Magnification: x 3.000. (From REESE, 1965)
layer remained after SHIBUYA'S treatment. Even more unintelligible is the as-
sumption that the cilia were not damaged. It has been established in numerous
studies that olfactory cilia extend to the outer surface of the mucus layer. Removal
of half of this layer implies by necessity that the greater portion if not all of the
cilia were broken off. A brief glance at Fig. 12 showing the mucus layer with
embedded cilia is sufficient to show this. The obvious conclusion to be made from
SHIBUYA'S experiments is that the EOG disappeared because of the destruction of
the cilia (cf. OTTOSON, 1970). Actually SHIBUYA'S finding provides an elegant
demonstration that the olfactory cilia represent the chemosensitive elements of
the receptor cell.
It is today generally agreed that receptor potentials are more resistant to the
action of local anaesthetic than impulse activity. In the early experiments on the
S JIb. Sc-nsory Physiology. Vol. IV/I
rabbit is was found that cocaine did not block the response (OTTOSON, 1954). This
suggested that the potential did not represent summated nerve activity. However,
the evidence obtained in the rabbit was not conclusive since uniform treatment of
the entire olfactory area could not be achieved. In the frog, however, this could
easily be done. It was found that the potential remained almost unchanged after
treatment of the mucosa with cocaine in concentrations which blocked the impulse
activity of the olfactory nerve fibres (KIMURA, 1961). Additional evidence showing
that the olfactory nerve fibres do not participate in the development of the EOG
has been obtained in experiments with antidromic stimulation of the olfactory
nerves (OTTOSON, 1959b). In the frog the olfactory fibres are aggregated into one
single nerve which is easily accessible within the cranial cavity. By stimulation of
this nerve all the olfactory fibres can be antidromically activated. If the fibres con-
tribute to the response obtained from the mucosa with odorous stimulation one
would expect this latter potential to be reduced or eliminated during antidromic
activation of the olfactory nerve. No such effect has been found. This clearly
shows that no significant fraction of the response originates from the conducted
activity in the afferent nerve fibres. All the experimental evidence thus suggests
that by the criteria generally used for the identification of receptor potentials the
response obtained from the olfactory mucosa is the receptor potential of the
olfactory organ.
Before it can be definitely concluded that the EOG represents the generator
potential of the olfactory organ it has to be proved, however, that the activity in
the olfactory nerve and higher centers is related quantitatively and in time course
to the electrical changes in the olfactory membrane. Evidence for the relation
between the EOG and the discharge of the olfactory nerves is difficult to obtain
because of the small dimensions of the nerve fibres. As will be described in more
detail below it is possible to record the impulse traffic in thin strands of the
olfactory nerve. In this way it has been demonstrated by KIMURA (1961) that the
impulse activity closely follows the EOG. There is also a close relation between the
EOG and the activity in neurons of secondary order (DaVING, 1964).
The conclusion that the EOG represents the generator potential has been
questioned, mainly on basis of the above mentioned report by SHIBUYA. The
arguments raised have been met earlier in two articles (OTTOSON, 1963; OTTOSON
and SHEPHERD, 1967). In spite of this SHIBUYA'S experiments have recently been
referred to as evidence against the conclusion that the EOG is the generator
potential of the olfactory organ (MOULTON and BEIDLER, 1967; TAKAGI, 1967). It
appears necessary therefore to direct attention once more to some of the most
obvious deficiencies of SHIBUYA'S experiments and conclusions.
SHIBUYA recorded the impulse activity of a small strand of the olfactory nerve
of the tortoise. The area of the olfactory membrane innervated by the fibres was
mapped by antidromic stimulation of the nerve strand. As described above a small
piece of absorbant paper was then placed onto this area. After removal of the
paper 8HIBUYA found that the EOG in the treated area had disappeared whereas
impulses could still be recorded from the strand. He therefore concluded that the
EOa did not rcprcBcnt thc gcncrator potcntial. Hc diBcardcd thc fact that thiB con-
clusion was disproved by KIMURA'S demonstration (1961) that the nerve response
decreases and disappears in parallel with the EOG when the receptor activity is
Origin of EOG 115
blocked with volatile narcotics such as ether or chloroform. He also ignored all
evidence by other authors (cf. OTTOSON, 1956-1970; KIMURA, 1961; DOVING,
1964, 1965, 1966a, b; MAC LEOD et al., 1966; DRAKE, JOHANSSON and VON SYDOW,
1969) showing that the EOG is of olfactory nature and has all the properties of a
receptor potenti<l,l as known from studies on other sense organs (cf. EYZAGUIRRE
and KUFFLER, 1955; BOECKH et al., 1963; LOEWENSTEIN and MENDELSON, 1965;
OTTOSON and SHEPHERD, 1965). An obvious explanation to the reported findings
would be that the impulses recorded derived from fibres coming from regions
outside the treated area. This could easily have been confirmed by subjecting the
entire sensory epithelium to the treatment. SHIBUY A ignored to do this obvious
control experiment and claimed that the disappearance was due to removal of the
mucus. However, he left unexplained that the EOG disappeared though only half
of the mucus layer was removed. If the EOG was produced by the mucus it should
have remained. Furthermore he claimed that the cilia were almost undamaged by
the treatment although observations on the living epithelium (cf. REESE, 1965)
clearly show that the cilia extend to the surface of the mucus and that removal of
half of this layer consequently is bound to cause severe damage to the cilia. The
most likely explanation to the disappearance of the EOG in the treated area would
therefore be that the transducer elements were removed. SHIBUYA also disregarded
the fact that the EOG is only obtained in the sensory region, while there is mucus
over the respiratory as well as over the olfactory region of the nasal mucosa. He
overlooked the fact that the EOG is closely related in magnitude and time course
to the strength and physico-chemical properties of the stimulating odour and that
the response exhibits adaptation, fatigue and other features which would not be
the case, if the potential was a purely physical phenomenon.
TAKAGI (1967) has questioned that the EOG is the generator potential on basis
of the finding that the bulbar waves did not always conform to the positive poten-
tial or the off-effects produced in the receptor layer with ether or chloroform. It is
difficult to understand the logic of this reasoning since it has been demonstrated by
TAKAGI and co-workers (cf. TAKAGI and SHIBUYA, 1960b, c) that the potentials
evoked with ether and similar substances differ greatly from one region of the
sensory epithelium to another. The congruity or absence of congruity between the
peripheral response and the bulbar waves therefore of necessity would depend on
from which part of the mucosa or the bulb the recording is made. To explain
the EOG, TAKAGI (1967) has developed a hypothesis according to which the EOG
is produced by the supporting cells in the mucosa. This hypothesis would appear
to be refuted by TAKAGI'S and his co-workers own finding that the EOG disappears
after degeneration of the receptors while the supporting cells remain intact.
As has been emphasized above the EOG has all the characteristic properties of
a receptor potential. It is a graded non-conducted response the time course and
amplitude of which is closely related to the parameters of the olfactory stimuli. The
fact that the response is restricted to the olfactory region of the mucosa provides
direct evidence of its olfactory nature as does also the finding that the response
disappears when the olfactory receptors degenerate. The finding that the response
is abolished when the cilia are removed strongly suggests that the olfactory trans-
ducer action takes place in the cilia. It would thus appear that the solution of
many of the challenging problems related to receptor specificity and odour
s
ll6 D. OTTOSON: The Electro-Olfactogram
discrimination have to be sought for in the functional properties of the ciliary

membrane.
III. Relation to Stimulus Characteristics

A. Relation to the Dynamic Characteristics
of the Stimulating Air Current
It is a basic requirement for the analysis of receptor functions that the para-
meters of the stimulus are well defined. It would appear that such a requirement
would also be necessary for the quantitative analysis of olfactory receptor function.
The olfactory stimulus is carried by an air current to the olfactory membrane. It
is obvious therefore that knowledge not only of the stimulus concentration but
also of the distribution of the material and the dynamic characteristics of the air
current is essential for the evaluation of the response. This fact has generally been
overlooked in olfactory research.
Studies on the relation between the "waveform" of the stimulating air current
and the response of the mucosa have provided evidence that the potential changes
closely follow the waveform of the stimulus. The obvious conclusion to be drawn is
that the electrical response of the olfactory membrane faithfully reproduces the
dynamic characteristics of the stimulus (OTTOSON, 1956).
B. Responses to Different Types of Odorous Substances

In recording action potentials from units of the olfactory bulb, ADRIAN (1953)
observed that the responses evoked by different substances exhibited different
Fig. 13. Responses to different types of odorous stimuli. A, to amyl acetate. B, to butanol
(0.C5 M). C, to oil of cloves. Volume of stimulating air: 1 cc. Vertical line in C: 1 mV. Time
bar: 1 sec. (From OTTOSON, 1956)
Relation to the Properties of the Stimulating Substances 117
temporal characteristics. Thus he found that esthers as amyl acetate produced a

discharge characterized by a sudden onset and a rapid decrease while substances
with an "oily" smell gave responses which increased relatively slowly and had a
relatively long duration. Similar differences have later been reported by other
authors (C. MOZELL and PFAFFMANN, 1954).
The differences in time course of the excitatory processes evoked by different
stimuli are directly reflected in the shape of the potential recorded from the
mucosa. The records in Fig. 13 show the potentials set up by stimulation with
amyl acetate (A), butanol (B) and oil of cloves (0) (OTTOSON, 1956). Since the time
course of the response varies with the strength of the stimulus the concentrations
were adjusted so that responses of identical magnitudes were evoked. It is seen
that the rising phase of the responses to these three substances do not differ
significantly from each other, whereas there are marked differences between the
falling phases. No systematic study has so far been carried out on relating the
temporal characteristics of the EOG to the physico-chemical properties of different
stimuli. It would appear that a study on a great number of substances should
provide insight into the relationship between their physico-chemical properties and
their action on the olfactory receptors.
C. Relation to the Properties of the Stimulating Substances

In studies of the function of sense organs, recording of the impulse discharge in
single primary afferent fibres has generally been a useful method to obtain infor-
mation about the response of the end organs to various stimuli. The small diameters
of the olfactory nerve fibres have made this approach extremely difficult in olfac-
tory research. Until recently most of the information available about the relation
between the properties of odorous components and their stimulating action on the
olfactory membrane therefore derived from subjective measurements. The demon-
stration of the receptor potential of the olfactory organ has, however, provided a
tool for quantitative measurements of the effects evoked by odorous substances of
the end organs.
Homologous substances have generally been used in studies on the relation
between the physico-chemical properties and the olfactory efficiencies of odorous
compounds. The reason for this is that there is a gradual change in physical proper-
ties as a homologous series is ascended while the chemical properties remain
unaltered. A number of such series has been studied by using the EOG as an index
of the stimulating potency. In this way it has been demonstrated that there is a
regular relation between the odorant potency and chain length of a number of
homologous compounds including primary aliphatic alcohols, aldehydes and
ketones (OTTOSON, 1958). A similar relationship has been found in studies on
rejection thresholds in insects (DETHIER and YOST, 1952) and also applies to
olfactory thresholds in dogs (for ref. see OTTOSON, 1963).
Studies of the EOG have shown that there is an inverse relation between water
solubility and stimulating potency of the first members in the serie3 of primary
aliphatic alcohols. A similar relation was observed by BACKMAN (1917) in human
studies and has later also been demonstrated in insects. These observations strongly
support the view that the action of odorous compounds on the olfactory membrane
lI8 D. OTTOSON: The Electro-Olfactogram
is related to their partition coefficients. With increasing chain length the vapor
pressure also changes. Another way of analyzing the effect of vapor pressure is to
determine the partial pressures of substances of equal stimulating efficiency. When
this is done for primary aliphatic alcohols it appears that there is a linear relation
between the partial pressures and the saturated pressures when the values are
plotted on double logarithmic scales (Fig. 14). This relation is of particular interest
since the relative saturation of the vapor of a substance in the vapor phase repre-
sents its thermodynamic activity. It has been suggested that the thermodynamic
activity should be used as a measure of the potencies of certain biologically active
2 -CHpH
-SHsOH
-CfipH
D...'" -C4HgDl:i
01
a
..J -c;;HIlOH
0 -C6H1PH
-C,Hl)H
-I -~HlpH
Fig. 14. Relation of partial vapour pressure (P,) to saturated vapour pressure (P.) of primary
aliphatic alcohols of equal stimulating effectiveness. (From OTTOSON, 1958)
compounds. Studies in insects have shown that for a limited number of the mem-
bers of homologous substances the olfactory stimulating efficiency is a function of
the thermodynamic activity. Similar observations have also been made in the frog
by using the EOG as a measure of the activity elicited in the receptors. A com-
parison of the responses produced by primary aliphatic alcohols shows that
0 4 - Os have approximately the same stimulatory efficiency while the first three
members in the series are less effective. This difference is of interest since the lower
members in the series are infinitely soluble in water and either insoluble or only
slightly soluble in oil while the opposite is true for the higher members. This sug-
gests that for substances of similar partition coefficients the stimulating potency
may be a function of the thermodynamic activity. It is obvious, however, that the
effect of an odorous substance on the olfactory receptors cannot be attributed to
one single factor of the stimulus. The close relation between the stimulatory
efficiency and the thermodynamic activity on the one hand and the solubility
properties on the other suggests that these two parameters represent properties of
primary importance with respect to the ability of a given substance to excite the
receptors. It is likely that there are other contributing factors such as the effect of
certain functional groups and the steric configuration of the molecules (cf. BEETS,
1964).
Quantitative Analysis of Odor Mixtures 119
It would be reasonable to assume that if data were available on the effect of a

great number of substances it would be possible to define the relative influence of
the various molecular parameters of the stimulus. This would then also give
insight into the basic mechanisms of the excitatory processes. Olfactory research
has mainly been focused on problems related to the discrimination of different
odorous substances and comparatively little attention has been paid to the
mechanisms of excitation. If the basic mechanisms underlying the excitatory action
of odorous compounds on the olfactory receptors were known, there would be a
greater chance to find the solution of the problems of odour discrimination.
D. Quantitative Analysis of Odor Mixtures

In daily life the stimulus to which the olfactory receptors are exposed is a mixture
of odorous compounds. In general the various components are present in very low
concentrations and their contribution to the odour complex is not easily deter-
mined. In studies on the odour composition of aroma complexes of food items the
ingredients are usually separated by gas chromatography and their odours identified
by subjective methods. In this way it is possible to determine the qualitative
properties of the components of a complex odour. The quantitative contributions
of the different ingredients to the intensity of the odour complex can be determined
by using the EOG as a measure of the olfactory potencies of the individual com-
pounds as they pass out of the gas chromatograph (OTTOSON and VON SYDOW,
1r--21
L---21 I~I li""d I~I l
I~I
~ ---L- ,
xl
9J 65 00 65 60 55 :IJ 25 20 15 10 5
M ,nul~5
Fig. 15. Gas-chromatogram of a concentrate of the aroma of black currants. Inserted: EOG
elicited by the eluated components. (From OTTOSON and VON SYDOW, 1964)
1964). Figure 15 shows the results of such an analysis of the compounds of a

concentrate of black currants. The electrical responses evoked by the different
components are shown in the inset. The heights of the electrical responses give an
indication of the relative intensities of the ingredients. Their absolute contribution
to the total odour intensity of the composite concentrate can be calculated from
the concentration values as given by the gas chromatograph. It is thus possible by
combining gas chromatographic separation methods with recording of the EOG to
obtain quantitative data about the odour potencies of the components of a complex
mixture of odour-components.
IV. EOG and Olfactory Input to the Bulb

A. EOG and Impulse Activity of the Olfactory Nerve Fibres
It has been demonstrated in studies of single end organs such as the muscle
spindle (KATZ, 1950; SHEPHERD and OTTOSON, 1965) and the crustacean stretch
receptor (EYZAGUIRRE and KUFFLER, 1955) that the generator potential determines
the impulse frequency of the afferent nerve. A corresponding analysis of the activity
of the olfactory receptors would require recordings from single units. The only
possibility for doing this appears to be by intracellular recordings from the cell
body of the olfactory receptor cell. If such recordings could be made it would be
possible to study more closely the relation between the transducer action and the
afferent impulse message. Although this has not yet been possible we still have
some information about the general relationship between the activity of the
receptors and the discharge of the olfactory nerve. It has been shown by KIMURA
(1961) in recordings from the frog that the activity in a strand of the olfactory
nerve closely follows the EOG. With increasing strength of the stimulus the nerve
activity increases in parallel with the changes in amplitude of the EOG. When the
nasal mucosa is stimulated repetitively with brief puffs of odorized air the impulse
activity and the EOG suffer the same amount of reduction. As described earlier the
EOG evoked by a continuous flow of odorized air consists of an initial dynamic
phase followed by a static phase. The activity of the nerve exhibits closely similar
characteristics. Following the onset of stimulation there is a vigorous discharge
which coincides with the dynamic phase of the EOG. As the EOG declines to its
static phase the impulse activity also decreases and is then maintained at a constant
level during the ensuing static phase of the EOG. With termination of stimulation
both activities decline in parallel. The close correlation between the activity of the
nerve and the EOG as demonstrated by KIMURA provides strong evidence that the
information carried by the impulse message closely reproduces the excitatory
process in the peripheral receptor sheet.
There is little doubt that we would know considerably more about the receptor
mechanisms if the diameters of primary olfactory fibres were not so small. The fact
that there are about 10 fibres per fL2 in a cross-section of the olfactory nerve would
appear to preclude the possibility of recording the activity of single units. It was
therefore a considerable advance in olfactory research when GESTELAND (GESTE-
LAND, 1961; GESTELAND, LETTVIN, PITTS, and ROJAS, 1963; GESTELAND, LETTVIN,
and PITTS, 1965) succeeded in recording the activity of single units within the
olfactory mucosa. In their study of the responses obtained with different substances,
GESTELAND and co-workers also recorded the EOG. An illustrative example of their
results is shown in Fig. 16. The olfactory membrane was stimulated with musk
and the EOG was recorded together with the impulse activity of a single unit. As
can be seen the impulse discharge appears shortly after the onset of EOG; it
increases rapidly in frequency with increasing amplitude of the EOG and then
declines gradually with the fall of the EOG. In other units the impulse discharge
exhibited a different pattern in relation to the time course of the EOG. This is
explained by the fact that the EOG represents the total activity of all the receptors
thrown into activity. Depending on differences in sensitivity and in time at which
EOG and Olfactory Bulb Activity 121
the receptors are reached by the stimulating molecules different units will be
activated at different times. It would therefore be premature to assume that the
response of a single sensory element should always follow the total response as
represented by the EOG.
Fig. 16. EOG and single unit activity of frog olfactory mucosa. Response to stimulation with
musk. (From GESTELAND et al., 1963)
B. EOG and Olfactory Bulb Activity

1. Slow Bulb Potential and Induced Waves
In most vertebrates the analysis of the relation between the activity of the
receptors and that of the secondary neurons in the olfactory bulb is hampered by
the difficulties encountered in recording the peripheral and the central responses
Fig. 17. EOG (lower trace) and slow bulbar potential (upper trace). Recordings ofresponses to
stimulation with 0.001 M (A), 0.01 M (B) and 0.1 M (e) butanol. Vertical bars: 1 mY. Upper
bar relates to the bulb response; lower bar to EOG. Time bar: 1 sec. (From OTTOSON, 1959b)
simultaneously. In the frog no such difficulties are met and the responses at differ-
ent levels of the olfactory system can easily be recorded simultaneously. As
illustrated in Fig. 17 stimulation of the olfactory mucosa with a brief puff of
odorized air gives rise to a slow potential change in the olfactory bulb. Super-
imposed upon this potential there are regular oscillations of 8-12 per sec. As can
be seen there is a close correlation between the bulbar response and the EOG in
terms of changes in height and time course with increasing stimulus strength. The
experimental analysis of the properties of the slow potential indicates that it is
produced in the dendritic network within the glomeruli while the induced waves
appear to arise as a result of synchronous activity of the secondary neurons. Thus
antidromic activation of the mitral neurons leaves the slow potential unaffected
whereas the waves are abolished. The oscillatory potentials superimposed upon the
slow potential are not governed by the rhythmic activity of the receptors as shown
by the fact that the bulbar waves are always present while the receptor potential
only rarely exhibits rhythmic waves. When the rhythmic waves of the EOG
appear they usually differ in their general time relation and frequencies from the
bulbar waves. This suggests that the rhythmic activity of the secondary bulbar
neurons is the result of the integrative activity of the bulb.
Recent experiments by LEVETEAU and MAC LEOD (1966a, b) have provided
important contribution to the understanding of the relation between the peripheral
and bulbar potentials. They recorded the EOG from the unexposed olfactory
epithelium together with the potentials developed by single glomeruli. The
glomerular response is a slow monophasic or diphasic potential which appears with
a latency of about 40 msec after the onset of the EOG. Of particular interest is the
finding that individual glomeruli exhibit specific sensitivities so that one glomerulus
may respond to one type of stimulus but not to another. It has been suggested
(cf. LE GROS CLARK, 1957) that the fibres from receptors with similar sensitivity
properties are directed to particular glomeruli. The results obtained by LEVETEAU
and MAC LEOD (1966a, b) are the first experimental evidence for this hypothesis.
The individual olfactory receptors and nerve fibres are extremely difficult to
record from, while the glomeruli are more easily accessible. If the afferent fibres
from receptors of similar sensitivity properties are directed towards particular
glomeruli recording of the response of individual glomeruli would provide the same
information as recordings from single receptors. Recordings from the glomeruli
with the technique developed by LEVETEAU and MAC LEOD would have the ad-
vantage that the olfactory membrane is left intact. The observations made by
LEVETEAU and MAC LEOD therefore offer a new and interesting possibility for a
systematic analysis of the mechanisms underlying olfactory discrimination.
2. Unitary Activity
ADRIAN (1953) was the first to study the discharge of single units within the
olfactory bulb. In recordings with an electrode pushed into the bulb of the rabbit
he observed that there was a burst of impulses at each inspiration of room air.
When odorous substances were added to the inspired air the discharge became
more intense. Subsequent studies have demonstrated that a great portion of the
bulbar neurons are inhibited during olfactory stimulation. The records in Fig. 18
show the EOG and the responses obtained from two bulbar units when the
olfactory mucosa was stimulated with butanol. The unit in A responded with a
burst of impulses while the activity of the unit B was completely inhibited. Most of
the units which are activated by a given stimulus respond with increasing frequency
as the strength of the stimulus is increased. Simultaneous recordings of the EOG
and the responses of such units show that the discharge frequency increases in
EOG and Olfactory Bulb Activity 123
direct relation to the increase in amplitude of the EOG (DOVING, 1964). In other
units the response pattern varies in a less regular way with the strength of the
stimulus.
The analytical work of SHEPHERD (1963a, b) and RALL and SHEPHERD (1968)
has provided an important contribution to the understanding of the functions of
the bulbar neurons. These studies have emphasized the important role of inhi-
bitory processes in the transmission of olfactory signals. It is interesting to note
that there appears to be a kind of lateral inhibition between mitral cells analogous
Fig. 18. Excitatory (A) and inhibitory (B) effect on bulbar units induced by olfactory stimula-
tion. Lower trace shows EOG. Recordings from frog's olfactory bulb and mucosa. (From
D6vING, 1966a)
to that in the Limulu8 eye. It is clear from the work of RALL and SHEPHERD that
the effect of the afferent input to a given unit depends on a complex interaction
between excitatory and inhibitory influences.
The interaction between bulbar neurons may explain their characteristic
behaviour during stimulation, as shown by DOVING'S work (1964). For instance
stimulation with a given substance at weak strength may excite an unit while
strong stimulation with the same substance produces inhibition. Units which are
inhibited during stimulation usually discharge when stimulation is terminated. In
studies of the response characteristics of bulbar units these off-responses are
difficult to distinguish from the excitatory on-responses if the exact time course of
the stimulating action of the odorous substance on the olfactory membrane is not
known. By recording the EOG simultaneously with the discharge of the bulbar
unit the type of response can easily be identified and related to the excitability
process in the receptors.
The observations by DOVING (1964, 1965) clearly show that the pattern of the
response of a bulbar unit to a given substance may vary with the concentration of
the stimulus. In order to compare the effects of different substances on a population
of bulbar units it is often essential that the stimulating efficiency is monitored so
that it is the same for all substances to be tested. As shown by DOVING (1966a, b)
the EOG is a useful tool for the control of the strength of the stimulus. The results
obtained by DOVING from studies of substances of homologous series demonstrate
that neighbouring substances in a series have more similar olfactory stimulative
properties than substances which are widely separated in the chain. It is interesting
to note also the close correlation between the results obtained by DOVING on frogs
and those by ENGEN (1962) in psychophysical studies of similarity estimates in
human. It would appear from these results that the basic mechanisms of olfactory
discrimination in humans and in the frog are not very different.
v. EOG and Psychophysical Measurements

Comparative Studies of the Frog's EOG and Subjective Odor Intensity in Human
A primary concern in sensory physiology is to establish the correlation between
neural activity and sensation. As early as 1927, ADRIAN and MATTHEWS pointed
out the close similarity between the discharge in the eel's optic nerve and the
perceptual intensities of illumination. Later studies have provided evidence of a
similarly close correlation in other sensory systems. Thus it has been demonstrated
by BORG, DIAMANT, STROM, and ZOTTERMAN (1967) that perceptual taste inten-
sities are faithfully reproduced in the impulse activity of the chorda tympani nerve.
FROG
Slope =0.38
2 3
Log!
Fig. 19. Frog EOG and subjective odor intensity in human in relation to concentration of the
stimulus. Left diagram: magnitude estimation of n-butanol at different concentrations. (From
JONES, 1958). Right diagram: magnitude of frog's EOG evoked with different concentrations of
n-butanol. (From OTTOSON, 1956)
As already mentioned the amplitude of the electrical response of the frog's

olfactory organ is a power function of the strength of the stimulus (Fig. 19, right
diagram). The exponent of this function is 0.38 when butanol is used as test
substance. It is of particular interest to note that the psychophysical response to
the same substance is also a power function (Fig. 19, left diagram), the exponent
of which is 0.40 (JONES, 1958). This suggests a close agreement between the electri-
cal events in the frog's olfactory organ and those underlying the sensory evaluation
of odour intensities in man. This view gains further support from recent experi-
ments by VON SYDOW and co-workers (VON SYDOW, 1968; DRAKE, JOHANSSON,
VON SYDOW, and DOVING, 1969). In one series of measurements they compared
electrophysiological and psychological data for different concentrations of methyl
benzoate. The EOG obtained from the frog's olfactory mucosa was used as an
index of the peripheral neural activity. Values for subjective odour intensities were
obtained in psychophysical tests by using a magnitude estimation scale. The results
are illustrated by the diagram in Fig. 20. As can be seen the psychophysical data
fit a power function with an exponent of 0.28. The electrophysiological data also
fit a power function with an exponent of 0.32. The correlation between these two
functions is represented by a straight line with a slope of 1.16, the ideal being
1.00. In another series of ll1easurell1cnts DRAKE et al. (1969) compared the data
obtained from measurements of four substances by using a given solution of
benzaldehyde as standard solution. The pooled data from these series provide
EOG and Psychophysical Measurements 125
further evidence of the correlation between the EOG in frog and the perceived odor
intensity in man. These findings suggest that results obtained in the frog may be
directly applicable to humans with respect to intensity functions. This would also
imply that the EOG may be applied as a biological test for quantitative determina-
tion of odor intensities.
In recent years interest has been focused on the relation between subjective
sensations and the underlying neural activity at different levels of afferent path-
ways. At present time there is evidence available from two levels of two different
A,-________________~ Br-----------------~
2 log log
psych physiol
Slope-0.28
2S~~
~ log head space conc
,....,
log head space conc
2 3 2 3
C
log
physiol
2
Siope- L16
r - 0.98
log psych
o
Fig. 20. Psychophysical and electrophysiological measurements of odor intensities for different
concentrations of m9thyl benzoate. A, data from psychophysical measurements. B, measure-
ments of EOG. C, correlation diagram between psychophysical and electrophysiological data.
(From VON SYDOW, 1968)
sensory channels showing that there is a close congruity between the electrical activity
and the perceptual experience as far as intensity functions are concerned. The
above mentioned experiments by BORG et al. (1967) show that the impulse activity
in the peripheral pathways is closely related to the perceptual intensity to various
taste stimuli. At the cortical level there exists a similar relation between the
perceived intensity of tactile stimulation and the evoked cortical potentials (FRAN-
ZEN and OFFENLOCH, 1969).
As far as receptor functions are concerned there is indirect evidence that the
peripheral intensity functions are faithfully reproduced at the cortical level. As
has already been mentioned OSTERHAMMEL et al. (1969) have succeeded in record-
ing the human EOG. In collaboration with FRANZEN (FRANZEN,OSTERHAMMEL,
TERKILDSEN, and ZILSTORFF, 1970) they later measured the subjective intensity
functions of the same persons and under identical stimulus conditions. A com-
parison of the obtained psychophysical data and the electrophysiological record-
ings showed that there was a high degree of correlation between the EOG and the
subjective intensity of odour. The conclusion to be made on basis of these data
would therefore be that the fundamental interrelationship between the strength of
the stimulus and the intensity of the perceived sensation is determined by the
transducer process in the receptor membrane.
Fig. 21. Schematic diagram of olfactory receptor cell
VI. Concluding Remarks

Receptors are often designated as biological transducers which convert the
energy of a given type of stimulus into a coded message of nerve impulses. The
transduction process involves the production of a local current which is known as
Concluding Remarks 127
the generator potential. This potential generates the afferent nerve discharge by
which information is conveyed to the brain about the nature and strength of the
stimulus.
The transducer cell of the olfactory system is a bipolar neuron with hairlike
filaments protruding into the mucus covering the epithelium. This small neuron is
able to detect and identify minute amounts of substances impinging upon the muco-
sa. How the cell carries out this function has for long been a challenging problem to
physiologists and biochemists. It seems likely that the first step in the excitatory
process involves the absorption of the molecules on the membrane of the receptor.
This stage may include an orientation of the molecules so that they fit into certain
sites of the membrane. The excitatory process may also be preceded by an inter-
action between the stimulating agent and the macromolecular system of the sensory
membrane. The ultimate effect of the odorous particles on the receptors is a
reduction of their membrane potential. This change is the first step in the excitatory
chain of events which at present is amenable to direct electrophysiological studies.
This electrical reaction is the generator potential of the olfactory organ, the
electro-olfactogram.
The electrical response of the olfactory organ exhibits the same general charac-
teristics as homologous potentials recorded from the simple invertebrate eye and
those obtained from single sensory endings such as the eccentric cell of the Limulu8
eye, the muscle spindle and the decapsulated Pacinian corpuscle; it is a purely
monophasic potential with an initial transient phase followed by a decline to a
static level during maintained stimulation. Compared with corresponding potentials
in other sense organs the response of the olfactory membrane is relatively slow.
This is explained by the specific conditions underlying the olfactory stimulation.
The stimulating particles have to pass through a layer of mucus before they reach
the receptors. The time course of the excitatory process therefore will be a function
of the number of particles which reach the receptor per unit time and the time
elapsing before they become inactivated. All experimental evidence support the
view that the primary reaction between the stimulating particles and the receptor
cell takes place in the membrane of the olfactory hairs.
One of the important problems to be solved is that of the properties required to
make a substance an olfactory stimulating agent. We know that certain conditions
with regard to vapour pressure and solubility properties have to be fulfilled. We
also know that the steric configuration is important. What is needed is to bring all
the present data together to a synthesis which could form the basis for a working
hypothesis which could be tested with different methods. It would appear that if
we could answer the question of why one substance is odorous while a closely
similar one is not, then we would also be in a much better position of solving the
problems related to the mechanisms underlying odour discrimination.
It is of particular interest to note the striking resemblance between the
behaviour of the EOG with increasing stimulating intensity and the relation be-
tween subjective odour intensity and stimulus strengths. For a given substance
the correlation may approach the ideal value of 1.0. This suggests that data
obtained in quantitative measurements in the frog's olfactory organ may be valid
for human. Another interesting functional feature of the olfactory organ as revealed
by the EOG is the relation between the amount of odorous material and the
reaction elicited in the receptor membrane. For a short stimulus the olfactory
membrane measures the absolute amount of odorous material, i.e., it behaves like
the eye to variations in intensity and duration of illumination.
The EOG represents the activity of receptors of different specificity which are
activated by the odorous stimulus. Though the response provides valuable infor-
mation of the function of the olfactory membrane it tells us relatively little about
the properties of the individual receptor units. It seems that a precise knowledge
of the functional characteristics of the olfactory receptors can only be gained by
recordings of the receptor potentials of individual elements and the afferent
impulse discharge in their fibres. This will be the challenge for future electro-
physiological work on olfactory receptor functions.
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g.
Chapter 6
Neural Coding in OHactory Receptor Cells

ROBERT c. GESTELAND*, Evanston, Illinois (USA)
With 3 Figures
Contents
Unit Techniques. . 133
Ensemble Techniques 134
Insect Olfaction. . . 139
Marine Invertebrate Olfaction. 141
Marine Vertebrate Olfaction . 142
Terrestrial Vertebrate Olfaction . 142
Effects of Odors on other Tissues 146
Conclusions . 147
References . . . . . . . . . . 148
One goal which the electrophysiologist pursues is to discover the relation

between inputs to a particular cell or section of the nervous system and the changes
in temporal patterns of electrical neural activity which result from those inputs.
Each pair of events which an animal can distinguish as being different must
certainly be represented by a different pair of patterns of nerve cell activity. This
must hold for the receptor cells (in the case of the vertebrate olfactory organ
neurons) and for all units higher in the nervous hierarchy. Efferent connections
from other parts of the nervous system to the olfactory receptor neurons are absent,
hence we expect that changes in activity patterns of these neurons will depend
only upon changes in smells presented to the animal, assuming a stable internal
milieu. This assumption may not hold in fact since odorous stimulation may
modify receptor function through sympathetic control of air flow through the
nose and through circulating endocrine substances. If our goal were to be achieved
completely we should be able to look at the activity of primary neurons and identify
the stimulus or stimuli. That is what we mean when we say we understand the
neural code.
We can imagine two very simple kinds of codes. The first of these would repre-
sent a particular odor sensation or a particular molecular stimulant by a response of
a particular cell. For the other simplistic code, all the cells would respond to all odors
or molecular species and each different odor or molecule would evoke a charac-
teristic pattern of spike activity in each of the nerve cells. All cells would respond
* Supported in part by U.S. N.I.H. Grant No. l-ROlNB06063-02, U.S. Air Force Con-
tract Nr. F3361567-Cl497, and U.S. Army Grant No. DAARO-D31124-G991.
Unit Techniques 133
identically. If either of these two situations occurred we should find it fairly simple
to devise a decoding book for any particular animal by recording the responses to
a number of stimuli. At the present time we are not in a position to make a unique
description of how chemical stimuli are represented as primary receptor cell
responses except possibly for some contact chemoreceptors in invertebrates and
certain specialized receptors for airborne stimuli in insects. It does not seem likely
that simple coding schemes apply. I therefore will consider the several ways of
studying the activity of primary receptor cells and the sorts of information about
codes that have been gathered using these methods.
We can make two large groups of the various techniques currently in vogue.
One group distinguishes activity of single nerve cells or of a few nerve cells electri-
cally close to each other. The other group measures primarily properties of a
population of neurons and gives little information about how any particular cell
responds. It is quite impractical to study simultaneously the individual responses
of a great number of single cells. We would like to ask our questions in such a way
that the code we find is significant to the animal. That is we should like to find a
code which distinguishes each different input which the animal distinguishes and
which is identical for each input which is behaviourally identical to the animal.
Unfortunately this appears most remote as the largest body of information about
distinguishable olfactory sensations comes from psychophysical studies on humans
and essentially all of the electrophysiology comes from organisms which are not
very closely related to man and for which behavioural evidence is limited.
Unit Techniques
Separate cells have been studied using metal micro electrodes recording extra-
cellularly, pipette microelectrodes recording intracellularly and extracellularly and
hook and suction electrodes recording from a very small filament of the nerve in
which only one or a few cells are active. Metal microelectrodes which record only
extracellularly have provided the major part of the scant information on single cell
responses in the vertebrate nose. Pipettes used extracellularly have been similarly
useful recording from insect noncontact chemoreceptors. The results with respect
to the coding question are remarkably consonant and are described in later sections.
The advantages of extracellular recording are that minimal cell damage occurs and
that studies are possible on very small cells. Microelectrodes used in this fashion
allow relatively easy isolation of activity of one or two neurons.
There has been one report published of successful intracellular recording from
vertebrate olfactory receptor cells (AOlIT and TAKAGI, 1968), none in invertebrate
chemoreceptors and one on the effects of odorous substances on invertebrate
ganglion cell activity (ARVANITAKI et al., 1967). The prospect is that these methods
will answer questions about generator mechanisms, but the techniques are only
now being perfected and no results have yet had any impact our understanding of
receptor ionic events. The certain damage done when cells as small as olfactory
receptor cells are penetrated suggests that this technique is not likely to be very
useful for coding studies. The possibility exists that changes in relative intervals
between successive spikes carries significant information about the particular
stimulating substance. Anything which disturbs spike intervals even slightly will
134 R. C. GESTELAND: Neural Coding in OUactory Receptor Cells
probably distort the real response. In the frog's nose, for instance, anoxia and
strong and irritating stimuli irreversibly convert cells which ordinarily respond
differently to different stimuli into cells which are generally and non-specifically
irritable. Intracellular recording techniques will certainly be useful and will be
pursued with great enthusiasm since membrane mechanisms are of particular
current interest to physiologists. They do not seem to be the most useful methods
for coding studies.
An important technique for studies of insect contact chemoreception has been
to use as a recording electrode a pipette filled with the stimulus solution slipped
over a sensory hair. These receptors are analogous to taste receptors in vertebrates
and will not be considered in this review. A useful body of information on chemo-
receptors in marine invertebrates has also been obtained. These are probably also
analogous to taste receptors. Receptors analogous to vertebrate and insect olfac-
tory cells have not yet been identified physiologically in marine invertebrates.
There is good behaviorial evidence that such exist.
Several studies of vertebrate olfaction have used gross electrodes to record
from a very fine filament dissected away from the main part of the first or fifth
cranial nerves (BEIDLER and TUCKER, 1955; KnroRA, 1961; TUCKER, 1963a, b).
Responses of one or a few active fibers can be followed. Some coding information
can be obtained this way. However, it is often difficult to be sure whether a spike
of a particular amplitude is coming from a single axon or from two axons coupled
to the electrode symmetrically. These studies have not been extensive. The
technique is potentially important as it has produced the best information on
stimulus coding in mammalian dorsal root c-fibers, nerves of similar properties to
the vertebrate olfactory nerve.
Ensemble Techniques
Ensemble methods sample the activity of a large number of receptor cells
simultaneously with a single electrode. These methods are easier to use than single
cell techniques, of much greater variety, and probably do little to disturb the func-
tion of the cells. All suffer by requiring unverifiable assumptions in their interpre-
tation.
The most obvious and the most commonly used ensemble method consists of
recording with a gross electrode from either a whole nerve or a filament of the
olfactory nerve (n. I), the accessory olfactory nerve (n. Ia), or the trigeminal nerve
(n. V) (MOULTON, 1963; MOZELL, 1964, 1967a; TUCKER, 1963a, b). The technique
is also useful in studying the chemical sensitivity of other nerves and it has been
applied with interesting results to the ventral nerve cord of the cockroach (Roys,
1958) and to a branch of the fifth nerve innervating the cornea of the eye of the
frog (DAWSON, 1962). Usually the increase in base line width evoked by a stimulus
is recorded photographically, the total number of spikes is counted, or the signal is
passed through a summator network with a decay time-constant of the order of a
few seconds to display relative changes in total electrical activity of the nerve
filament on the hook. This technique depends upon the assumptions that either a
stimulus evokes excitation or no response or, that, if it can cause inhibition either
the ratio of excited cells to inhibited cells is the same for all stimuli, or that the
inhibitory response does not involve a significant fraction of the receptor popu-
lation.
A second ensemble technique for the vertebrate nose is to record the potential
at the surface of the olfactory bulb where the principal component of the voltage
recorded is due to activity in the terminals of the olfactory receptor cells (OTTOSON,
1959a, b). These terminals are presynaptic to the glomeruli. All second order and
higher nervous elements in the olfactory system are located deeper in the olfactory
bulb. The early component of the evoked wave has a shape and time course nearly
identical to the voltage recorded from the receptor cell layer in the nose (OTTOSON,
1959c).
The third ensemble signal event which can be recorded is called the electro-
osmogram or electro-olfactogram in vertebrates and electroantennogram in insects
(abbreviated E.O.G. and E.A.G. respectively). The E.O.G. is discussed in detail in
another chapter of this volume. Since I claim that it is a direct sign of receptor cell
activity in this paper I will briefly indicate the mechanistic argument upon which
this claim is based. Both the E.O.G. and the E.A.G. have been called generator
potentials by many people working with them. There can be no serious question
that they are causally related to the generator events but since they are recorded
by extracellular electrodes they can hardly be throught to be the generator event
itself. It is easy to distort the recording of such external signs without disturbing
the current flow across the excitable membrane, hence experiments which show
that distortion of extracellular voltages does not distort cellular responses
can not be taken as serious evidence against a causal relationship between the
E.O.G. and receptor cell activity (SHIBUYA, 1964). In particular the E.O.G. is
recorded by placing a reversible electrode on the surface of the olfactory mucosa
and measuring the voltage between it and an indifferent electrode elsewhere on the
animal (HOSOYA and YOSHIDA, 1937; OTTOSON, 1954). The E.A.G. is recorded by
placing a micropipette somewhere on the insect antenna and recording the voltage
between it and a gross electrode on the base of the antenna or elsewhere on the
body of the insect (SCHNEIDER, 1957). It can also be recorded by a micropipette
inserted through the cuticle where it will respond to unit activity as well (SCHNEI-
DER and BOECKH, 1962; SCHNEIDER, 1963). The E.O.G. and the E.A.G. voltage
waveforms evoked by an odor are quite similar. They consist primarily of a
negative-going deflection, often accompanied by smaller negative and positive
waves, the origins of which are a subject of considerable controversy.
Two sorts of extracellular voltage changes can be expected when the chemical envi-
ronment of irritable tissue is changed. One sort consists of various electrochemical
phenomena occurring in extracellular fluid and is not uniquely related to cell function.
The other sort consists of voltage drops across the resistance of extracellular fluid due
to current flow through activated receptor cell membranes. In the nose, these two
classes of phenomena can be separated in a simple way (GETCHELL and GESTEL.AND,
1971). Receptor cells are capable of supplying a considerable amount of energy and
have a source impedance lower than the electrochemical processes in typical
extracellular fluids. Therefore a recording amplifier with low input resistance
responds primarily to the receptor cell events. Further, electrochemical events
occur everywhere there is extracellular fluid, whereas the current flow from
receptor cells is greater nearer the receptor cells. Therefore an electrode touching
136 R. C. GESTELAND: Neural Coding in Olfactory Receptor Cells
the mucosa surface records larger receptor cell generated voltages than it does if it
is withdrawn slightly pulling with it a mucus strand. If the nasal cavity is water-
filled, the electrochemical events are displayed by raising the electrode up into the
water so that it does not touch the mucosa (GETCHELL, 1969a).
The electrochemical events in frog extracellular fluid cause a positive-going
voltage waVe when a puff of a smell is directed at the fluid near the electrode,
independent of the sort of electrode used. It rises and falls exponentially. This
wave is negative if the concentration of substance in the fluid near the recording
electrode is decreased.
The polarity of the principal component of the receptor cell-generated voltage
drop is such that the mucosa surface goes negative. This indicates that positive
current is flowing extracellularly from the depths of the mucosa to the surface, i.e.
that the basement membrane is becoming positive with respect to the surface. This
is what we would expect for the usual excitatory generator event in a receptor cell
where sodium ions enter the chemically sensitive distal portion of the cell near the
mucus air interface and positive charge flows out of the cell in the region of the
axon hillock or initial portion of the axon where the spike is generated. This is
shown diagramaticly in Fig. 1.
o.e.
hm
=:=~~~ax.
7S
T
Fig. 1. Extracellular electrodes measure a voltage change (V.og) across the olfactory epithelium
(o.e.) when an odor is introduced into the air over it. This highly simplified schematic shows one
receptor ceIl (r.c.) with its distal process (d.p.) and cilia (c.) lying in the mucus (m.) and the
cell axon (ax.) lying under the basement membrane (b.m.) projecting centrally. If the stimulus
causes the membrane of the distal process to become more permeable to a particular species of
positive ion and if the concentration of these ions is higner outside the cell than inside,
current flow is as indicated by the a.rrows (i). V will be the product of the extracellular current
and the extracellular resistance. For the case shown here, the polarity of the measured voltage
thus will be such that the upper electrode touching the mucus surface will go negative with
respect to the lower one
Byzov and FLEROVA (1964), by measuring the rate of change of the E.O.G.
voltage with depth, show that excitatory current enters the distal process of the
vertebrate receptor cell at the cilia base and leaves the cell deeper, near the nucleus
or basement membrane. Furthermore they show that the large negative E.O.G.
wave and the small positive wave sometimes seen have the same rate of change
with depth and thus the current sources responsible for each must occur at
approximately the same place on the cell. The components of the E.O.G. waveform
which have been recorded from vertebrates are similar to what would be expected if
the excitatory and inhibitory events were like the events occurring in the lobster
stretch receptor. Here the excitatory process consists of an increase in the per-
meability of the membrane to an ion whose distribution is far from equilibrium
with the potential across the membrane, i.e., sodium ions, while the inhibitory
process is due to increased permeability of the membrane to an ion whose distribu-
tion is close to being in equilibrium with the resting potential of the cell membrane,
i.e., potassium or chloride (EYZAGUIRRE and KUFFLER, 1955; KUFFLER and
EYZAGUIRRE, 1955). The principle effect of stimulating the inhibitory process there-
fore is to introduce a Jow impedance region on the distal part of the receptor cell
while stimulation of the excitatory mechanism introduces current sources and an
accompanying impedance decrease. During inhibition the membrane is driven
toward a certain potential regardless of the cell potential preceding inhibition.
Thus inhibition could be either depolarization or hyperpolarization. This has been
shown to be the case for the early positive component of the E.O.G. wave also
(GESTELAND et al., 1965). Experiments in which the extracellular concentrations
of various ions in the frog's nose are changed also imply that the positive component
of the E.O.G. is a sign of the olfactory stimulus acting upon the membrane to
increase the membrane permeability to chloride or potassium ions (TAKAGI et al.,
1966; TAKAGI et al., 1968).
The E.O.G. and similarly the E.A.G. can therefore be taken as signs of activity
of receptor cells. It is not possible to decide what part of the E.O.G.waveformis
contributed by what component of the receptor cell population if the population is
not a homogeneous one with respect to the effects of various stimulating substances.
Furthermore, if it is true that a particular stimulus affects the excitatory process
on some cells and the inhibitory process on other cells it is not possible to separate
these two phenomena in any simple way. This is because the excitatory processes
are mostly additive current sources while the inhibitory processes are essentially
attenuative shunt paths and these two processes do not combine linearly under
most situations. The sorts of E.O.G. waves which have been described are shown
in Fig. 2. They include an early positive-going "on" wave, an early and much
larger negative-going "on" wave, a negative-going wave occurring at the cessation
of a stimulus, a positive-going wave following cessation of the stimulus by some
time, i.e. a positive after voltage following a negative "on" wave, and an inverted
early positive "on" wave. These waveforms are easily accounted for in terms of the
two kinds of receptor processes by postulating that a particular stimulus can evoke
both excitatory and inhibitory processes and that the time constants of each
process are independent of each other and stimulus-related. Thus pyrrole evokes
an inhibitory event that has a rapid turn-on and a rapid decay upon cessation of
the stimulus and an excitatory event that has a slower turn on and lasts con-
siderably after the stimulus has ended. The negative "off" response thus is the
result of rapid cessation of inhibition leaving the excitatory current to manifest
itself fully. As stimulus intensities are changed the relative amplitudes of various
,...---,
A G
,----,
V
B
E ____ ~q- _________________
Fig. 2. EOG waveforms of the various sorts evoked by different odor puffs delivered to the
frog's olfactory mucosa. All of the sweeps are 10 seconds in duration. The duration of the
stimulus is indicated by the horizontal bracket over each response. The responses are tracings
of oscillograms. Vertical calibration bar at the top left is 1 millivolt with an upward deflection
indicating that the surface of the mucosa is positive with respect to an indifferent electrode.
A. The positive "on" response evoked by methanol. B. The negative "on" response evoked by
a low concentration puff of anisole. C. The response to pyrrole consisting of a positive "on"
deflection, followed by a negative "on" deflection. At cessation of the stimulus a negative "off"
wave occurs. Under certain circumstances the "off" response may be the only obvious deflec-
tion (see Fig. 3 c). D. The positive after voltage which occurs following a strong anisole stimulus.
E. The positive "on" response can be inverted when the stimulus evoking it is paired with
another stimulus. The upper dotted trace is the response to a short puff of n-butanol; the
lower dotted trace is the response to a long stimulation puff of pyrrole. When the two are
delivered together at the times indicated by the brackets, the solid line represents the resulting
response. The butanol positive "on" wave is changed into a negative-going event
components of the E.O.G. can vary in very complicated ways. This does not
necessarily mean that the cells are behaving in such complicated ways, only that
the nonlinear combinations of the external signs of inhibition and excitation pro-
cesses with different time constants are very complicated.
Insect Olfaction 139
One other indication of the activity of the ensemble of receptor cells in ver-
tebrates can be got by measuring either the transfer impedance between a current
injection electrode touching the mucosa surface and a recording electrode near to
it or the two-terminal impedance of the mucosa by injecting a small a-c current
through a low impedance electrode and recording the voltage which results
(GESTELAND, 1967). These impedance changes hold the same sort of relationship
to the coding process as does the E.O.G. There would be hope of a unique inter-
pretation of the impedance changes only if the receptor cell population were
homogeneous. This does not seem to be the case. However patterns of impedance
change, like the E.O.G., do differ for different stimulating substances and to a
certain extent may be useful in deciphering the code. The impedance change
technique is a particularly sensitive extracellular indicator of receptor cell action.
Damage due to noxious stimuli and anoxia shows up first on the impedance change
trace. Very small changes can be measured by using coherent detection techniques
which effectively average over a large number of cycles of the measuring signal.
The receptor cells are excited electrically by the a-c measuring signal if it is too
large. When the applied voltage rises above the cell threshold, the mucosa im-
pedance drops and electrical excitation then shows up as distortion of the sine
wave at its peak. This provides a useful indication of the range of measuring signals
which can be applied without seriously altering the receptor thresholds.
Insect Olfaction
By far the largest collection of data on single unit responses to airborne stimuli
comes from the work of SCHNEIDER and his associates on insect antennal receptors.
It is summarized in the papers by BOECKH et al. (1965) and SCHNEIDER (1969). They
have recorded from the single cells of the trichoid sensilla of moths, beetles,
grasshoppers, flies and cockroaches, from pit sensilla of flies, grasshoppers and
bees, and plate sensilla of bees and wasps. Cells associated with the pit sensilla of
honey bees are excited by carbon dioxide, changes in the moisture content, or
changes in temperature of the air stream. The remaining antennal receptor cells in
the organisms respond to either of two classes of odorous stimuli. One class
stimulates the cells called "generalists" which are analogous to the cells of the
vertebrate olfactory sense which have been so far studied. The other class stimu-
lates the cells called "specialists". The generalists are cells which respond to a
great variety of odors with moderate sensitivity. These odors have no particularly
obvious adaptive significance to the anima1. The specialist cells are those which
respond with high sensitivity to a small group of compounds which are closely
related to mating stimuli or to food smells of particular significance to the species.
The pattern of intervals between spikes for the specialists are relatively simple. An
appropriate stimulus evokes a rapid burst at the onset of the stimulus declining to
a lower rate while the stimulus is maintained and falling back to the pre-stimulus
rate when the stimulus is turned off. The cells mayor may not have a resting
discharge. Generalist receptor cells have been found in the trichoid sensilla of
moths and the plate sensilla of honey bees. Spike firing patterns appear to be less
regular for the generalist than for the specialist cells or the cells which respond to
atmospheric factors other than odor.
Generalists mayor may not have a resting discharge. Excitatory firing can be
phasic, tonic, phasic-tonic, or can occur in bursts. The phasic-tonic firing pattern
is most often seen. Inhibition can occur either by slowing the resting discharge or
by slowing a discharge evoked by an excitatory stimulus. Latencies and decay
times are variable from cell to cell and from stimulus to stimulus. The odor code of
the generalists is holistic. That is, there is no simple stimulating factor possessed
by various stimulating substances. Therefore the stimuli can not be ordered in any
simple way which will describe relative stimulating effectiveness. For any particular
receptor cell some substances excite, some substances inhibit and some substances
have no effect. Using these same substances to study another cell in the same sort
of sensilla in the same animal some will also excite and some inhibit and some have
no effect but the lists will not be ordered in the same way. The most adequate
description seems to be that if more than a few stimulating substances are used, no
two cells seem to be alike and a substance that excites one cell may inhibit another
or have no effect. Another substance which also excites the first cell may excite,
inhibit or not affect the second cell. BOECKH et al. (1965) conclude that generalist
receptors can not be ordered into a few groups of response types for any particular
animal. There is considerable overlap between the substances which affect one cell
and the substances which affect another.
The specialist cells respond to a particular category of compounds usually in an
excitatory way. Inhibition is not a commonly seen response. There are large
numbers of specialist cells in any particular creature with identical response pat-
terns. The carrion receptor of the blow fly responds strongly to carrion odor,
amines, mercaptans, and hexanol. It does not respond to straight chain fatty acids
either saturated or unsaturated. The carrion receptor of the carrion beetle responds
strongly to carrion odor, amines and mercaptans, and less strongly to straight
chain fatty acids with chain lengths between six and ten carbon atoms. The
grasshopper responds strongly to hexanal which has a green smell, does not respond
to hexanol and responds strongly to saturated C3, C4, C6, and C7 straight chain
fatty acids. It responds less strongly to C8 saturated fatty acid and CI8 un-
saturated fatty acid as well as hexenol and hexenylformate. A plate receptor from
the honey bee which is specialized for and responds strongly to the queen substance
(9-oxo-decanoic acid) responds strongly to hexenal and 04, C5, and C6 straight
chain fatty acids. It responds less strongly to the C8 fatty acid and to 9-hydroxy-
decanoic acid and does not respond to hexenal and hexenol. Thus the substances
which stimulate the specialist receptors of one species will often stimulate these
receptors in others, and these receptors tend to respond not only to a particular
pheromone or food odor but also to substances with related chemical structures.
Selective adaptation does not seem to occur in the specialist receptors. When the
receptor unit is fatigued for one appropriate stimulus it is fatigued equally for all
others.
For the generalists a substance which is excitatory for a cell is usually more
effective at a particular concentration than it is on a cell which responds in an
inhibitory manner to that substance. The maximum firing rate for insect olfactory
receptors is in the range of 500 impulses per second. This is very much faster than
vertebrate olfactory receptors. It is also interesting to note that the insect olfactory
receptors are derived embryonically from the dermal cells, unlike vertebrate
Marine Invertebrate OHaction 141
olfactory receptor cells, which are neural. The limited amount of information
collected by investigators not associated with SCHNEIDER'S group tends to confirm
the sorts of responses described here. MAYER (1968) has described responses of
specialist cells of Triatoma. YAMADA (1968) has found on-off responses to olfactory
stimuli in the cockroach central nervous system unlike the periphery, where only
on-type (excitatory) and off-type (inhibitory) responses occur.
Complex E.A.G. waveforms with both positive and negative waves occur
(BOECKH, 1962). Since a particular stimulus may excite some cells and inhibit
others with independent time patterns it is not surprising that this is so. H the
firing rate of the cells of a particular sensillum excited by a stimulus is plotted as a
function of time, the curve has in most cases the same shape as the E.A.G. recorded
from the sensillum. There is an initial peak followed by a plateau during stimu-
lation followed by a decline to the baseline after the stimulus is turned off. BOECKH
(1967) discussed the relationships between the E.A.G. and cell firing patterns.
For the specialist receptors particularly clear relationships exist between the
slow waves and the spike response. For instance, in Bombyx for the E.A.G. recorded
between the ends of the antenna, the generator potential recorded by a micro-
electrode from a bombykol receptor and the mean impulse frequency from that
cell are identical functions of the stimulus concentration over more than five log
units (BOECKH et al., 1965). Such simple relationships probably do not hold for the
generalist receptors.
There is good evidence that there is structural basis for differential responses of
insect olfactory receptors. In Bombyx (the silkworm moth) the female has no
E.A.G. in response to bombykol, nor have single units responding to bombykol
been found. Bombykol evokes an E.A.G. and responses in some receptor cells at
very low stimulus strengths in the male. Behaviourally the male reacts strongly to
bombykol, the female does not, and both the male and female react to a variety of
other odors. Thus the female is lacking a specific receptor type capable of respond-
ing only to bombykol and closely related substances (SCHNEIDER, 1963).
Marine Invertebrate Olfaction

Published studies of nervous responses from primary chemo-receptor units in
marine invertebrates have all been concerned with receptors thought to be analog
to the contact chemoreceptors of insects and "taste" receptors in man. There are
behavioral observations that certain marine invertebrates also have receptor
organs analogous to the olfactory sensors in insects. CASE (1969) has suggested that
the Annelids have such receptors on their labial palps and that the antennulles of
some crustacea may contain such organs. These structures are anatomically
distinctive in that there are many more receptor cells associated with each surface
receptor structure than for the contact chemoreceptors. Contact chemoreceptor
organs in marine invertebrates will not be discussed here except to note that they
commonly respond to temperature, mechanical deformations, CO 2, amino acids,
some amines and some dipeptides (HODGSON, 1958; BARBER, 1961; CASE, 1964;
LEVANDOWSKY and HODGSON, 1965). These receptors can often distinguish be-
tween d and l forms of a compound. Tactile, temperature and chemical sensitivity
are sometimes found in one receptor cell. Patterns of spike activity are usually
142 R. C. GESTELAND: Neural Coding in OUactory Receptor Cells
relatively simple, often a phasic-tonic type of response with relatively little scatter
in spike periods. However there is a report of "off" responses, slow "on" responses,
and long lasting decay of the excitatory responses (LAVERACK, 1963). Reports of
inhibition are few.
Marine Vertebrate OHaction

There is an extensive literature on behavioral responses of marine vertebrates
to olfactory stimuli. The performances are often remarkable in terms of sensitivity
and ability to make fine discriminations. For the teleostei and elasmobranchii
there are no significant physiological studies of the responses of receptor cells in
the nose.
Terrestrial Vertebrate OHaction
Single receptor cell responses in the vertebrate nose have been recordable
beginning circa 1960, as for insect receptors. The evidence as to how different
stimuli are encoded as patterns of nervous activity is remarkably like that for
insects except that no specialist fibers have been found. The olfactory receptors in
the vertebrate, like the insect generalists respond to many different smells and in
diverse ways. Studies using ensemble recording techniques imply that some kinds
of order exist.
Of the studies using micropipette electrodes which have been published only
the study of SHIBUYA and TUCKER (1967) on the vulture reports responses to
several different olfactory stimuli. Most often they saw excitatory responses to
their several stimuli. However at least one clear case of inhibition occurred. Patterns
of spike intervals during a response were often complex or irregular, and of course
phasic with respiration.
TAKAGI and his co-workers have reported unit responses using extracellular
micropipettes, paying attention to the sorts of nervous response rather than
stimulus-response relationships. In their early reports they identified three kinds
of activity, "on" responses, "off" responses, and "on-off" responses in receptor cells
as well as in second order neurons (SHIBUYA et al., 1962; TAKAGI and OMURA, 1963;
SHIBUYA and SHIBUYA, 1963).
Metal microelectrodes which can record rather easily from single axons of the
olfactory nerve and from receptor cell bodies have been used to compare the
effects of different stimuli (GESTELAND et al., 1963; GESTELAND et al., 1965;
MATHEWS and TUCKER, 1966; ALTNER and BOECKH, 1967; O'CONNELL and
MozELL, 1969). These studies (on frogs and turtles) show that any particular nerve
cell will respond to many odors. There seems to be no simple common chemical
property or subjective odor quality for those substances which stimulate a parti-
cular cell. Substances which are chemically or organoleptically very similar may
differ violently in how they affect a particular cell. Substances which are quite
different from each other may affect a particular cell almost identically. Any
particular substance may excite a cell, may inhibit an on-going discharge, may
cause excitation after the stimulus has been turned off, may cause inhibition after
the stimulus has been turned off, may have a long lasting effect starting at the
onset of the stimulus and continuing long after turn-off, or may change the pattern
of firing of the cell, i.e. may markedly alter the distribution of time intervals
Terrestrial Vertebrate OUaction 143
between successive spikes. As in the generalist receptors in insects, the code for a
particular cell can be searched for in terms of gross changes in cell activity.
Stimuli can be arranged as a continuum where at the head of the list we put that
substance which causes the strongest excitation of the cell and at the end of the
list the substance which causes the strongest inhibition of the cell and in between
rank order those substances from stimulating through no effect to inhibiting. If
the same procedure is now followed for a second cell using the same stimuli a
different rank order will be obtained. In general, no two cells seem to rank order a
group of stimuli in the same way. These responses are quite repeatable and if
stimulation intensities are kept within normal physiological ranges the responses
can be repeatably elicited over a long period of time. Any particular response is not
very strongly contingent upon what happened before it. If the strength of the
stimulus is changed, i.e. if the number of molecules per second delivered to the nose
changes, the gross pattern of the response is usually unchanged. That is, what was
excitatory is still excitatory. A weaker stimulus evokes a lower increase in firing
rate if it is an excitatory one. In addition, the temporal patterns of the intervals
between successive spikes tends to be preserved. A glance at the spike record
shows the same sort of grouping and timing of relative quiet periods even though
the number of spikes associated with each of the varying epochs of the response
may change. This is illustrated in Fig. 3. Occasionally one smell changes the response
patterns evoked by other smells for long after. It is not clear whether this is a
pathological change or whether receptor cell response can be a function of its
recent past. Often, the response to a mixture of two smells is not any simple
combination of the responses evoked by each smell when presented separately.
There is no agreement as to how significant are inhibitory phenomena in coding
odor quality. Ofthe 101 receptor cells in the frog's nose exposed to each of 4 odorants
by O'CONNELL and MOZELL, 47 were excited, 1 was inhibited and 53 did not
respond. This same distribution occurred for each of the 4 different esters used as
stimulants. No two cells were exactly alike in their responses. MATHEWS and
TUCKER found only excitatory responses in the tortoise, testing 40 cells with
30 odors at two concentration levels. I have found inhibition almost as commonly
as excitation in frogs but have no careful counts or statistical analysis to verify this
statement. Irregularities of spike intervals, low maximum firing rate and typical
resting discharges of the order of 1 per second make it difficult to distinguish
between inhibition and no response. For anyone stimulus the response frequency
and response latency as a function of stimulus concentration varies from cell to cell.
These functions also vary from stimulus to stimulus for any particular cell. Early
results of studying the responses of single cells to a variety of odors led to a pre-
diction that a small number of different categories or kinds of receptor cells could
be found with respect to their responses to different chemical stimuli. As these
studies continued and the number of cells sampled increased the probable number
of categories of cells increased. More surprising than this was the fact that when
two cells responded similarly to a large number of substances, one substance could
always be found which would clearly distinguish between the two cells. That is, it
would strongly affect one cell and inhibit or not affect the other at all. Furthermore
any two odors however similar, if they were not identical, could be tried on several
cells in succession and it took only a short search to find a cell which would clearly
144 R. C. GESTELAND: Neural Coiling in Olfactory Receptor Cells
,....., "
lJ- JIIIII11Jml&
1\uU- JJlln'!!1 'u- lilt
I! II II
V- ~
II II11
,\t.J ,.,1,1 II
Fig. 3 a. Benzaldehyde Fig. 3 b. Nitrobenzene
Fig. 3 a-c. Spikes recorded with extracellular me

tal microelectrodes from single axons of frog olfac.
tory receptor cells. The signal from the electrode is
passed through an amplitude selector to eliminate
baseline noise and is then added to the signal from
a large salt bridge electrode touching the mucosa
surface. The result is an EOG trace with spikes
superimposed. Horizontal brackets above traces in
dicate stimulus duration. All sweeps are 10 seconds
long. Vertical calibration varies from trace to trace
and is not the same for EOG and spike components.
Polarity of the spikes is arbitrary while the EOG
is positive upward in all cases. Spikes of different
amplitudes in one trace indicate responses from dif
ferent cells. The five responses in Fig. 3a are evoked
Fig. 3 c. Pyrrole from cells in five different frogs by 1 second puffs of
benzaldehyde. The five responses in Fig. 3 b are evoked
by 1 second puffs of nitrobenzene which has an odor
very similar to that of benzaldehyde. Spikes appear to occur at preferred times after the onset of
the stimulus. These times are characteristic for the stimulus and are similar for similar stimuli.
Fig. 3 c shows the responses of different cells to pyrrole. Characteristic burst timing occurs for
all of the responses with the exception of the center trace. Here the unusual case of a regular.
interval excitatory response is shown
Terrestrial Vertebrate Olfaction 145
distinguish the two odorous substances. The single generalizing statement that
seems to apply is that receptor cells in the frog's nose are organized in such a way
as to "look" at the odor world in as many different ways as possible. As for insects
generalists, the code appears holistic.
The single unit studies have led to interesting speculations about receptor
mechanisms but to very little useful information about coding of odors. It has not
been possible to make a satisfactory match between any of the psychophysical
theories of odorous properties of molecules and the physiological responses of
primary receptor cells. One difficulty may lie in the fact that odor theories are
built upon attempts to correlate odor properties as perceived by man with physical-
chemical properties of organic molecules whereas most of the electro-physiological
studies of coding are on frogs. GETCHELL (1969a, b) recently has asked what
substances will affect receptor cells in the frog. In addition to those substances
which smell, many other substances act like odors ifthey can get to the receptors.
By stimulating the nose with "odors" dissolved in a fluid stream flowing through
the nasal cavity, substances which do not have significant vapor pressure can be
tested (TUCKER, 1963a; TUCKER and SHIBUYA, 1965). Single unit recordings from
receptor cells and second order cells in the olfactory bulb, slow voltage changes
measured at the surface of the olfactory mucosa, slow voltage changes at the
surface of the bulb, and impedance changes at the olfactory mucosa all indicate
that many soluble odorless substances stimulate the olfactory receptors (GETCHELL,
1969 a, b.) The responses are in no way distinguishable from the responses to smells in
air or dissolved in water. It appears futile, for the frog at least, to sort chemicals into
two groups one of which smells and one of which does not and then search for those
chemical properties which distinguish the smelly group from the non-odorous
group. The chemical properties of the receptor membrane will have to be looked
for using physiological and chemical techniques rather than psycho-physical ones
in the absence of detailed behavioral data on animals which can be used for
electrophysiological studies.
Since studies of single units show great divergencies of properties and hold
little hope of revealing what regularities might exist, other measures of responses
of the primary receptor system loom large in their importance. The small sizes of
olfactory receptor cells and their great numbers precludes sampling a significant
fraction of the receptor cells using single cell techniques in any animal and attempt-
ing to sort these into functional groups or types.
MOZELL (1964, 1967a, b), using the frog, has recorded summated extracellular
action potentials in small twigs of the first nerve coming from various portions of
the receptor organ. He finds spatial sorting of odorant substances, i.e., one area of
the mucosa more sensitive than another to a subgroup of olfactory stimuli. The
spatial pattern depends more on what stimulus is applied to the nose than how
strong it is. Different stimuli differ from each other with respect to which area of
the mucosa they affect most strongly. MOZELL suggests that this is a sort of
chromatographic separation occurring in the extracellular mucus layer which acts
to increase the resolution or discrimination ability in the nose. The technique has
been applied to rather large bundles of the olfactory nerves as well and it is found
that the time course of the summated spikes is a strong function of the particular
stimulus being used and tends to parallel the time course of the E.O.G. For any
10 IIb. Sensory Physiology. Vol. IV/1
given stimulus the amplitude of the summated peak varies approximately with the
log of the stimulus concentration over a range of few log units and there is a clear
threshold value and saturation value (KIMURA, 1961; TUCKER, 1963b; TUCKER
and SHIBUYA, 1965).
The E.O.G. voltage recorded across the mucosa varies in a well behaved way
also. Its time course is dependent on the particular stimulating substance used
and varies regularly with thermodynamic properties of the substance (OTTOSON,
1956,1958). It has a saturation value for the phasic or initial peak and a saturation
value for the tonic or maintained plateau and some groups of substances can be
sorted from others merely by looking at the shapes of the E.O.G. waveforms. This
response, like the summated spike response of the first nerve, does not show high
enough discrimination to infer the coding scheme for odors. At best it can be said
that a certain amount of regularity exists and that the search for a unique relation-
ship or set of relationships between molecule properties and responses of the
ensemble of receptor cells should not be futile.
Electrical impedance changes of the receptor cell layer as a function of odorous
stimulation have been measured. These changes can be separated into two com-
ponents with temporally independent time courses. This implies that at least two
kinds of receptor events are activated by a stimulus, as was inferred from E.O.G.
waves. For odor puffs of a given duration and of a strength such that the E.O.G.
and impedance changes were neither near threshold nor near the saturation point,
eighteen different smells evoked discriminantly different impedance change pat-
terns. Whether or not these differences would merge into a continuum when data
from a larger number of odors is got is not clear (GESTELAND, 1967).
Effects of Odors on other Tissues

In a surprising paper ARVANITAKI et al. (1967) investigated the effects of a
number of different substances possessing an odor not generally thought to be
active on nerve membranes. These substances were applied to areas of cell body
membranes in which there are no synapses in the ventral ganglion of Aplysia. Some
substances were depolarizing and led to spike generation. Some substances produced
hyperpolarizing currents. Effects of multiple stimuli were additive. Different cells
responded to the same substances in different ways. Any particular substance
always had the same effect on any particular cell. There were specific patterns in
the spike firing. Each different substance appeared to evoke its own pattern of
responses in a particular nerve cell. Whether or not olfactory receptor organs are
merely high surface area versions of "irritable" membrane typified by the Aplysia
ganglion cell is not clear at the present time. It is also clear that no biological
tissue including the nose has been exposed to a wide enough variety of chemical
reagents and physical conditions such that it is possible to state its response
properties. Roys (1958) found that cells in the ventral ganglion of the cockroach
responded to a variety of substances generally thought to act only on spezialized
chemoreceptor organs. The branch of the fifth nerve which has sensory bare nerve
endings in the cornea of the frog's eye has two sorts of fibers. The fast myelinated
fibers respond to touch. The slow unmyelinated fibers are chemosensitive. When
stimulated with amyl acetate or phenyl-ethyl-alcohol bursts of spikes are evoked,
Conclusions 147
reaching a peak firing rate in a few seconds, then declining slowly (DAWSON, 1962).
BEIDLER (1965) compares the sensitivity of the olfactory nerve to the fifth nerve
chemoreceptory fibers. He reports that the fifth nerve is more sensitive to the
flowery order of phenyl-ethyl-alcohol while the olfactory nerve is more sensitive to
foul smelling mercaptans. The fifth nerve responds slowly, reaching its maximum
response after the olfactory nerve has adapted.
Conclusions
In spite of impressive technical advances which now enable direct measures of
the responses of olfactory receptors, we still know little about the coding of odors.
ADRIAN (1953) speculated that the only way in which a nose could distinguish more
than a few general types of molecules would be if there were a difference in
sensitivity of the individual receptor cells. That these differences exist has been
amply demonstrated, so much so that chaos appears to substitute for code
(LETTVIN and GESTELAND, 1965; GESTELAND et al., 1968).
It is worth asking whether we would recognize "the code" if we stumbled onto
it. BULLOCK (1968) has listed possible nervous coding schemes including latency
modulation, burst duration modulation, probability of firing variation, variation
of microstructure (relative timing of successive spike intervals), several kinds of
frequency modulation, and channel selection code (which axon fires). These
schemes can be separated and identified when the appropriate stimulus is simple.
(Many of the examples of these codes come from the fish with sensory organs
peculiarly sensitive to electric currents.) With odors as the stimulus the problem is
more difficult because we do not know how to define an odorous substance in terms
of its physical properties. The notion of "primeness" of some property of an odor
has been particularly evasive. If we adopt an operational approach to decoding we
can describe some experiments which we would wish to do. These would include:
1. The Search for the Properties of the Receptor Sites. To accomplish this we
would record from a single cell and try various stimuli which would elicit maximal
excitatory response. Then we would make small chemical variations in this "most
effective" substance to find out how stimulating effectiveness varied with molecular
architecture. We would look for multiple modes (this is a hill-climbing technique
and an area can have more than one hill), and if we found them, study the chemical
properties in the vicinity of each stimulus maxima. We would pursue a similar
course to find maximally effective inhibitors. Then we could try pairs on this cell
to see whether these very effective stimuli compete for the same site on the receptor
cell or react at separate sites. The search would then proceed to another receptor
cell to see if it had the same kinds of receptor sites. After sampling enough cells we
could guess whether receptor cells can be grouped by types, whether there are one,
several or many kinds of active sites on the cell, and maybe even discover how
excitatory and inhibitory substances interact with each other. This is a very
difficult experiment (to record from a single cell for a long time is unusual) and no
existing experimenter has broached the question with this sort of Teutonic
thoroughness.
2. The Search for Stimulus Equivalents. This might appeal to some as a code
breaker. With careful conditioning work we might find those substances which
10'
behaviorally evoke the same response from an animal, then analyze the spike
patterns of cells or ensembles of cells to find out what parameters of the response
are invariant for stimulus equivalents. This fits nicely with our rationality but
defies the intuitive feeling that we can tell the difference between smells from very
similar chemical compounds. We have observed how difficult it is to exactly
duplicate a particular odor. And we find receptor cells which seem best described
by the statement that no two seem to be alike. The search for stimulus equivalents
may prove to be a very long job.
3. The Search for Common Signal Parameters from Different Cells. We could
study the response of first one cell to various concentrations of a particular odor,
then another cell, then another. After collecting many such responses we could ask
if there is any property of the time intervals between spikes of the responses of
different cells which is common for the stimulus. We then would try another
stimulus and see if it had characteristic time interval relationships. This is a
search for the code directly, ignoring the molecular properties of receptor sites and
the behavioral responses of the animal. It also looks like a very long job.
The last of these approaches seems particularly interesting. No effort has yet
been made to find orderly structures in the time sequence of either vertebrate
olfactory cell activity or insect generalist cell responses. The most obvious property
of signals from both organs is the lack of regularity in the spike patterns. The
maximum firing rates are low. Resting discharges of both are very low, thus
inhibition acting alone can not transmit much information. If spike interval
modulation is of any biological significance it should be under the circumstances
that hold here.
It may be possible to find psychophysiological evidence for holistic coding. If
each cell carries a different representation of odor space, the effect of disabling a
portion of the receptor population should be to reduce ability to distinguish
between similar odors. Sensitivity should not be much changed and no distortions
of odor sensation should occur.
We know nothing about how the nervous system sorts smells into the categories
which we feel must exist.
References
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- Comparison of slow potentials evoked in the frog's nasal mucosa and olfactory bulb by
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their responses to gustatory stimuli. BioI. Bull., Woods Hole 115,490-507 (1958).
SCHNEIDER, D.: Electrophysiological investigation on the antennal receptors of the silk moth
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- Electrophysiological investigation of insect olfaction. In: Olfaction and Taste I, Y. ZOTTER-
MAN, ed. Oxford: Pergamon Press 1963.
- Insect olfaction: deciphering system for chemical messages. Science 163,1031-1037 (1969).
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Insektenantenne. Z. vergl. Physiol. 45, 405-412 (1962).
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- WYSE, G. A., KITAMURA, H., ITo, K.: The roles of sodium and potassium ions in the
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TUCKER, D.: Physical variables in the olfactory stimulation process. J. gen. Physiol. 46,
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- Olfactory, vomeronasal and trigeminal receptor responses to odorants. In: Olfaction and
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Chapter 7
Nonolfactory Responses from the Nasal Cavity:

Jacobson's Organ and the Trigeminal System
By
DON TUCKER Tallahassee, Florida (USA) Approved D. T.
With 8 Figures
Contents
I. Introduction . . . . . . . . . . 151
II. The Vomeronasal System . . . . 153
A. Electrically Recorded Responses . 154
B. Vomeronasal Epithelium and its Distribution in Turtles and Mammals. 160
C. Stimulus Transport to the Organ. 161
III. The Trigeminal System . . . . . . 164
A. Electrically Recorded Responses. 165
B. Subjective and Reflex Responses 166
1. Origin of the Concept of Pure Olfactory Stimuli . 167
2. Do Pure Olfactory Stimuli Exist? 169
IV. Functional Anatomy. . . . . . . . . . . . . . . . 171
A. Autonomic Efferents . . . . . . . . . . . . . . 171
B. Are there Mesencephalic Nucleus V Fiber Endings in Nasal Mucosa? 172
V. Vomeronasal, Trigeminal and Terminal Nerve Relationships 173
A. The Nervus Terminalis System. 173
B. The Vomeronasal System 174
VI. Summary 176
References . . 177
I. Introduction
The purpose of this chapter is to describe organ systems, other than the ol-
factory, which respond to chemical stimulation of the nose. In human experience
this would be principally the trigeminal system. However, in most common
laboratory animals the organ of Jacobson (vomeronasal organ) is rather well-
developed and must play a role the nature and scope of which are relatively
undetermined. References have been selected to provide a wide range of viewpoint
and good coverage of the literature.
152 D. TUCKER: Nonolfactory Responses from the Nasal Cavity
Fig. 1 (from Fig. 5, F. BOJSEN-MoLLER, Anat. Rec. HiO, 17, by permission of author and
Wistar Institute of Anatomy and Biology). Nasal septum. Four week old rat. PAS (periodic
acid-Schiff) stained whole mount. The apex nasi on the right, choana at the bottom on the left
below the indicated position of the bony lamina terminalis (It). The olfactory region is deline-
ated by Bowman's glands, which are visible as delicate dots arranged in linear patterns between
the fila olfactoria. Notice the trigeminal innervation in the olfactory mucosa. Mucous glands
may be seen collected in one area in the lamina propria of the respiratory mucosa (lower center)
and they all empty into Jacobson's organ. nJ nerves ofthe organ of Jacobson. ro regio olfactoria.
rp rami nasalis posteriores and nervus nasopalatinus. It lamina terminal is. oj organ of Jacobson.
x 10
Fig. 2 (from Fig. 3, F. BOJSEN-MoLLER, J. Anat. 101, 324, by permission of author and
Cambridge University Press). Mucosa of the right side of the nasal septum from a newborn
piglet. OS04 stained whole mount. The anterior nasal glands situated in the respiratory region
are revealed by this stain and they exit by ducts in the vestibule and upper and front parts of
the respiratory region. nJ nerves of the organ of Jacobson. gl glandulae nasales anteriores.
rm rami nasales mediales from nervus ethmoidalis anterior. rp rami nasales posteriores and
nervus nasopalatinus. oj organ of Jacobson. x 3
The Vomeronasal System 153
Jacobson's organ (PARSONS, 1959) and its neural termination in the accessory
olfactory bulb is a system that is clearly part of the olfactory system, proper.
Embryologically, both the olfactory and vomeronasal organs are derived from an
ectodermal placode separate from the neural crest. Although the organ of Jacobson
is absent or rudimentary in the human adult, it is clearly demonstrable during
intrauterine development. An interesting parallel is the complete absence of an
olfactory organ and nerve in the toothed whales. However, it has been recognized
that the so-called anterior cerebral ganglion in the dolphin is derived from the
embryonic olfactory bulb (SINCLAIR, 1966). The baleen whales do have olfactory
nerves to mucous membranes lining olfactory pouches, which hang downward and
have slit-like orifices. It has been said that the Eocene ancestors of whales under-
went a gradual loss of smell and that this loss was accompanied by an excessive
development of the trigeminal apparatus (KELLOGG, 1928).
The trigeminal apparatus is well-developed in the human. The massive fifth
cranial nerve provides afferentation to the facial region and the oral and nasal
cavities. Trigeminal sensory qualities are usually considered to be pain, tempera-
ture, touch and proprioception. The 1st cranial nerve is accepted as subserving the
modality of olfaction, but it has long been recognized that odorants also stimulate
Vth nerve afferents. Such trigeminal responses are usually considered as merely
part of the common chemical sense (PARKER, 1922; MONCRIEFF, 1967). HARPER
et al. (1968) repeatedly emphasized the importance of discrimination between
olfactory and trigeminal components in the description of odorous sensations.
Aside from introspection, it is not evident how this might be done, but this
emphasis is tacit recognition of the role of trigeminal responses in odorous sen-
sations.
Investigation of different animal species leads to considerable problems relative
to anatomical details. However, anatomical esoterica of the nose are of great
practical importance to the experimenter who would study responses that are not
strictly olfactory and are doubtless of importance to one who would study only
olfactory responses.
Figs. 1 and 2 aid in the orientation of the reader to the topography of nasal
nerves and glands. The rat olfactory mucosa extends quite far forward dorsally
and is richly innervated by branches from the maxillary division of the trigeminus
in addition to the numerous bundles of fine olfactory fibers. The vomeronasal
nerve is evident as several strong white bundles extending anteriorly to the stained
Jacobson's glands, which all empty into the organ; a region which is also supplied
with fibers from the maxillary nerve. The preparation of figure 2 illustrates dorsal
innervation from the ophthalmic division of the trigeminus by way of the internal
rami of the ethmoidal nerve, some fibers of which characteristically reach the tip
of the snout. Although not shown, maxillary fibers also extend far forward,
notably, to the region of the nasopalatine duct just behind the incisors. Normally,
in the human, the nasopalatine duct is secondarily obliterated.
II. The Vomeronasal System

Jacobson's organ is present in the majority of amphibians, reptiles and mam-
mals, but there is great species diversity (NEGUS, 1958). It is a tubular organ in the
rabbit, with a small opening on the anteroventral septal wall. In the cat it opens
directly into the nasopalatine canal, which is transmitted through the hard palate
via the incisive foramen. PARSONS (this volume) would restrict the name J aco bson's
organ to those forms in which the vomeronasal epithelium is enclosed in an
outpocketing of the nose. Therefore; in turtles, one would refer simply to the
vomeronasal epithelium, which is freely exposed in the nasal cavity. The accessory
olfactory bulb, wherein occurs the first synapse in the vomeronasal pathway, may
be relatively large in lizards and snakes; it exceeds the main olfactory bulb in the
black snake (CAREY, 1967). Generalizations are unsafe, however. Vis a vis primates,
STEPHAN (1967) measured the volumes of olfactory and accessory olfactory bulbs
and various related structures in 51 species of insectivores and primates and found
that the accessory bulb is well-developed in all prosimians, varies greatly in new-
world monkeys and is absent or very small in old-world monkeys.
A. Electrically Recorded Responses

ALTNER and MULLER (1968) recorded electro-olfactograms (OTTOSON, this
volume) from the olfactory and vomeronasal epithelia in lizards. In the Squamata
the organ opens directly into the mouth, having no connection with the nasal
cavity in adults, and it is likely that the sensory epithelium of the lizard was
exposed by removal of the apposing "mushroom-shaped" body.
ADRIAN (1955) recorded synchronized activity in the vomeronasal nerve of
rabbit in response to abnormal stimulation (pressure applied laterally against the
enveloping Jacobson's cartilage). TUCKER (1963b) recorded neural activity from
vomeronasal nerve twigs in response to inhaled odorants in the gopher tortoise,
but not in the rabbit. However, responses occurred after maneuvers were made to
cause applied substances to be sucked up into the rabbit's organ of Jacobson.
The successful application of conventional recording techniques to olfactory
type nerve requires the use of small nerve bundles, since the unmyelinated fibers are
extremely fine (GASSER, 1956). Discrete action potentials can be distinguished in
the baseline activity of some bundles approximately ten microns in diameter.
During the response to insufflation of benzyl amine vapor as shown in Fig. 3, there
was too much temporal overlapping for units to be discernable. The puff technique
was used to test with many odorous substances, the strength of stimulation being
adjusted by controlling the amount and quickness of squeezing of the polyethylene
bottles containing odors. Simultaneous recording from olfactory and vomeronasal
nerve twigs revealed that some substances are consistently more effective for
olfactory receptors and others are more effective for vomeronasal receptors
(TUCKER, 1961). Graded stimulation frequently reveals one of the receptor systems
responding first to a substance, but at higher intensity the other type responding
more strongly as illustrated in Figs. 4 and 5. Appreciating the problem of stimulus
control with the insufflation method, one may notice in Fig. 6 that the vomeronasal
receptors respond better to the lower molecular weight fatty acids and that the
olfactory receptors respond better to the longer chain acids. Similar results hold
for the aliphatic alcohols (TUCKER, 1963b).
Chemoreceptor responses in the gopher tortoise (Gopherus polyphemus) were
studied by means of an olfactometer with variation of odorant concentration and
Electrically Recorded Responses 155
nasal flow rate (TUCKER, 1963a). For different choices of odorant it does not seem
predictable in which order vomeronasal, olfactory and trigeminal responses will
appear with variation of these important parameters. Vomeronasal responsiveness
l~ ____________ ~ ___________ \~ ____ ~ ____ ~ _________
l~ ____________ ~ __________ ~,~ __________ ~ ____ ~ __
--'------- ---------,~---------~--~--
_______________
O,_lm
__v ~~________________________
...._- - - - -100msec
Fig. 3. Continuous oscillographic record from a small twig of tortoise vomeronasal nerve,
showing discrete or unit nature of baseline activity and the response to a puff of benzyl amine
directed into the naris. Amplifier bandpass was 1.5 to 500 hertz between the half-amplitude
response points. Calibration signal - 0.1 mV. Time marks - 1, 10, 50, and 100 msec
tends to resemble trigeminal more than olfactory, but this is not true for amyl
acetate as comparison of Figs. 7 and 8 will show. The vomeronasal responses are
more phasic in nature, giving rise to "on" and "off" responses. None of the acetate
esters that were tested were potent stimuli for the vomeronasal receptors.
..v
i
o e.
d
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Fig. 4. Graded puff stimulation with ammonia - vomeronasal (above) and olfactory (below)
responses in each pair of traces. Amplifier bandpass was 7 to 500 hertz between half-amplitude
response points. Calibration signals are 50 Il-V and time marks are 0.1 and 1.0 sec
0.1 sec
50~N
Fig. 5. Graded puff stimulation with tert-butyl alcohol - vomeronasal (above) and olfactory
(below) responses in each pair of traces. Amplifier bandpass was 7 to 500 hertz between half-
amplitude response points. Calibration signals are 50 Il- V and time marks are 0.1 and 1.0 sec.
The "waves" in the one vomeronasal record represent an interaction between receptors more
often seen in the olfactory epithelium
v 1,
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Decanoic
0 '"____________ .,.WIjo,L.~_IIf/IN'r~'~ .~,~~',., , 'i ,PT '; Ipdf
Fig, 6, Moderate puff stimulation with fatty acids - vomeronasal (above) and olfactory
(below) responses in each pair of traces, From top to bottom, stimuli are formic, acetic,
propionic, n-butyric, iso-valerie, n-valeric, hexanoic, heptanoic, octanoic, nonanoic and
decanoic acids, Calibration signals are 50 fLV and time marks are 0,1 and 1,0 sec
, O~__~~____~_~__~l~l~'__~~~
~
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_O_____.....:\ _J,_!-_ _ __\.,~I

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o to avoid
damag ing preparation
100 V
----~~
. ,~~
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o 0.1 0.32 1.0 3.2 10 32
Choanal ai r flow rate - MLlsec
Fig. 7. Integrator (fullwave rectifier and lowpass filter) records of tortoise olfactory and
vomeronasal nerve twig responses to amyl acetate. Nasal air flow was on two min and off two
min alternately. During the on periods odorant was introduced 5, 10, and 30 sec, beginning on
each half min. From left to right the air flow was zero (control) and 0.1 to 32 ml(sec in one-half
log steps. From top to bottom, for each pair of traces, the amyl acetats concentration was zero
(control) and 0.1 to 100% of vapor saturation (20 0 C) in one-half log steps
0
0 T
~~
0
'-""-""- L v . - -\__ .. ~.1~ ~ ~
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10
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Stimuli omitted
to avoid
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Choanal air flow rate -MUsec
Fig. 8. Integrator records of tortoise olfactory and trigeminal nerve twig responses to amyl
acetate. Nasal air flow was on two min and off two min alternately. During the on periods
odorant was introduced 5, 10, and 30 sec, beginning on each half min. From left to right the
air flow was zero (control) and 0.1 to 32 mljsec in onehalf log steps. From top to bottom, for
each pair of traces, the amyl acetate concentration was zero (control) and 0.1 to 100% of
vapor saturation (20 C) in one half log steps. The initial phasic components in the trigeminal
0
record at the highest concentration (last trace) were due to large fiber responding which did not
occur at lower concentrations
B. Vomeronasal Epithelium and its Distribution in Turtles

and Mammals
The similarity of the morphology of the vomeronasal and the olfactory sensory
epithelia has long been recognized. Nevertheless, there are some interesting dif-
ferences. READ (1908) for example, was not able to demonstrate terminal hairs
(cilia) on vomeronasal receptors and ALLISON (1953) affirmed that in a rabbit
histological preparation showing olfactory and vomeronasal epithelia in the same
section, only olfactory hairs could be seen. The contention by LE GROS CLARK
(1957) that rabbit vomeronasal receptors do have the terminal hairs is made
explicable by recent electron microscopic revelation of microvilli. Olfactory type
cilia and centrioles are rare in vomeronasal receptors of the slow worm Anguis
tragilis (BANNISTER, 1968), the gopher tortoise Gopherus polyphemus and the box
turtle Terrapene carolina (GRAzIADEI and TUCKER, 1968), while on the other
hand, microvilli of somewhat varying morphology are commonly present. Curiously,
the olfactory mucosal pigmentation attracts much attention (KOLMER, 1927;
ALLISON, 1953; NEGUS, 1958; MONCRIEFF, 1967; MOULTON, this volume), whereas
pigmentation is seldom mentioned in discussion of the "olfactory epithelium" of
the organ of Jacobson. In the live tortoise viewed with the dissecting microscope,
the exposed vomeronasal mucosa is pale gray in comparison with the rich yellow
of the olfactory mucosa. Bowman's glands are characteristic of the olfactory
mucous membrane in all terrestrial vertebrates (KOLMER, 1927; ALLISON, 1953),
but the arrangement of Jacobson's glands and organ in the typical mammalian
form is completely different, with secretion entering the more posterior portions
of the tubular organ from glandular ducts and apparently exiting through the
anteriorly placed pore-like opening. Jacobson's glands may exist collectively as
the medial nasal gland in animals without a Jacobson's organ.
The gopher tortoise has well-developed medial nasal glands housed in promi-
nent lateral projections of the nasal septum, which present the appearance of
baffles to the incurrent air streams. Dissection of fresh material reveals presence of
pigmentation in the common duct which TUCKER (1961, 1963b) took to be indicative
of sensory epithelium. However, in surgically exposed preparations, vomeronasal
nerve response to odorous stimulation was observed at the instant copious flow of
secretion was observed issuing from the pore-like opening. Mapping of electrically
induced responses indicated that the pale gray mucosal area on the anterior
aspect of the baffle, part of which can be seen by looking directly into the naris of
an intact animal, is innervated by at least part of the vomeronasal nerve. The
opening of the medial nasal gland is at the dorsal edge of this patch of sensory
epithelium, which is thus bathed by the issuing secretions. Although the medial
nasal gland is comparatively smaller in the box turtle, SHIBUYA (unpublished)
showed through mapping experiments that the vomeronasal epithelium is much
more extensive than in the gopher tortoise.
It was obvious that the inability to record vomeronasal nerve response to
inhaled odorant in the rabbit was due to the inaccessibility of the sensory epithelium
in the organ of Jacobson (TUCKER, 1963b). However, RODOLFO-MASERA (1943)
described in several mammals what he proposed to call a septal olfactory organ,
which was usually situated near the beginning of the nasopharyngeal passage. It
Stimulus Transport to the Organ 161
was olfactory-like mucosa including the presence of Bowman's glands, but the
sensory fibers joined the dorsally coursing vomeronasal nerve. The organ was best
developed in the guinea pig, could be demonstrated only in the fetal rabbit and
was present in an unidentified species of "Didelphys." In one experiment on the
opossum Didelphys virginiana, SHIBUYA and TUCKER (unpublished) recorded
neural activity from a twig of the vomeronasal nerve located near its termination
in the accessory olfactory bulb in response to each odor inspiration. PLANEL (1953)
mentioned the organ of Masera without citing any references. BROMAN (1920)
figured a "Riechepithel-Insel" in the newborn mouse in correspondence with
RODOLFO-MAsERA'S (1943) description.
The vomeronasal epithelium of turtles is variable (PARSONS, this volume),
when extensive, often appearing on ventral and lateral locations. This caused con-
siderable difficulty in earlier times, for many felt strongly that the organ of Jacob-
son must be medially situated because of its embryonic position in the developing
nasal apparatus. However, SEYDEL (1896) studied land and water dwelling species
and deduced that the vomeronasal epithelium is freely exposed in the nasal cavity
of turtles. VON MnIALKOVICS (1899) rejected this conclusion and identified the duct
of the medial nasal gland as the organ of Jacobson and pointed out its similarity
to the mammalian organ. However, ZUCKERKANDL (1910) examined a specimen of
Emys europaea and could not agree with VON MmALKOVICS that the duct was
lined with sensory epithelium. As recently as 1926 (VON NAVRATIL) the views of
VON MmALKOVICS were defended based on material from Emys europaea and in
1930 (NEMOURS) through a study of Pseudemys elegans. In many turtles a distinct
accessory olfactory bulb is not present, thus contributing to the confusion.
There seems little doubt that the bulk ofthe vomeronasal epithelium is exposed
in the nasal cavity of most, if not all, turtles. If some sensory epithelium were
incorporated in the duct of the medial nasal gland, it would appear to be a rela-
tively minor portion. One should caution about the possibility of individual dif-
ferences in this regard, in addition to the well known problem of species differences.
C. Stimulus Transport to the Organ

The vomeronasal epithelium of Chelonia can obviously receive stimuli in the
same manner as the olfactory epithelium. However, the ventral location of the
former suggests that it would be bathed by water drawn in or expelled through the
nose, while the dorsally situated olfactory chamber would likely contain an air
bubble.
ZUCKERKANDL (1908) reviewed the literature on Jacobson's organ and remarked
that there was little lack of unfounded speculation in comparison with solid
anatomical fact. Much of that speculation was about the function of the organ.
BROMAN (1920) related precisely the essential points of this speculation to introduce
his own research results. He took the position that the organ is obviously olfactory
in function and concluded that it is adapted to smell odors dissolved in glandular
secretions (aqueous solution). The long-hypothesized pumping ability of the
mammalian organ was invoked. HAMLIN (1929) mentioned only the negative
experimental results of BROMAN and demonstrated a pumping effect by injecting
11 Rb. Sensory Physiology, Vol. IV;l
adrenalin into the circulation. He also placed more emphasis upon the presence of
air in the lumen of the organ, although not so overwhelmingly as NEGUS (1958)
indicated.
The anatomical observations that were made by BROMAN (1920) are elegant
(BOJSEN-M0LLER, 1964). In particular, he showed that the lateral nasal gland and
practically all the glands situated in the respiratory part of the nasal mucosa
empty into the front of the nose along with the lacrimal duct and that an excess of
secretions should accumulate in the nasal furrow, or gutter, in which lies Jacobson's
organ. His physiological experiments, however, are not so convincing. The pre-
sence of india ink within the lumen of Jacobson's organ was demonstrated in some
guinea pigs and dogs after instillation within the naris. When not found, this was
taken as evidence of the speed with which the organ could be cleared by flow of
the secretion from its glands, a function which he considered analogous to that of
the glands found at the bottom of the moat of the circumvallate papilla of the
tongue. The pumping mechanism depended strongly on the similarity of the lateral
part of the organ to erectile tissue. The variable relation of the opening of the
organ to the nasopalatine canal prompted the conclusion that odors might come
to it from the nose, from the mouth, or in some instances, from either or both. The
nasopalatine canal is secondarily obliterated in the horse and in the cow the
organ opens onto the palate almost independently of the nasopalatine canal, which
suggested that action of the tongue tip might be important in transferring stimuli
in this instance. In rodents he noted varying degrees of development of palatal
musculature around the soft part of the nasopalatine canal and he remarked on
the innervation by the nasopalatine nerve. In lizards and snakes there was no sign
of a swell body, of course, but he thought that slight displacements of the mush-
room body were possible, thereby changing the volume of the lumen of Jacobson's
organ which opens into the mouth of these forms. In snakes and bifid-tongued
lizards, which flick their tongues in and out, he emphasized his belief that the
tongue is an important accessory organ to the organ of Jacobson, serving to
transfer odorant from outside the mouth. Interestingly, he stressed his impression
that animals with the ability to trail posessed a well-developed Jacobson's organ
and a well-developed olfactory organ.
BAUMANN (1929) performed a variety of experiments on the tracking ability
of the European viper and emphasized the use of its tongue flicking behavior.
KARMANN (1932) showed that snakes actually touched surfaces with their tongue
tips and that surgical removal of the tips disrupted trailing performance. In
lizards and a snake he induced animals to eat, bit or touch with their tongue
objects covered with lamp black and in all cases demonstrated subsequently the
presence of particles in the lumen of the organ. These results were interpreted as
confirmation of BROMAN'S deductions. In two species of snakes closely related to
that studied by KARMANN, NOBLE and CLAUSEN (1936) obtained differing results.
The eyes were covered with blackened tape, the nostrils were filled with cotton
plugs smeared with vaseline and sealed with collodion, the tongue was amputated
caudal to its bifurcation and the Jacobson's organs were cauterized, all in various
combinations. With the tongue and Jacobson's organs intact or with only the
olfactory organs intact, an animal could follow food trials, but better in the latter
instance. A series of similar experiments was performed in a bifid-tongued lizard
Stimulus Transport to the Organ 163
(NOBLE and KUMPF, 1936). These animals performed better when both olfactory
organs and Jacobson's organs were functioning.
WILDE (1938) sectioned the vomeronasal nerves in common garter snakes and
found that they would not feed, which apparently contradicts the results of
NOBLE and CLAUSEN who found that garter snakes with cauterized Jacobson's
organs fed freely. However, WILDE used pieces of scalded earthworms as food and
mucus from such worms for chemical stimulation. He claimed that a garter snake
attempts to swallow its own tail if the tongue touches a drop of such mucous
solution placed on it (WIEDEMANN, 1931, presented photographs of the European
grass snake swallowing a cigar and grasping a human finger, each of which had
been covered with frog mucus). Garter snakes with their olfactory nerves severed
did feed. For either a strike response or seizure of food, contact with either the
tongue or lips was required. Amputation of portions of the tongue indicated that it
was sufficient for stimulating substances to be left near the orifices of the vomero-
nasal organs. BOGERT (1941) showed that removal of the tongue in rattlesnakes of
various species eliminated a stereotyped defensive response posture, normally
elicited by the presence of ophiophagus king snakes or even by sticks that had
previously been rubbed on their backs.
PRATT (1948) has shown that in lizards there is a continuous fluid flow due to
the cilia of the epithelium on the mushroom body and the border of the choanal
fold. He found soot particles in Jacobson's organ of Lacerta vivipara within one-
half minute of their deposition on the choanal fold of a pithed animal in normal
orientation with the lower jaw removed. Actually, he distinguished four degrees of
specialization, culminating in those forms in which the tongue is deeply forked
and probably inserted into the duct of Jacobson's organ, the choanal folds are
absent and the lacrimal duct opens into the duct of Jacobson's organ. The constant
association of the opening of the lacrimal duct and the duct of Jacobson's organ in
Squamata was emphasized by BELLAIRS and BOYD (1950). Snakes might represent
a further degree of specialization. It was noted that the greater vascularity of the
vomeronasal mucosa could make possible significant changes in the volume of the
lumen of Jacobson's organ.
BURGHARDT (1967) has used water extracts from various animals to study the
food acceptance behavior of newly hatched snakes. Interesting comparative dif-
ferences were reported. KUHLENBECK et al. (1967) repeated the suggestion that
the protrusible tentacle of Gymnophiona, the least familiar order of Amphibia, is
used in tracking behavior. It has been observed that some species will follow
objects that have been in contact with earthworms. In lungless plethodontid
salamanders, which frequently touch surfaces with their nose, fluids apparently
are drawn up the nasolabial grooves and over the vomeronasal epithelium by the
action of intranasal cilia (BROWN, 1968).
These bits of evidence suggest the possibility that the Jacobson's organ might
perform the function of detecting chemical compounds which are not sufficiently
volatile to be detected readily with the olfactory organ. Such conjecturing does
not necessarily apply to all species, of course. Let us examine an enigmatic finding
made by BENJAMIN (1960). Using a preference conditioning technique with rats,
bilateral ablation of cortical taste area and coagulation of thalamic taste nucleus
caused transient or permanent impairment of quinine discrimination. With the
11*
use of shock avoidance conditioning technique, temporary loss occurred with the
combined lesions followed by a return to normal thresholds. Then, however,
removal of olfactory bulbs produced a marked and permanent increase in "taste"
thresholds. This experiment shows that animals can be forced to reveal sensory
capabilities not demonstrated initially. The quinine solutions may well have con-
tained odorous contaminants and thus have been smelled with the aid of the
olfactory organ. However, the bulbar removal also included the accessory olfactory
bulb and one has BROMAN'S suggestion of "mouth smelling." There is no obvious
reason why quinine might not stimulate the vomeronasal receptors. Furthermore,
there is evidence that the attractive principle for snakes in earthworm mucus is
nonvolatile, being heterogeneous macromolecules in excess of 5,000 molecular
weight (SHEFFIELD et al., 1968).
Some electrically recorded responses have been obtained in the anesthetized ra b-
bit (TUCKER, 1963b), but the hypothesized accessory mechanisms exploited by the
interested animal for transporting stimuli to the organ were nonfunctional. Those
results do not support the deduction of MILSTEIN (1929) that the rabbit's organ of
Jacobson responds on each inspiration and is the most peripheral receptor of
olfactory substances. Surgical access was gained through extraction of the upper
incisors. Plugging of the openings to the organs delayed the "olfactory reflex" to
citral for two or three inspirations. The "trigeminal reflex" to toluene was elicited
immediately, but the pneumogram indicated considerable breathing during
application of the nose mask, unlike during the control records. The tongue
flicking of snakes is abolished by minimal doses of anesthetics. The commoner
lizards would appear to be much more promising for neural recording, as a ciliary
transport mechanism would likely be much more resistant to immobilzing pro-
cedures.
III. The Trigeminal System

The trigeminal nerve is comparatively large in most animals and may primi-
tively have represented three segmental divisions. The mandibular, maxillary and
ophthalmic divisions correspond to the assumed first, second and third segments.
The ophthalmic contribution to the nasal mucosa is the most accessible, surgically.
In common laboratory mammals the ethmoidal nerve (ophthalmic division) is
transmitted through one or more of the dorsal-most foramina in the cribriform
plate; it is given off in the orbit as the major branch of the nasociliary nerve,
enters the cranial cavity through the ethmoidal foramen and courses over the
olfactory bulb embedded in the dura mater. The maxillary division's posterior
nasal rami and nasopalatine nerve, branches of the sphenopalatine, are much more
difficult to get at. However, a lateral approach might be feasible in some animals
for the laterally situated innervation. Photographs of excellent dissections
illustrating nasal innervation in dog, cat and man are to be found in the contribu-
tion by READ (1908). Comparative studies of lizards and snakes (MALAN, 1946;
PRATT, 1948; BELLAIRS and BOYD, 1950) are helpful for the nasal trigeminal
innervation of these forms and MATTHES (1934) treats representatives of all ver-
tebrates.
A. Electrically Recorded Responses

A transorbital approach was used by BUT and KLIMOVA-CHERKASOVA (1967)
in the cat. Bursts of action potentials were recorded from the ethmoidal nerve and
a branch of the maxillary nerve in correlation with the breathing rhythm. Oc-
clusion of the opposite naris increased the discharges and occlusion of the side
recorded from caused a low frequency of discharge not correlated with breathing.
An increase of response occurred when wadding soaked with ammonia or ether
was brought to the nares, although for ether, this was observed only with the
ethmoidal nerve. Ethmoidal nerve recording in the rabbit yielded similar results
(TUCKER, 1963b). However, prominent spikes from mechanoreceptors were very
much in evidence during manipulation of the tip of the nose. Responses to chemical
stimuli of concentrations considerably below vapor saturation appeared to be
mediated by much smaller, slowly conducting afferent fibers. The lowest velocities
determined experimentally were below 1 m/sec.
The first report of responses recorded from olfactory nerve with natural
stimulation (BEIDLER and TUCKER, 1955) was somewhat ambiguous concerning
the fact that both olfactory and trigeminal responses were illustrated. Records in
which individual spikes are clearly distinguishable were from ethmoidal nerve
higs. In vitro preparations of nasal mucosa of the opossum and the rabbit were
investigated. Many fine bundles of myelinated nerve fibers are visible to the
unaided eye in the periosteal side of the opossum mucosa, but in the rabbit they
are obscured by copious white connective tissue. Trigeminal responsiveness per-
sisted for a few hours, but olfactory nerve twigs were generally unresponsive.
However, an anterior septal region in the rabbit is quite thin and the olfactory
mucosa makes a gradual transition to the respiratory type in this region. Olfactory
twigs obtained from this part of the rabbit in vitro preparation were responsive to
odors. Although trigeminal units in this preparation exhibited various patterns of
responsiveness to different odorants, including those of coffee and asafetida (BEID-
LER, 1965), it was deemed preferable to study in vivo preparations.
Many odorants give rise to neural responses in freely respiring anaesthetized
rabbits. The trigeminal threshold concentration for phenylethyl alcohol is usually
lower than the olfactory threshold. Amyl acetate showed the greatest difference
between olfactory and trigeminal threshold concentrations, the upper limits for
each being roughly 10-3 and 10-1 of vapor saturation at 20 C, respectively
(TUCKER, 1963b). However, responses commonly are highly dependent upon the
depth and quickness of inspiration.
A systematic study of chemoreceptor responsiveness in the gopher tortoise
(Gopherus polyphemus) revealed that differences in complexity of nasal geometry
playa strong role in the nasal flow rate dependence of responses (TUCKER, 1963a,b).
Respiratory movements are infrequent in this animal while under anaesthesia,
therefore the importance of flow rate was studied by cannulating the choana and
drawing air through the nasal cavity (one side only) at various flow rates. With
amyl acetate there was a range of flow rate in which the neural responses did not
increment further. For these conditions, olfactory and trigeminal responses were
consistently observed at levels somewhat below lO-5 and lO-l of vapor saturation,
respectively. With benzyl amine the flow rate dependence of neural responses was
166 D. TUCKER: Nonolactory Responses from the NasaI Cavity
much stronger, and at all concentrations the trigeminal response appeared before
the olfactory response. The trigeminal response concentration range was much
greater than with amyl acetate (Fig. 8) and a benzyl amine threshold could not be
readily determined because of the lack of a sharp approach towards zero response
level. Olfactory responses were always small to butyric acid and were depressed
below the resting or background level at higher concentrations. On the other hand,
trigeminal responses to butyric acid were much more vigorous. These responses
were all obtained with multifiber twig preparations with the responses being
quantified by the integrator, or moving-average, technique as shown in Fig. 8_ It
would be interesting to study the coding of trigeminal unit responses in vivo.
Chemical responsiveness of trigeminal corneal innervation by the ciliary nerve
was studied by DAWSON (1962) in the bullfrog. The afferent fibers which responded
to touch were larger than those which responded to aqueous solutions of amyl
acetate or phenylethyl alcohol. On the basis that the aqueous solubility limit of
14 ruM for amyl acetate corresponds to vapor saturation, the reported amyl
acetate ocular threshold concentration for the bullfrog approximates that for
nasal trigeminal nerve endings in tortoise and rabbit.
B. Subjective and Reflex Responses

An opinion common among experimental psychologists is that phenylethyl
alcohol, coffee and asafetida are pure olfactory stimuli. Also, it seems to be com-
monly held that trigeminal and olfactory components of sensation can be separated
with the use of psychological methods. The bases for such opinions have several
sources in the literature. PARKER'S (1922) book is often cited for the comparison of
olfaction, gustation and common chemical sense. An early study of reactions of the
restrained dogfish (SHELDON, 1909) showed that essential oils in aqueous solution
applied to the nostril provoked the same behavioral response as acid, salty and
bitter substances. Section of the olfactory crura did not abolish the response,
which was shown to be mediated by the maxillary nerve. MONCRIEFF'S (1967) book
stresses the noxious character of trigeminal stimulation. However, electro-
physiological results suggest otherwise at lower concentrations, and investigations
of human subjective responses are not in agreement. Amyl acetate below trigeminal
threshold concentration is useful for achieving only olfactory stimulation, e.g., in
behavioral study of the pigeon (HENTON et al., 1969).
MONCRIEFF (1955) described a method for comparing the relative thresholds of
olfactory, nasal trigeminal and ocular irritations in man. The subjectivity of the
discrimination between the olfactory and trigeminal irritations, as named in this
instance, is apparent. The results are summarized in Table l.
Table 1. MONCRIEFF'S ordering of threshold stimulating effectiveness of irritants
Substance Increasing sensitivity
Acetic anhydride trig. olf. eye General tendency

Formaldehyde trig. olf. eye for critical time
Ammonia all equal of contact of
Triethyl amine trig. eye olf. odorized air with
adsorbent to increase
Subjective and Reflex Responses 167
Using different methods, KATz and TALBERT (1930) found the order for allyl
alcohol to be ocular irritation, nasal irritation and odor, in terms of concentrations
which were not greatly different. The nasal irritation appeared several seconds
after a single inspiration (which is a startling and painful experience at high con-
centration). They found ocular but not nasal irritation for iso-amyl acetate,
although it is difficult to imagine that no nasal trigeminal afferents responded or
would not have if a 10 second exposure had been used as for the eyes. Under their
tabulation of physiological effects they noted that it produced tickling in the
throat and coughing in higher concentrations. A revealing experience is the inha-
lation of aerosols of odorants which in the vaporous state produce only mild and
pleasant sensations, e.g., phenylethyl alcohol.
Much early work on nasal reflexes was described by VON SKRAMLIK (1925a) in
the Handbuch der normalen und pathologischen Physiologie. Three kinds of responses
from organs situated in the nose are possible; motor, secretory and vasomotor.
More extensive reflex responses are respiratory, cardiac, vascular and secretory in
nature. The relative importance of olfactory and trigeminal innervation in eliciting
these reflex responses was a subject of active investigation. The so-called diving
reflex, still studied today, could be elicited with many different kinds of stimuli.
The trigeminus seemed to be considerably the more effective with odorants.
However, electrical stimulation of the olfactory bulb changed respiratory rhythm
as well as did presentation of odorants so dilute that it was considered stimulation
of the trigeminus could not have taken place!
1. Origin of the Concept of Pure Olfactory Stimuli

ALLEN (1928) wished to find a volatile substance which would stimulate the
olfactory nerve alone, so that the reflex pathways could be traced without the
necessity of having to section the trigeminal and possibly other cranial nerves in
each experiment. He defined an olfactory animal as one in which the trigeminal
nerves to the nasal region have been cut and the vagus and other nerves supplying
the lower respiratory areas are not functioning. However, this term was used
subsequently without close adherence to the definition. He showed that in the
rabbit no variation occurred in respiration or circulation during inhalation of very
strong ammonia vapor when the olfactory bulbs were isolated from the brain by
section, the nasociliary and maxillary nerves were cut in the orbit, and the vagi,
depressors and cervical sympathetics were sectioned at the level of the larynx.
One maxillary nerve functioning in isolation was much more effective than one
nasociliary nerve in affecting respiratory rate, pulse rate and blood pressure. He
concluded that sectioning the nasociliary and maxillary division of the trigemini
was as effective as severance of both trigeminal roots intracranially in producing
the so-called olfactory animal.
ALLEN (1929) studied the effect of inhaled vapors on respiration and blood
pressure in normal human subjects and in one who had been rendered anosmic by
an extensive skull fracture. According to the X-ray examination, fracture lines
extended into the vault of the pharynx and to the sphenoid and ethmoid sinuses.
The anosmic could recognize some substances through sensations referred to his
nose and there were other substances which caused changes in his respiration
although he was unaware of their presentation. The changes in blood pressure
during inhalations followed very closely those of respiration. The results from
normal subjects were most consistent for strong irritants. The following quotation
conveys more information than would a summary of details.
"The inhibitory respiratory reaction obtained from inhalants in human subjects
under anaesthesia and in some records from non-anaesthetized subjects is apparently
the olfactory-trigeminal respiratory reflex from inhalants in man. This is identical
to the olfactory-trigeminal reflex in rabbits. Different respiratory changes some-
times occurring in unanaesthetized subjects may come from psychical stimulation."
A species comparison among rat, guinea pig, rabbit, dog and man was made
with nerve-intact animals, some in which the nasal trigeminal innervation was
interrupted and others in which the olfactory bulbs were isolated from the brain
(ALLEN, 1936). Considerable species differences were noted, with dog being most
like man, for whom pure olfactory odors were lavender, anise, cloves, asafetida,
butyric acid, wintergreen and xylol (xylene). ALLEN reported that there are appar-
ently few, if any, vapors which affect respiration solely over the trigeminal nerves
in any mammal. In the rabbit without nasal trigeminal innervation, xylol and
benzol (i.e., xylene and benzene) produced powerful effects on respiration, usually
arresting it for a while and often eliciting cries! It was stated that:
"strong irritants were generally insufflated and stimulations were limited to
the olfactory or trigeminal nerves by severing one or the other and inserting a
tracheal cannula towards the lungs and a laryngeal cannula into the pharynx."
ALLEN (1937) studied olfactory and trigeminal conditioned reflexes in dogs in
which the nasociliary and maxillary nerves were cut in orbit and olfactory bulbs
transected through frontal sinus exposures, respectively. For some of the odorants,
a trigeminal conditioned reflex could not be established, whereas all were effective
over the olfactory nerve. Practically all of the vapors which altered respiration
over the olfactory or trigeminal nerves were shown to be capable of producing a
conditioned reflex from stimuli received over the same nerves. Various controls
were introduced, e.g., some dogs were blinded. One interesting observation was
that a blinded dog with a trigeminectomized snout was greatly handicapped over
a simply blinded dog in ascending a flight of stairs. Also, such a dog experienced
great difficulty in eating soft food from a pan.
"One dog gradually reduced the length of his snout by one half through
pawing food from his nostrils."
A series of thirteen papers on olfaction by ELSBERG and various collaborators
are all to be found in Vol. 4 of The Bulletin of the Neurological Institute of New
York. ELSBERG et al. (1935a) varied the volume and pressure of the gas phase in
their well-known blast injection apparatus. They very nearly deduced the import-
ance of nasal flow rate, but concluded by emphasizing their instrumental variable
of pressure in the test bottle. ELSBERG et al. (1935b) introduced the stream injec-
tion test to study the trigeminal effects of odorous substances, because the blast
injection method did not yield results which differed materially from those obtained
with nasal inhalation or sniffing. Most of the tests were made on specially trained
individuals. Odorous air was streamed through the nose of the subject who
breathed regularly from the mouth, indicating the time of occurrance of sensations
such as itching, stinging, burning, warmth, coolness or pain, when lacrimation
occurred, the side affected and who also estimated intensity on an arbitary rating
scale. The quality of sensation was often characteristic of the substance used. The
Subjective and Reflex Responses 169
pain caused by benzyaldehyde differed from that produced by xylol (xylene).

Tests were made on individuals with complete anosmia and they were found able
to distinguish between trigeminal sensations produced by different odors. Nasal
flow rate was varied with the result that trigeminal effects increased with increase
of the streaming rate. The only substances examined that apparently did not
affect the trigeminus when injected into the nose at the rate of 10 l/min were
coffee, musk ketone and phenylethyl alcohol. They called these substances pure
olfactory stimulants. Marked variations in the degree of stinging or painful sen-
sation occurred during the period of stream injection of other odorants.
2. Do Pure Olfactory Stimuli Exist 1

Electrical recording evidence cited above indicates that there are distinctly
different olfactory and trigeminal threshold concentrations for some compounds.
However, trigeminal responses to coffee and phenylethyl alcohol observed in
rabbit preparations cast doubt on the conclusion that the human olfactory nerve
mediates all subjective sensation to these compounds, especially for all extremes of
stimulating conditions.
The dependence of neural responses on nasal flow rate is illustrated in Fig. 8
for amyl acetate and much more marked dependence was found for butyric acid
and benzyl amine (TUCKER, 1963a, b). Notice that the trigeminal response has a
long build-up time in comparison with the phasic-tonic nature of olfactory
response. In freely respiring anaesthetized rabbits trigeminal responses often waxed
and waned during prolonged administration of strong odorants. The waning
sometimes appeared to correlate with the appearance of copious secretion.
ALLEN (1928, 1929, 1936, 1937) did an impressive amount of work on natural
and conditioned reflexes, aimed at resolving the roles of olfactory and trigeminal
afferents in responding to odorants. The importance of concentration was obviously
not appreciated and consequently the methods of stimulation were not controlled
adequately. For example, a cotton ball soaked with so many drops and held a
certain number of centimeters in front of the nose results in highly variable
stimulation. Classification of a group of compounds as being purely olfactory
stimulants, but with the assertion that some have a greater arousal effect than
others, seems forced. The writer has recorded from a small filament of the ethmoidal
nerve in rabbits which were otherwise intact and under urethane anaesthesia; pre-
sentation of concentrated xylene (xylol) caused massive trigeminal responses and
frequently crying of the animal. Thus, although ALLEN (1936) claimed that crying
occurred in rabbits without nasal trigeminal innervation, it is clear that xylol is
not a pure olfactory odor. The claim that there is much species variation, with dog
being most like man and rat most unlike, needs validation. Nevertheless, without
recourse to electrophysiology, ALLEN'S general methods would appear to form a
logical approach.
With electromyography, it was found easier to inhibit the spontaneously
firing motor units of leg muscles in the rabbit than it was to influence respiration
or pulse rate (ANDERSON, 1954). Ether was ineffective after section oithe trigeminal
nerves intracranially. Coffee and asafetida were effective in intact animals.
Mechanical stimulation of skin of the face and ear also strongly inhibited spon-
taneously firing motor units in the rabbits, which were lightly anaesthetized with
170 D. TUCKER: NonoIactory Responses from the Nasal Cavity
urethane. By contrast, electromyographic responses (positive, not inhibitory) were

obtained from arm and leg muscles in human infants without any visible move-
ment in response to weak electrical stimuli on the cutaneous receptive field of
"nerve trigeminus parts II and III" (HoPF et al., 1964). Stimulation of other
regions was ineffective unless the stimulus was very strong.
An approach that avoids the deterioration of nerve-sectioned animals (ALLEN,
1937) was the implantation of cannulae to permit reversible blocking with the
injection of xylocaine into the trigeminal sensory ganglia (STONE et al., 1966).
During the blocked state there were no changes in respiration or heart rate to the
odorants presented and only in some of the records can one discern a change in
cortical EEG or olfactory bulb activity. Most striking, however, were the changes
in baseline activity. The respiratory frequency became very much slower and the
EEG had the appearance of high amplitude slow wave activity often associated
with the state of deep sleep. Later, STONE et al. (1968) concluded that the response
of rabbits to odorants is an "olfactory-trigeminal" response with the trigeminus
exerting an influence through contrifugal pathways to the olfactory bulb.
Curiously, those working with rabbits have often failed to mention active
sniffing movements, which occasionally occur even under anaesthesia. WELKER
(1964) noted that in normal rats sniffing occurred in bursts of 5 to 11 sniffs per
second and that this behavior persisted unchanged after bilateral ablation of the
olfactory bulbs. However, unlike controls, the bulb ablated animals did not react
aversively to xylol-soaked cotton balls. Sniffing sounds have been monitored
electronically to study responses of rats to odorants (TEICHNER et al., 1967).
Sniffing is a means of increasing the inspiratory nasal flow rate. Flow rate was
found to be a first order variable for trigeminal, olfactory and vomeronasal
responses in experiments of the "stream injection" type on tortoises in which
odorant was caused to appear without altering the nasal flow rate (TUCKER, 1963a).
Such dependence was especially striking for benzyl amine, with the trigeminal
response always appearing before the olfactory response and at increasingly lower
flow rates with increase of odorant concentration. However, at the highest flow
rate and concentration used, the olfactory response to benzyl amine was greater
than that observed for any other odorant. While it was practical to estimate a
trigeminal threshold concentration for amyl acetate (Fig. 8), this simply represents
a particularly favorable combination of odorant and sensory nerve. The dream of
finding an odorant that is purely olfactory in its stimulating capabilities is still
unrealized.
VON SKRAMLIK (1925b) listed about fifty compounds as olfactory stimuli on the
basis of inability of subjects to localize which naris received the stimulation.
However, he had many reservations and described various sensations accompany-
ing the olfactory sensation. For example, in many subjects phenylethyl alcohol
caused a very weak stinging and citronellol was distinctly harsh. For the con-
siderably larger number of odorants which could be localized, a variety of ac-
companying sensations was described and also the places to which they were
projected. These were thought to be the cues utilized in localization.
VON BiKESY (1964) found that human subjects could localize fairly accurately
the angular direction of odorous sources in analogy to directional hearing. How-
ever, it has been suggested that his results were not due solely to "pure" olfactory
Autonomic Efferents 171
information and are actually evidence that man has considerable ability to
localize the source of trigeminal stimulation (SCHNEIDER and SCHMIDT, 1967).
IV. Functional Anatomy

The oHactory organ occupies a posterodorsally situated diverticulum of the
nasal cavity in many forms of vertebrates above the amphibians. The rest of the
nasal cavity forms the respiratory passageway, excepting sinuses, which may be
complicated with elaborate structures to condition the inspired air. Most of the
interior nasal trigeminal innervation seems to go to the respiratory mucosa.
although in the traversal of oHactory fields some trigeminal fibers apparently
terminate there. The whole of the nasal mucosa may profitably be regarded as a
chemoreceptive organ, in which the respiratory and oHactory parts have quite
different, but overlapping response properties. Attempts to localize where smell
sensations are projected to, VON SKRAMLIK (1925b), can reveal a considerable
subjective space in the nose.
A. Autonomic Efferents
The nasal cavity is not a static structure. Its dimensions can be varied princi-
pally by autonomic mechanisms and this results in variable access to receptive
structures, particularly the oHactory organ (TuCKER, 1963a, b). The resulting
modulation of oHactory response, for the same stimulus at the level of the naris,
seems to be caused by trigeminal response to the same odorant. The motor struc-
tures are vascular and perhaps other smooth muscle in the instance of the tortoise.
The extent of nervous control of the various types of nasal glands (BOJSEN -M0LLER,
1964, 1967) is unknown.
Sympathetic and parasympathetic efferents are distributed with many of the
peripheral branches of the trigeminus. MALCOMSON (1959) demonstrated for the
first time, so far as he was aware, vasomotor and secretomotor activity of the
ethmoturbinal region in the cat's nose. He implicated the ciliary ganglion for the
parasympathetics. Conditions may be different in the rabbit, however, for TUCKER
and BEIDLER (1956) found that sectioning of the cervical sympathetic and seventh
cranial nerves in that animal abolished all of the efferent activity in the ethmoidal
nerve. Responses to electrical stimulation of the greater superficial petrosal nerve
(TuCKER, 1963b) would implicate the sphenopalatine ganglion. Section of the
vidian nerve did not abolish all sympathetic activity in the ethmoidal nerve bundle.
The amount of sympathetic activity, in particular, often followed reflexly the
afferent trigeminal responses to odorants. Presentation of amyl acetate below
trigeminal threshold and above oHactory threshold often caused a momentary
reduction in the level of sympathetic activity. Parasympathetic activity was
inhibited with increasing depth of urethane anaesthesia and sympathetic activity
increased dramatically, often with a large component of synchronization with
respiration. This was found to be caused reflexly by a mucous rattle in the naso-
pharynx.
B. Are there Mesencephalic Nucleus V Fiber Endings

in Nasal Mucosa?
In view of the finding presented earlier that ethmoidal afferents responding to
odorants seemed to be of small caliber, it is interesting that WINDLE (1926) found
that the nasociliary nerve in the cat had an unusually high percentage of small
myelinated and unmyelinated nerve fibers relative to other branches of V. In con-
junction with this result, CORBIN (1940) unexpectedly found degenerating fibers
in the ethmoidal nerve following lesions of the mesencephalic nucleus of V.
They were smaller than those seen in maxillary and mandibular nerve branches. In
both of these studies control experiments were performed to remove sympathetic
fibers. There is a large body of literature on the mesencephalic root and nucleus of
the trigeminal nerve, the cells of which are generally recognized as being sensory
in nature. These atypically situated sensory cells serve the function of propriocep-
tion of oral structures and often they have been suspected of the same role for the
extrinsic eye muscles.
CORBIN (1940) related his major anatomical finding to PFAFFMANN'S (1939)
recording of action potentials from the superior alveolar nerves in response to blunt
pressure applied to the teeth and gums and postulated that mesencephalic root
fibers mediated masticatory reflexes. CORBIN and HARRISON (1940) found that
magnificant responses could be recorded from a 22 gauge wire electrode inserted
into the nucleus upon depression of the cat's lower jaw. Stretch of the extrinsic
ocular muscles through rotation of the eyeball failed to produce potentials in any
part of the mesencephalic root, thus dealing a blow to the school of thought that
the mesencephalic nucleus subserved ocular proprioception. With the mandibular
nerve cut to eliminate jaw closing reflexes, blunt pressure stimulation of the teeth
and hard palate was effective, but no stimuli could be found that were effective
for the ethmoidal nerve field. It is interesting to speculate that the smaller ethmoidal
fibers may be the chemosensitive fibers. SHEININ (1930) studied mesencephalic
nucleus cell types in the dog. He concluded that his large type A cells were pro-
prioceptors, by analogy with the dorsal root ganglia cells that innervate muscle
spindles, but he was clearly uncertain about the other large class of cells, which were
mostly small. However, PEARSON'S (1949) opinion was that the frequent com-
parison of mesencephalic V cells to peripheral sensory ganglia neurons had a
misleading influence.
Much of the confusion evidently stems from species differences, which seem to
be considerable. Muscle spindles have been found in the eye muscles of man and
chimpanzee, but not in the monkey. However, they are much more numerous in
the cloven-hoofed ungulates (WHITTERIDGE, 1960). On the other hand, recogniz-
able muscle spindles have not been found in the tongue of the cat nor in the lateral
pterygoid muscles of the cat and monkey (SMITH and MARCARIAN, 1968), but they
are present in the tongue of the rhesus monkey (BOWMAN and COMBES, 1968).
MANNI et al. (1968) found a cellular pool in the trigeminal sensory ganglion con-
taining soma of the afferent fibers from spindles of extraocular muscles in pig and
sheep, however they were unable to get similar responses from the cat. They con-
cluded that responses recorded earlier from the trigeminal mesencephalic nucleus
and attributed to the primary cell bodies of proprioceptive fibers of extraocular
The Nervus Terminalis System 173
muscles (WHITTERIDGE, 1960) were really from second or third order neurons.
Proprioceptors in the masticatory muscles of cat are probably represented by large
cells in the mesencephalic V nucleus and responses from these, as well as from
tooth and tongue have been mapped, but it remains to be shown that the tooth
and tongue responses are evoked from soma of primary afferents (SMITH and
MARCARIAN, 1968).
N one of these studies indicate the functional modality of the mesencephalic V
fibers in the ethmoidal nerve. However, there seem to be no recent studies which
would support or discount their existence. The trigeminal system is relatively
large and complex. It is quite important in the behavior of higher vertebrates, yet
much of its afferent responding appears not to reach the level of conscious sen-
sation.
V. Vomeronasal, Trigeminal
and Terminal Nerve Relationships
Those who have had a course in comparative anatomy are likely to recall
demonstration of the nervus terminalis in the shark by gross dissection. This nerve
to the nasal capsule appears to be quite distinct from the olfactory nerve and to
persist in the evolutionary sequence of vertebrates. Ontogenetic and phylogenetic
observations of the nerves to the nose and hypotheses about function are collected
here.
A. The Nervus Terminalis System

MCCOTTER (1913) succeeded in demonstrating the nervus terminalis in the
adult dog and cat, but not in opossum, rat, rabbit, guinea pig and sheep. JOHNSTON
(1913) tabulated a number of findings obtained from the study of adult fishes and
amphibians and embryonic reptiles and mammals. He proposed that the close
association of the nervus terminalis in amphibians, reptiles and mammals with the
vomeronasal organ suggests an influence of the terminalis on the differentiation of
Jacobson's organ in the fish-like ancestors.
The nervus terminalis is poorly understood. Histological studies seem to
indicate the presence of both sensory and motor components. PEARSON (1941)
found it difficult to follow the central roots of the vomeronasal nerve in a number
of human embryos which he studied and thus easy to understand the failure of
some earlier workers to recognize the presence of two nerves in the vomeronasal
and terminalis nerve complex. The ganglion terminale was formed by migration of
cells and fibers from the olfactory placode in the region of the vomeronasal organ
anlage and there was further evidence of cellular migration into it from the fore-
brain. The roots of the nervus terminalis penetrated the ventromedial surface of
the forebrain caudal to the attachment of the olfactory bulb. Peripherally, it
extended to the vomeronasal organ and the field of the anterior ethmoidal nerve.
Cells also migrated from the vomeronasal organ and were arranged in groups
around the nerve fibers supplying it, i.e., those of the vomeronasal nerve, terminal
nerve and the nasopalatine branch of the trigeminal nerve. Quite similar results
were obtained for the nasal distribution of the nervus terminalis in the rabbit by
174 D. TUCKER: NonoHactory Responses from the Nasal Cavity
HUBER and GUILD (1913) and in the cat and other mammals by LARSELL (1918).
The latter thought there was evidence for free nerve terminations in the epithelium
of the septal mucosa and of Jacobson's organ belonging to afferent neurons of the
nervus terminalis. However, READ (1908) was unable to find free nerve termi-
nations in the cat's vomeronasal organ, although she demonstrated them in both
olfactory and respiratory portions of the nasal mucosa.
LARSELL (1950) cautioned that both in the peripheral plexus and intra-
cranially there is the possibility of confusion with fibers from other sources.
However, he reiterated that the fibers of the nervus terminalis appear to stain
differentially with the pyridine silver method. Bipolar ganglion cells and small
multipolar neurons of autonomic type were recognized. Sensory roots of the nervus
terminalis were claimed to be in the septal nuclei, olfactory lobe and the posterior
precommissural region. The supraoptic region was suggested as the source of pre-
ganglionic fibers to the autonomic component. The postganglionic fibers were said
to innervate Bowman's glands and, apparently, blood vessels of the olfactory
mucosa. Afferent nerve endings were found in the olfactory epithelium and they
differed in appearance from endings of the presumed trigeminal nerve in the
respiratory mucosa. KOLMER (1927) has a famous drawing illustrating trigeminal
nerve terminations within the olfactory epithelium. However, the writer knows of
no supporting evidence from electron microscopy for the existence of such termi-
nations. CAUNA et al. (1969) recently demonstrated unmyelinated nerve terminals
in human respiratory mucosa between ciliated epithelial cells and within the
lamina propria, i.e., on both sides of the basement membrane.
It would be of much interest to have electrical recording experiments in
forms in which the nervus terminalis can be suitably demonstrated. There is
always some question as to what one may be picking up in bundles of nerve
identified as vomeronasal, trigeminal or even olfactory. A case in point would be
the organ of Masera, in which olfactory-type mucosa containing Bowman's glands
apparently sends axons into the vomeronasal nerve.
B. The Vomeronasal System

MCCOTTER (1912) made generally known the connection between the mam-
malian olfactory bulb and the organ of Jacobson. His drawings graphically
illustrate the fact that in some common mammals one can obtain two or three
inch lengths of olfactory-type nerve. The unusual course was illustrated of the
vomeronasal nerve of the guinea pig under the olfactory bulb to make a ventro-
lateral approach to the accessory olfactory bulb. The connection between J aco bson's
organ and a definite part of the olfactory bulb was already well known in other
forms, asZUCKERKANDL'S (1910) references to the area vomeronasalis and olfactory
fossa of the olfactory bulb in squamates and urodeles indicate. He was concerned
with why the nerve makes a lateral approach in amphibians and a medial approach
in the other forms. One must wonder at how differently he might have been in-
fluenced had he used a rat or rabbit instead of guinea pig embryo, in which he saw
"RiechnervenanJagen" extending from the medial wall of the nasal pit towards the
lateral wall of the olfactory bulb. MCCOTTER (1919) took up the question in the
turtle Chrysemys punctata and the bullrog Rana catesbeiana and presented very
The Vomeronasal System 175
iniormative illustrations of wax plate reconstructions (In MCCOTTER'S publication

some figure legends were interchanged).
Bundles of myelinated fibers, presumably trigeminal (e.g., KERKHOFF, 1924),
are intimately associated with bundles of "olfactory" fibers distributed to Jacob-
son's organ. Little has been made of their function.
As was discussed earlier, BROMAN (1920) felt he had demonstrated that the
Jacobson's organ epithelium of terrestrial vertebrates was adapted to function in
the watery fluid medium of nasal secretions and he concluded that the organ is
simply the old water smelling organ of vertebrates after adaptation to a land life.
Those who claim that his conclusion is vitiated by the presence of a secretion from
Bowman's glands bathing the olfactory organ must grant that all mucous mem-
branes are moistened.
HERRICK (1921) dismissed BROMAN'S conclusion rather scathingly and, as it
turned out, was championing the much older hypothesis that the vomeronasal
apparatus was differentiated in connection with the opening of the posterior nasal
aperture and the consequent passage of olfactory media from the mouth cavity
into the nasal sac. The earliest enunciation of this concept at length was probably
by SEYDEL (1895). He pointed out that Bowman's glands are associated with the
regio olfactoria from amphibians onward, whether they are water or air breathing
animals. Likewise, in both instances, the prominent association of accessory glands
with the vomeronasal organ was emphasized. The layers of secretion over the two
kinds of olfactory epithelium were thought to perform similar functions; protection
against irritating particles from air or water and an intimate medium for reception
of odorants. Stimuli were carried into the vomeronasal organ by force of the
expiratory stream. Increasing specialization was noted in the amphibians, ulti-
mately to result in loss of the functional connection to the choana of higher forms.
The hypothesis of SEYDEL (1895) was supported and updated by BRUNER
(1914). HERRICK (1921) saw a parallel increase in specialization of the forebrain in
the amphibian series, culminating in a specific nerve and accessory olfactory bulb
with a fiber tract projecting to the anuran amygdala. These relations suggested that
the mediation of olfactory, gustatory and perhaps other excitations arising from
within the mouth was the original integrating physiological factor leading to
formation of the amygdala, but the amygdalar complex of higher forms persists in
the absence of the vomeronasal organ (man) or the entire olfactory organ (dolphin,
which retains the nervus terminalis). A brief discussion of more recent work on
comparative neurology of the amygdala is by SCHNITZLEIN (1967). SCALIA et al.
(1968) observed degeneration in amygdalar regions on both sides after unilateral
lesioning of the olfactory bulb in the frog, but they were unable to verify presence
of an efferent projection from the accessory bulb as described by HERRICK.
Although there seems to be a general opinion that no fish has a demonstrable
vomeronasal organ and nerve (BERTMAR, 1965), references have been made to an
accessory olfactory bulb in the African lungfish Protopterus annectens (RUDEBECK,
1945; SCHNITZLEIN, 1967) and in the paddlefish Polyodon spnthula (STORY, 1964).
In such instances an accessory olfactory bulb is recognized on the basis of segrega-
tion of morphological elements, which are typically not so well organized as in the
olfactory bulb proper. However, one must wonder about the peripheral connec-
tions. A recent report (l\frCHAEL and KEVERNE, 1968) claims that by lack of effect
176 D. TUCKER: NonoIactory Responses from the Nasal Cavity
of section of the vomeronasal nerves, sexual pheromone reception in the rhesus

monkey was shown to be olfactorily mediated. A great service could be rendered
by describing the operation and demonstrating the vomeronasal nerve.
The discovery of distinct histochemical differences between the main and
accessory olfactory bulbs of guinea pig and hamster (WITKAM, 1967) is very inter-
esting. In laboratory rats it was found that the size of the main olfactory bulb
decreases markedly during the second year of life, whereas the accessory bulb
suffers no significant diminution (SMITH, 1935). HUMPHREY (1940) found that the
human accessory bulb reached the peak of its development at the time fetal
movement begins, after which atrophication sets in. Interestingly, the olfactory
bulb itself seemed to reach a peak in development in later fetal life, to be followed
by some regressive changes. Although absent in many, a complete vomeronasal
system has been demonstrated in some species of bats (MANN, 1961), but the con-
tention that the nerve fibers are myelinated can hardly be given credence. Were
there some myelinated terminalis fibers present? Some aspects of the bat's
behavior and accessory nasal structure were interpreted as favoring the transport
of stimuli to the Jacobson's organ during sexual activity.
VI. Summary
A vertebrate above fishes in the evolutionary scale is likely to have two other
chemoreceptor systems in the nose than the olfactory, proper. The vomeronasal
(organ of Jacobson) system is of unknown function, but morphologically it is
parallel or accessory to the olfactory system. Trigeminal innervation of the nose
has long been known to be responsible for a "common chemical sensitivity."
"Electrically recorded responses" have been studied in "the Vomeronasal
System" of lizards, turtles and rabbits. Inhaled odorants are effective in turtles.
"Vomeronasal epithelium and its distribution in turtles and mammals" discus-
ses confusion of the turtle medial nasal gland with the organ of Jacobson typical
of other reptiles and mammals, which is an outpocketing of the nasal cavity with
an opening either in the nose or onto the roof of the mouth. In turtles, both the
vomeronasal and olfactory sensory epithelia are exposed freely in the nasal cavity.
"Stimulus transport to the organ" considers evidence that a variety of mecha-
nisms must be used in different types of animals. Anatomical evidence has led to
experimental manipulations and behavioral observations. A pumping ability due
to erectile tissue seems possible in many mammals, coupled with the possibility of
stimuli coming from the nose or from the mouth via the nasopalatine canal. A
series of specializations appears likely in lizards and snakes, ranging from an
extensive ciliary apparatus to the bifid tongue with fine tips which may enter the
openings to the paired organ.
"Electrically recorded responses" were obtained from trigeminal nerves to the
nasal mucosa in cat, rabbit and tortoise. In the latter, trigeminal responses
appeared at lower concentrations of some compounds than were required for
olfactory responses. A complicating factor is dependencc of nasal chemoreceptor
responses on inspiratory flow rate, a dependence which varies strongly between
different compounds.
References 177
"Subjective and reflex responses" were the objects of classical study. The
"origin of the concept of pure olfactory stimuli" is attributed to two workers. "Do
pure olfactory stimuli exist?" evaluates evidence concerning compounds that were
reputed to be pure olfactory stimulants and concludes negatively. However, the
existence of widely disparate olfactory and trigeminal threshold concentrations
for some compounds makes it possible to work in a range in which trigeminal
stimulation is absent, but vomeronasal stimulation is not necessarily absent.
"Functional Anatomy" suggests that the whole of the nasal mucosa should be
regarded as a chemoreceptive organ. "Autonomic efferents" are intimately related
anatomically and functionally to the trigeminal afferents, whose role in smell
sensation is ambiguous. Variations in accessibility of stimuli to the olfactory organ
seem to be produced reflexly by trigeminal stimulation. "Are there mesencephalic
nucleus V fiber endings in nasal mucosa?" speculates that cell bodies of the
chemoreceptive fibers might be found in the mesencephalic nucleus of the tri-
geminal nerve rather than in its external sensory ganglion, the semilunar or
Gasserian ganglion.
"Vomeronasal, Trigeminal and Terminal Nerve Relationships" introduces "the
nervus terminalis system," which is somewhat of an enigma. The terminal nerve
has been demonstrated in various fishes, amphibians, reptiles and mammals, often
in embryonic stages. During embryological development of higher forms the
terminal nerve is intimately associated with the vomeronasal nerve and, to a lesser
degree, with the nasopalatine nerve of the trigeminus. Considerable evidence
indicates that both sensory and motor components are present in the nervus
terminalis system, with specific suggestions of autonomic type innervation to
olfactory mucosal Bowman's glands and blood vessels.
"The vomeronasal system" may have been influenced in its evolution by the
nervus terminalis. One theory is that the vomeronasal organ is the old olfactory
organ of fish-like ancestors and therefore that the olfactory organ with Bowman's
glands is a more recent development. Another theory is that the vomeronasal
apparatus was differentiated in connection with the opening of the posterior nasal
aperture into the oral cavity. There are exceptions to the generalization that
Jacobson's organ is absent in some groups of mammals, such as Chiroptera and
higher Primates.
"References" cited are necessarily a small sample of the heterogeneous liter-
ature brought together in this chapter. However, extensive coverage is afforded
through their citations. Early historical aspects, for example, might be of interest.
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Chapter 8
Structure and Function of Higher Olfactory Centers

By
P. MAC LEOD, Paris (France)
With 5 Figures
Contents
Introduction . . . . . . . . . . . 182
Structure and Connections of the Olfactory Centers 184
Olfactory Bulb. . . . . . . 184
l. Peripheral Neural Plexus . 184
2. Glomerular Layer 185
3. Mitral Cells Layer . . . . 186
4. Granular Layer . . . . . 187
5. Accessory Olfactory Bulb. 187
6. Internal Wiring of the Olfactory Bulb 187
7. Efferent Connections of the Olfactory Bulb 190
8. Centrifugal Projections to the Olfactory Bulb 190
Anterior Olfactory Nucleus . . 191
Primary Olfactory Cortex. . . 192
Secondary Olfactory Projections 193
1. Thalamic Projections. . . . 193
2. Hypothalamic Projections . 194
3. Entorhinal and Amygdaloid Projections 194
4. Centrifugal Fibers to the Olfactory Bulb 194
Central Olfactory Function. . . . . . . . 196
Odor Discrimination . . . . . . . . . 196
1. Sensory Input to the Olfactory Bulb . 196
2. Discrimination at Glomerular Level 197
3. Discrimination at Mitral Cells Level 198
4. Discrimination at Cortical Level. 199
5. Behavioural Experiments. . 199
Olfactory Sources Localization. 200
1. Elementary Mechanisms . 200
2. Behavioural Experiments. 201
References . . . . . . . . . . 201
Introduction
The olfactory system appears to be completely developed in rodents, and no
more sophistication is encountered as one ascends the phylogenetic scale. On the
Introduction 183
contrary, a slight amount of involution of the olfactory cortex is observed in the

so-called "microsmatic" animals and especially Primates and man.
This situation allows for a general description of olfactory centers which applies
equally well to all mammals, including Primates. Ordinary laboratory animals
such as rat, guinea pig, rabbit and cat have been extensively studied and many
outstanding reports are found in the literature. Less detailed and complete data
are available concerning the dog, Primates and man.
The olfactory system may roughly be divided in three parts: a peripheral part
represented by the neuroepithelium and the olfactory bulb, an intermediate part,
the anterior olfactory nucleus, whose importance has been recently emphasized,
and a central part found mainly in the paleocortical area of projection. These three
segments are contiguously located at the rostral end of the brain. In lower macros-
matic mammals such as oppossum or hedgehog, the olfactory brain occupies about
one third of the total cortical area. In higher mammals, owing to an explosive
development of the telencephalon, this region becomes relatively very small
although somewhat larger in absolute size.
Olfactory central connections are peculiar in that they do not follow the
general pattern of other sensory inputs as they undergo a full paleo cortical pro-
jection before the final common thalamo-neocortical relay. A widely accepted idea
is that the chemical sense, in earlier aquatic vertebrates, was predominant amongst
other senses so that the olfactory brain was phylogenetically the first developed
and reached a very high level of efficiency. The design was so good that since the
beginning of amphibian and Terrestrian life, when visual cues became prevalent
and the optic brain enlarged, it was more efficient to add a final connection between
the former olfactory brain and the newer optic brain than to develop a new wiring
for the olfactory centers analogous to the henceforth general pattern.
Whether involved in alimentary, sexual or social behavior, all olfactory sources
raise first the same problem of identification and then the problem of orientation
within the corresponding concentration gradient. Provided a threshold amount of
odorant material is available these two processes must be performed efficiently
irrespective of the absolute intensity of the stimulus, which can vary tremendously
according to the distance between the source and the subject. A subsidiary com-
plication arises from the existence of an odorant background whose intensity may
be greater than the actual stimulus. This endows the olfactory detector with two
quite different tasks. One is to identify a given odour and to retain its identity
irrespective of its intensity and that of the background. The other is to orient the
animal's head and hence its body and course along the concentration gradient,
uphill or downhill, according to the positive or negative chemotactism involved in
the given case.
The identification task requires an extreme degree of both sensitivity and
selectivity, and it is a constant amazement to observe extreme performances in
this field; consider for instance a homing salmon or a dog picking up an old trail!
Even ordinary performances are outstanding as the recognition threshold of almost
every odorant lies in the p.p.m. range, which is seldom reached by our best physical
detectors such as gas-liquid chromatographs or mass spectrographs.
The complementary task of orientation was completely overlooked until recent
years. It implies a precise matching of odor intensity between the two nostrils,
184 P. MAC LEOD: Structure and Function of Higher Olfactory Centers
which is made possible by the twin structure of the whole olfactory system. The
accuracy of this intensity matching is in striking contrast with the estimation of
absolute intensity which remains quite inaccurate and is indeed almost useless in
normal behavior. The shortest way to locate is to ascend the concentration gradient
produced by the odorous molecules diffusing from the target. In order to determine
this gradient it is necessary to carefully compare the odour intensities of different
samples from the surrounding milieu. Of the two conceivable ways of sampling,
either successive or simultaneous, the second is far more efficient because it gives
the correct information as soon as the sensation of equality occurs, without needing
any memorization of the preceding evaluations. Moreover, any external or internal
biases of odor are equal in both nostrils and thus are nulled and cannot alter the
response. Once the direction of the gradient is known, it is only necessary to have
a gross indication of increasing or decreasing odour concentration by respect to the
movements of the animal relative to the source.
Structure and Connections of the Olfactory Centers
Olfactory Bulb
The olfactory bulb is an ovoid structure appended to the rostral end of the
telencephalon. It lies on the cribriform plate of the ethmoidal bone, close to the
olfactory epithelium to which it is connected by the numerous bundles of un-
myelinated fibers which, as a whole, constitute the olfactory nerve. The bulb is
organized in a series of concentric layers wrapped around a more or less vestigial
evagination of the lateral ventricle. The classical description given by earlier
anatomists, as reported by CAJAL (1911), mentions seven layers: the peripheral
neural plexus, the glomerular layer, the external plexiform layer, the mitral cells
layer, the inner plexiform layer, the granular layer and the periventricular layer.
As plexiform layers are merely connections between adjacent cellular layers,
and the periventricular layer is purely ependymal, we shall limit our description to
the four other layers.
1. Peripheral Neural Plexus

The incoming olfactory bundles are mixed up in a very dense felting of un-
myelinated fibers ensheathing the whole surface of the bulb into a continuous
mantle, thicker on the tip and the ventral aspect and thinner on the dorsal aspect.
It is quite impossible to find any topographical relationship between the point of
entrance of a given fiber and its termination into the underlying glomerular layer.
There are no synaptic junctions between adjacent fibers; accordingly, the sensory
message conveyed by each individual fiber remains undistorted until it reaches the
glomerular relay.
The physiological meaning of such a complex distribution of fibers is not clear.
It might be related to a functional grouping of analogue receptor axons towards
the same glomeruli. This hypothesis has been repetitively suggested (LE GROS
CLARKE, 1956) and partly substantiated by recent experimental evidence to be
referred to later.
Olfactory Bulb 185
2. Glomerular Layer
Olfactory glomeruli are well individualized spherical formations, 100 to 200 !1.
in diameter, closely packed in a simple, sometimes double regular layer. In cellular
stainings, they look almost empty as they contain but a few glial cells. The whole
available space is filled by a very dense agglomeration of fine fibers and synaptic
connections. This unique structure is exclusively found and always present in
olfactory centers not only of all Vertebrates but also of Insects. It appears to be
a fundamental morphological correlate of olfaction.
Afferent fibers to glomeruli are principally axons from the olfactory receptors
emerging from the peripheral plexus. There are also axonal branches from short
Fig. 1. Rat olfactory glomerulus. Electron micrograph X 28,000. Arrows indicate polarity of
synaptic contacts as presumed from their morphology: two axons from olfactory nerve
synapse on the central dendrite process; dendrite is presynaptic to another dendrite which in
turn is presynaptic to another dendrite that is also postsynaptic to an axon from the olfactory
nerve (after REESE, T. S., and M. W. BRIGHTMAN, unpublished data)
range horizontal neurons lying inbetween the glomeruli, as well as centrifugal

processes of the granules. The olfactory fibers are very fine and each of them
branches several times through the entire width of a glomerulus. They are terminal
and make numerous synaptic junctions with other fibers, but none with each
other (Fig. 1). All the synapses are centripetal and appear to be cholinergic on
examination of dimension and location of synaptic vesicles (DE LORENZO, 1963;
ANDRES, 1965).
The efferent fibers are main dendrites of mitral cells and also dendrites of tufted
cells and short range horizontal neurons. The main dendritic process of mitral cell
ends in the glomerulus as a dense terminal bunch which appears, in GOLGI stains,
to invade the whole glomerular space and bears countless synaptic buttons. These
synapses are chiefly olfactory nerve endings. Some of them, however, pertain to
short range interneurons and centrifugal granules processes and could be adrenergic
according to SHANTAVEERAPPA and BOURNE (1965) who found monoamino oxydase
activity by histochemical staining in the glomeruli of the squirrel monkey.
Beside the dendritic process of mitral cells, there is another quite similar
dendrite process emerging from the glomerulus, which belongs to a different cell
first described by CAJAL (1890) and named by him "tuftcd cell" because he failed
at that time to ascribe it any other particular feature.
Intermingled between the glomeruli are many horizontal interneurons with
short range axons and dendrites, interconnecting neighbour glomeruli in a dense
network. Electrophysiological studies by YAMAMOTO et al. (1963), MANCIA et al.
(1962), SHEPHERD (1963a, b) have clearly demonstrated that these interncurons
are inhibitory and subserve interglomerular reciprocal inhibition.
3. Mitral Cells Layer

The mitral cells are the more conspicuous cellular component of the olfactory
bulb; they are arranged in two or three densely packed arrays. The cell body is
20 fL or more in diameter and sometimes evokes a mitre by its general contour,
hence its name. Among the dendrites, there is always a single process of larger
diameter running without branching towards a single glomerulus where it gives
the characteristic bunch already mentioned. The existence of a single main and
primary process is a characteristic feature of the mammalian olfactory bulb. Mitral
cells are generally connected to more than one glomerulus in fishes, reptiles and
birds. Emerging from the lateral parts of the cell body, sometimes from the initial
segment of the primary dendrite, are two three or more secondary dendrites,
shorter than the primary one, whose mixed branches are the fundamental com-
ponent of the outer plexiform layer and never extend up to the glomeruli.
A thick axon emerges from the deep pole of the cell body, reaches the inner
plexiform layer and then runs caudalwards. It leaves many collaterals, some of
them being recurrent and directed towards the original and neighbouring mitral
and tufted cells dendrites into the outer plexiform layer.
The tufted cells are located midway between the glomeruli and the mitral cells.
Their dendritic processes are often connected to more than one glomerulus; their
axons are very tenuous and follow a route parallel to mitral ones as it was finally
demonstrated by LOHMAN and MENTINK (1969) after a long era of doubt and
controversy (cf. also: NICOLL, 1970).
Olfactory Bulb 187
According to their shape, dimensions, location, centripetal direction and long

range connections, the mitral cells are homologous to the large pyramidal cells of
more elaborated cortices.
4. Granular Layer
The granular layer occupies about half the total depth of the olfactory bulb
and contains the greatest number of neurons. The typical cell, the granule has a
rather small, round body and bears several dendrites and no axon. Instead, one of
the dendrites, the external process, crosses the mitral layer and blooms in the
outer plexiform layer, making numerous synaptic junctions with the lateral
dendrites of mitral cells (CAJAL, 1911). As recently demonstrated by HINDS (1969),
while all the granule dendrites are post synaptic to axon endings, the sole external
process is both presynaptic and post-synaptic to mitral dendrites making the cell
truly polarized in the centrifugal direction. Despite this fact, the external process
displays all the morphological features of dendrites, in particular irregular con-
tours with numerous spines and free ribosomes.
Apart from the granules, this layer also contains many short range inter-
neurons whose axonal and dendritic expansions remain in the limits of the granular
layer, and a wealth of passing fibers bundles with a general rostro-caudal direction.
These fibers are either large and myelinated or fine and unmyelinated, efferent as
well as afferent, and make connections throughout the whole bulbar structures,
including the glomerular layer.
5. Accessory Olfactory Bulb

This structure is well developed in lower mammals and absent in adult Primates
and man. Its sensory input comes from Jacobson's vomero-nasal organ via the
vomero-nasal nerve. Hemispherical in shape, it lies embedded into the dorsal
aspect of the main olfactory bulb. Its structure is analogous, in a simplified and
more archaic form, to the main olfactory formation. Vomeronasal nerve fibers end
in glomeruli by rather few ramifications; instead of mitral cells, there are smaller
starlike cells with dendrites connected to more than one glomerulus and axons
joining the mitral cells axons on their way to the lateral olfactory tract. The
granular layer is poorly represented by very small granules with thin external
processes. It receives a small contingent of the same centrifugal fibers as the main
olfactory formation.
The physiological meaning of the para olfactory functional unit constituted by
the vomeronasal organ associated with the accessory bulb is not yet clearly
understood, even in the Reptiles where it is fully developed. It could be an acces-
sory olfactory detector sensing the liquid content of the buccal cavity as a comple-
ment to taste (KAHMANN, 1932; WILDE, 1938; PLANEL, 1954; TUCKER, 1963).
6. Internal Wiring of the Olfactory Bulb (Fig. 2 and 3)

Although briefly mentioned in the preceding pages, the internal bulbar con-
nections may be worth summarizing in order to reach a better understanding of
the olfactory message. In a famous publication, ALLISON and WARWICK (1949)
gave a count of the main components of the olfactory bulb in the rabbit. This will
allow us to add a numerical dimension to our sketch.
The sensory input to the bulb comes from 50,000,000 receptors through an
equal number of olfactory nerve fibers ending into only 1900 glomeruli. Each
glomerulus receives thus an average of 26,000 olfactory fibers, realizing an utmost
information concentration. Through glomerular synapses the sensory input is
transferred to 45,000 mitral and 150,000 tufted cells, each mitral cell being con-
nected to only one glomerulus, and conveyed out of the bulb by their axons grouped
Fig. 2. Vertical connections in the mouse olfactory bulb. a, b, c olfactory glomeruli; d tufted
cells; e mitral cells; f mitral cells axon; g tufted cell axon; h mitral cell axon recurrent
branching; i, j granules; A glomerular layer; B outer plexiform layer; C mitral cells layer;
D inner plexiform layer; E granular layer; (after CAJAL, 1911)
into the lateral olfactory tract. It is noteworthy that a concentration of about

1,000 to 1 occurs between the olfactory input and the mitral output, that is to say
between the first and second neuron. The concentration is even 26,000 to 1 if, like
the author, one considers glomeruli as true functional units, a view which is
seriously supported by recent anatomical evidence from LAND et ai. (1970).
Several millions of granules directed parallel and opposite to the main afferent
pathway, represent a major negative feedback loop, as they receive excitatory
connections from axon collaterals of mitral and tufted cells and send in turn an
inhibitory output to the glomerular and outer plexiform layers mainly formed
respectively by the primary and secondary dendrites of the same neurons (YAMA-
MOTO et al., 1963; VON BAUMGARTEN et ai., 1962; PHILLIPS et ai., 1963; see also the
excellent review by OTTOSON and SHEPHERD, 1967). This feedback loop ensures
two basic actions. First, self-inhibition of mitral output resulting in a severe
Olfactory Bulb 189
firing frequency limitation, so that the output becomes relatively independent of

the input. Second, reciprocal inhibition between neighbour mitral cells resulting
in an enhanced contrast between adjacent channels, according to a general pattern
first stressed by HARTLINE, WAGNER, and RATLIFF (1956) for vision, and then by
MOUNTCASTLE (1957) for touch. In the case of olfaction, the reciprocal lateral in-
hibition is already initiated at the glomerular level by means of the interglomerular
short axon neurons. In addition, the feedback loop receives a massive centrifugal
Fig. 3. Horizontal connections in the mouse olfactory bulb. A glial cell; B, G granules; D glial
cell; E short range interglomerular interneuron; F long range interglomerular interneuron;
a axons; b axon branching of a tufted cell; (after CAJAL, 1911)
influence from the ipsilateral olfactory brain and contralateral anterior olfactory
nucleus joining a heavy exstrinsic contribution to the local inhibition.
This fundamental inhibitory loop is paralleled by a less important positive
feedback loop resulting from axon recurrent collaterals of mitral cells impinging
directly into the outer plexiform layer, thus sh:mting thc inhibitory granule path.
This activating, "reverberating" (CAJAL, 1911), circuit provides a way to the
spreading of excitation at the lowest levels of stimulation, when, lacking enough
input, the powerful inhibitory system cannot yet be thrust into action. This may
provide a key to the understanding of how certain detection thresholds can be so
low (PASSY, 1892; ZWAARDEMAKER, 1925; TEICHMANN, 1959; STUIVER, 1958;
LAFFORT, 1963).
As a whole, the olfactory bulb appears like a device able to detect very low con-
centrations of odorant molecules as well as to perform a constant high grade
discrimination throughout a broad range of increasing concentration while remain-
ing submitted to an overall control by centrifugal influences from higher centers.
190 P. MAc LEOD: Structure and Function of Higher Olfactory Centers
7. Efferent Connections of the Olfactory Bulb

The lateral olfactory tract is now accepted as being the only direct projection
of the olfactory bulb to the forebrain (POWELL et al., 1965). It is an important
bundle of fibers emerging from the mitral and tufted cells, first running caudal-
wards in the inner plexiform layer, then collecting, as they emerge from the bulb,
upon the ventrolateral surface of the external olfactory peduncle. They pass along
the lateral edge of the olfactory tubercle and spread over the whole prepiriform
lobe, including the periamygdaloid cortex (WHITE, 1965). During their course,
they emit numerous collaterals, making direct ipsilateral connexions with all the
subdivisions of the anterior olfactory nucleus (SCALIA, 1966), the olfactory tubercle,
the prepiriform cortex and the periamygdaloid cortex (LOHMAN and LAMMERS,
1967). There is also a contingent for the amygdaloid complex, restricted to the
cortical nucleus.
Because they are so fine, and hence difficult to follow in degeneration experi-
ments the contributions of the tufted cells axons to the lateral olfactory tract were
not definitely demonstrated until 1969 by LOHMAN and MENTINK, an idea first set
forth by VAN GEHUTCHEN and MARTIN (1891)!
No topographical relationship was ever found between the site of origin of the
lateral olfactory tract fibers and their cortical termination (WHITE, 1965).
8. Centrifugal Projections to the Olfactory Bulb

The olfactory bulb receives centrifugal fibers throughout its whole extent, and
from all the ipsilateral olfactory brain as well as from the contralateral anterior
olfactory nucleus. On the basis of present experimental data, three categories of
afferent fibers may be distinguished.
First, it is now established beyond doubt that centrifugal fibers are present,
though in a very small proportion, in the lateral olfactory tract (CRAGG, 1962;
POWELL and COWAN, 1963; DENNIS and KERR, 1968). These fibers correspond to the
network of thicker fibers described by CAJAL (1911). They arise from the total
projection area of the tract and are principally distributed to the granular layer.
However, many of them extend through the mitral cell layer to reach the outer
plexiform and glomerular layers. They do not penetrate into the glomeruli but
seem to end on the periglomerular neurons.
Second, the anterior limb of the anterior commissure appears to be the major
contributor of afferent fibers to the bulb. Being of various but generally fine
diameter, these fibers have a dual origin. They terminate in the granular layer,
without extending to the more peripheral layers (POWELL et al., 1965). The bulk
of them come up from the contralateral anterior olfactory nucleus, pars medi.alis
(LOHMAN and MENTINK, 1959; VALVERDE, 1964); these are the only contralateral
connections to the bulb. Other fine fibers, coming from the ipsilateral olfactory
tubercle join the anterior limb of the anterior commissure before entering the bulb,
and undergo the same distribution limited to the granular layer.
Third, recent experimental evidence has been reported by DENNIS and KERR
(1968) suggesting that some centrifugal fibers reach the olfactory bulb, even after
complete section of the lateral olfactory tract and of the anterior limb of the
anterior commissure. Not yet demonstrated histologically, these fibers should be
Anterior Olfactory Nucleus 191
very fine, arise from the deep aspect of the olfactory cortex and reach the bulb by
a central path, close to the ventricular cavity.
Anterior Olfactory Nucleus

The retrobulbar region is a transition zone of grey substance where the clearcut
organization of the olfactory bulb has disappeared and is not replaced by a true
cortical structure. Of the paleocortex it features only the typical superficial layer
of horizontal fibers but not the underlying stratification. It doesn't appear to be a
true subcortical nucleus as it is not well limited by white bundles but rather traversed
by many tangled fibers running in all directions. The neurons are of the poly-
morphous type, larger in the depth, smaller near the surface, especially in the external
part. It is thus easy to understand how CAJAL, who was somewhat obsessed by the
problems of cortical structures, although giving a careful and objectively correct
description of this region, failed to see anything else there than a degenerated
cortical border. It was only experiments with modern histological techniques,
associated with clever destruction, degeneration and electrophysiological experi-
ments that disclosed the individuality and importance of this long overlooked part
of the olfactory system, first named and described as such by HERRICK (1924). The
major contributors in this field were YOUNG (1936), LOHMAN (1963), VON BAUM-
GARTEN et al. (1962) ; VALVERDE (1964, 1965) and POWELL et al. (1965) who clearly
identified the different parts of the anterior olfactory nucleus and their relationship
to the anterior commissure whose description is henceforth bound to the latter.
According to LOHMAN and MENTINK (1969) one can divide the anterior olfactory
nucleus into a pars rostralis, a pars lateralis, a pars medialis and a pars externa.
The pars rostralis is located entirely inside the olfactory bulb and consists of
many clusters of large cells scattered along the ventricular walls. Their axons of
small calibre, proceed along the anterior limb of the anterior commissure, ending
by axo-dendritic synapses into the contralateral pars externa.
The pars externa is located external and anterior to the pars lateralis; it merges
rostrally into the granular layer.
The pars lateralis extends caudally towards the prepiriform cortex into which
it gradually blends. Alike, it receives a substantial projection from the lateral
olfactory tract on its emergence from the posterior edge of the olfactory bulb.
The pars medialis is in continuity with the pars lateralis through the pars
dorsalis and the pars ventralis and extends caudally without interruption into the
precentral frontal cortex. It contributes fibers to the contralateral olfactory bulb
via the anterior limb of the anterior commissure. These crossing fine fibers, thought
by CAJAL to be axons of the tufted cells, innervate the whole granular layer but do
not extend beyond it.
Another contingent of fine axonal fibers leaves the anterior limb on the
ipsilateral side and are distributed to the deep aspect of the whole cortical projec-
tion of the lateral olfactory tract, including the olfactory tubercle (SCALIA, 1966).
The origin of these fibers has not yet been precisely ascertained but it must be in
the anterior olfactory nucleus as they degenerate only when bulbar lesions extend
to the retrobulbar area.
The anterior olfactory nucleus appears therefore as the real origin of the
anterior limb of the anterior commissure, relaying the bulbar efferent impulses to
the opposite bulb and to the ipsilateral forebrain as well.
Primary Olfactory Cortex

The paleocortical area of projection of the lateral olfactory tract is located at
the rostral side of the telencephalon, just behind the insertion of the olfactory
bulb. It is limited to the antero-external border of the olfactory tubercle, the pre-
piriform area and the periamygdaloid area. To these truly cortical areas, one must
add the projection to the amygdaloid complex which is restricted to the lateral
olfactory tract nucleus and the cortical nucleus (POWELL et al. , 1963; SCALIA, 1966).
_l~O~
I ~'~M~---- ______, - - -- - - --------
- - ----------
L
Fig. 4. Schematic diagram of the fundamental connections in the cat primary olfactory cortex:
S superficial neurons; D deep neurons; I inhibitory synapses; E excitatory synapses; a axons;
(after STEVENS 1969)
The classical description of the olfactory cortex by CAJAL (1911) has been
recently renewed and enriched by STEVENS (1969). This cortical area (Fig. 4) is
characterized by the presence of a superficial layer of horizontal fibers gradually
diminishing in thickness from anterior prepiriform to periamygdaloid cortex and
made of the spreading fibers of the lateral olfactory tract. The tract fibers give off
many perpendicular collaterals and terminal branches to the underneath molecular
or plexiform layer where they make axo-dendritic synapses with the immediately,
subjacent pyramidal cells.
The deeper neurons of the olfactory cortex are rather large and densely packed,
so that in simple visual inspection of normally stained histological sections this
region appears as a darker, well limited zone. Being a typical paleocortex, it may
be laminated, on morphological as well as on functional ground, in no more than
two layers. The superficial half contains pyramidal cells of about 12 [1. in mean
diameter and whose maximum density is as high as 90,000 per cubic millimeter.
These cells emit numerous dendrites in the molecular layer and most of them in
addition present basal dendritic tufts which are more developed as one ascends the
phylogenetic scale, being maximal in man. Their axons have a perpendicular
downwards course through the deeper somatic layer to which they leave many
horizontal short range (100-200 [1.) collaterals; they then project into the infra
cortical white substance. The deeper half is made of larger polymorphic cells whose
mean diameter approximates 20 [1. and maximum density is about 30,000 per cubic
millimeter. The deep neurons axons are difficult to follow in Golgi material because
they branch in a very diffuse pattern; they seem not to leave the cortex. Axon
terminals from deep neurons are encountered on dendrites of other deep neurons,
on basal dendrites of superficial pyramidal cells and even in the molecular layer.
The axonal synaptic field of a given deep cell is far wider than that of a super-
ficial one, but keeps a local extension, thus conferring a columnar organization to
the olfactory cortex.
According to the electrophysiological study by BIEDENBACR and STEVENS
(1969a, b), an incoming olfactory volley results in a monosynaptic excitation of
the superficial neurons, invariably followed by an inhibition wave imputable to
the reflex of the deeper neurons. Indeed, evoked potentials following olfactory
tract stimulations, as well as intracellular recordings have definitely demonstrated
the inhibitory nature of the deep cells.
This negative feedback loop controlling the third neuron of the olfactory
pathway strikingly resembles the granule action on the secondary neuron and
should bear the same physiological meaning, namely output frequency limitation
and reciprocal inhibition.
Secondary Olfactory Projections

As the preceding description makes the primary olfactory cortex appear more
like a relay than like a final integrative projection area, our attention is now focus-
sed on the subsequent fate of the olfactory message. In as much as it is a relay, it
is a powerful one since, on a rough estimate, the outcoming information channels
are a hundred times more numerous than the incoming mitral fibers. Rather
multiplexed than further elaborated the olfactory message is hence dispatched over
two ascending, one lateral and one recurrent line; namely the thalamus, the hypo-
thalamus, the entorhinal and amygdaloid areas, and back to the anterior olfactory
nucleus and olfactory bulb.
1. Thalamic Projections
It will remain the deserts of POWELL et al. (1965) to have clearly shown the
projections from the primary olfactory cortex to the medial thalamus nuclei. There
is first an ipsilateral projection to the medioventral nucleus, the same recently
defined by BENJAMIN and AKERT (1959) as receiving direct taste input. From
this nucleus an ultimate connection to the contiguous fronto-lateral neocortex
13 Rb. Sensory Physiology. Vol. IV!l
makes this area look like the true primary cortical area of projection for the
chemical senses. There is also a bilateral connection to the medio-dorsal nuclei
through the intermediodorsal commissure; on their route, some fibers extend
dorsally into the lateral habenular nuclei on both sides. The thalamic mediodorsal
nuclei are known to be connected in turn to the orbito-frontal area of the neocortex,
which is considered as highly integrative.
In view of these thalamic connections, and despite the previous relay of mitral
fibers into the "primary" olfactory cortex, the olfactory pathway appears no
longer different from the other sensory pathways.
2. Hypothalamic Projections
The medially directed fibers emerging from the deep aspect of the prepiriform
cortex and from the olfactory tubercle lean to a caudal direction into the medial
forebrain bundle where they can be followed throughout the whole hypothalamic
region. Terminal connections occur in the lateral preoptic nucleus, in the lateral
hypothalamic area and farther back in the periinfundibular nuclei and nuclei
gemini of LUNDBERG (1962), but not in the suprachiasmatic nor in the peri-
ventricular nuclei.
These direct connections with olfactory cortex are very interesting in that they
place the hypothalamus at a distance of just two synapses from the peripheral
olfactory input (KANDEL, 1964). Other primary sensory systems can only influence
the hypothalamus through a far more indirect, hippocampal pathway (MORIN,
1950; CRAGG, 1961). LUNDBERG (1960) has recently confirmed the absence of
direct hypothalamic projection from any neocortical area. As shown below, these
direct olfactory projections are again substantiated by a relayed pathway through
the basal and lateral amygdaloid nuclei, so that the hypothalamus is actually the
main diencephalic partner to the olfactory cortex.
It now becomes quite clear why olfactory perceptions are so conspicuously
related to feeding (LE MAGNEN, cf. Chap. XIX) and reproductive (WHITTEN, 1958;
BRUCE, 1963) behaviour.
3. Entorhinal and Amygdaloid Projections

The entorhinal area of the piriform lobe is located just caudal to the prepiriform
and periamygdaloid areas. Careful anatomical studies (POWELL et al., 1965) have
revealed beyond any doubt that, contrary to the older view of CAJAL, more recently
shared by LE GROS CLARKE and MEYER (1947), by ALLISON (1953) and ADEY
(1953), this region does not receive direct connections from lateral olfactory tract
fibers, but instead a dual projection from the above mentioned areas and from the
olfactory tubercle (FERRER, 1969). The prepiriform - entorhinal projection is
twofold: in addition to the ordinary subcortical fascicles, there are also superficial
fibers running through the molecular layer. Associated with the deep pathway,
there is a heavy projection to the basal and lateral amydaloid nuclei; there is none
to the central amydaloid nucleus which is in close contiguity to the stria terminalis.
4. Centrifugal Fibers to the Olfactory Bulb

This already described projection (see p. 190, 8) is conveyed to the anterior
olfactory nucleus, pars lateralis and pars medialis and to the olfactory bulb by
centrifugal fibers in the lateral olfactory tract and by a distinct deep pathway as
well. The contribution of the olfactory tubercle to this projection seems largely
prevalent (FERRER, 1969). The projection to the anterior olfactory nucleus, pars
medialis, provides a pathway through the anterior commissure to explain the
~
~
% p.amyg
m.fb ortpx
omY:]d %
~
m.fb
011 tub.
HpPlr
oc ~cortPx
o o
2i n.aopxt
n.oa.rost~
1.0.1.
olf.bulb
_ _ _ _ _ _ 011 <'P' h
Fig. 5. This diagram is intended to summarize, hopefully better than a written text, the whole
hitherto reported olfactory connections network. acc. 011. b. accessory olfactory bulb; a. c.
anterior commissure; amygd. amygdaloid complex; gl. glomerulus; gr. granular cell; I. o. t.
lateral olfactory tract; m. I. b. medial forebrain bundle; m. mitral cell; n. o. a. ext. anterior
olfactory nucleus, pars externa; n. o. a. med. anterior olfactory nucleus, pars medialis; n. o. a.
rostr. anterior olfactory nucleus, pars rostralis; 011. tub. olfactory tubercle; p. amygd. cortex
periamygdaloid cortex; p. pir. cortex prepiriform cortex; thai., d. m. thalamus, nucleus medio
dorsalis; thai., v. m. thalamus, nucleus medio ventralis
negative bulbar potentials bilaterally evoked on unilateral stimulation of the

piriform cortex reported by CALLENS (1967) and by DENNIS and KERR (1968).
Although the fish olfactory bulb differs in several aspects from the same organ
in mammals, the thorough study conducted by DOVING and GEMME (1965, 1966)
and by DOVING (1966d) on the efferent influence of the lateral olfactory tract
IS
196 P. MAC LEaD: Structure and Function of Higher Olfactory Centers
upon the olfactory bulb of the burbot provides further evidence of how the third
order olfactory neurons control the second ones. D0VING and GEMME found that
unilateral stimulation of the olfactory nerve results in a bilateral efferent discharge.
In the absence of peripheral input, however, efferent units display a spontaneous
activity that may be influenced by extra olfactory stimulations such as touching
the skin. Moreover, when a bulbar unit is recorded, a shock to the contralateral
olfactory tract blocks the response to a shock on the ipsilateral olfactory nerve,
provided a precise time sequence is respected. Finally, it has been revealed that
efferent influence is excitatory for about 35% of the bulbar units, and inhibitory
for 60 %; only 5 % are indifferent. Conversely, an olfactory nerve volley excites
about 80 % of the same population, leaves 17 % unaffected while but 3 % are
inhibited.
The efferent connections from the olfactory cortex to the bulbs appear therefore
as a decussating, mainly negative feedback loop mediating a fine modulation of the
activity of each bulb under the influence of the opposite one as well as of remote
parts of the brain (cf. also KERR and HAGBARTH, 1955) (Fig. 5).
Central Olfactory Function
Odor Discrimination
As already quoted in the introduction, discrimination is the first step neces
sarily involved in any olfactory behaviour. In the ambient air odours are always
mixed, so that the relevant ones must be identified against a background of olfactory
"noise". Furt,hermore, there is contradiction between the natural situation where
odours individualized as single olfactory stimuli are chemically complex, often in
volving several hundreds of distinct components in somewhat variable amounts,
and the experimental situation where, aiming to find out correlations between odor
and molecular structure, one uses preferably the purest grade chemicals. Of course,
this discrepancy is easily dismissed on theoretical grounds since, in any case, a
given olfactory stimulation results in a given pattern of neuronal activation, and
obviously the same encoding applies to the olfactory message regardless of the
number of chemical components in the stimulus. Nevertheless, there is at the
present time a serious gap between analytical experimental situation and the
complexity of the daily olfactory world.
For the sake of experimental analysis, odour discrimination may be considered
under two different aspects: quality and intensity. Intensity will not be dealt with
in this chapter, olfactory psychophysics being fully accounted for by ENGEN in
another chapter. Similarly, no more than a short recall of peripheral coding will be
given since this question is discussed in details under different viewpoints by
GESTELAND, MOZELL, DAVIES and BEETS in other chapters.
1. Sensory Input to the Olfactory Bulb

It has become quite clear that the information coming from the receptors
sheet is encoded into a spatio temporal plattern of activation elicited by the chemi
cal stimulus among the 50,000,000 fibers of the olfactory nerve. The occurence of
Odor Discrimination 197
such a pattern depends only upon a differential response of the receptors to the
same stimulus; the degree of specificity of each individual receptor, owing to the
enormous number of available channels, may be quite low without impairing the
overall performance.
Moreover, intensity appears fundamentally associated to quality since each
channel conveys purely quantitative information; thus quality results merely from
patterning unitary intensities and the intensity from summing them.
2. Discrimination at Glomerular Level

As each glomerulus is in receipt of about 26,000 olfactory fibers (ALLISON and
WARWICK, 1949) the concentration of sensory information is maximal at this level.
All the histological features already reported concerning these specific olfactory
formations leads one to consider them as integrating devices, summing all the
information from sensory receptors, periglomerular cells and remote feedback
loops towards the mitral and tufted cells dendrites.
The summated synaptic potentials of single glomeruli elicited by chemical
stimulation of the olfactory epithelium have been recorded by LEVETEAU and
MAC LEOD (1965). It is an inside-negative slow potential initiated about 50 msec
after the onset of the corresponding E.O.G. and of similar time course. Although
biphasic, negative-positive responses are frequently found, in no instance has a
pure positive potential been recorded. The amplitude of the glomerular potential
increases with a steeper slope as a function of stimulus concentration than does the
E.O.G., and reaches a saturation plateau when the latter is only one third its
maximum amplitude (LEVETEAU and MAC LEOD, 1969b). This fact is worth con-
sidering in order to explain the well known limited number of subjective intensity
steps between threshold and maximum.
The most salient finding is that a given glomerulus responds selectively to a
series of chemicals in an "all-or-none" way (LEVETEAU and MAC LEOD, 1966,
1969a) irrespective of the stimulus intensity. The overall probability of response
of a given glomerulus to a given chemical approximates 60 %, but every possible
degree of individual selectivity has been encountered.
By considering the paired pattern of response of the whole glomerular sample
to the same couples of stimuli, it was possible to assess strong similitudes in
stimulant effectiveness of such pairs as, for instance, methanol-propanol, pyridin-
coumarin or decanol-amylacetate. If the first pair was not unexpected, the others
are quite disconcerting and will await for better times to be explained as none of
the nowadays suggested physico-chemical mechanisms of stimulus-receptor inter-
action may account for them. The least bad fit would be with MULLINS' (1955)
differential solubility theory.
A puzzling question that remains unanswered is whether or not receptor axons
of identical chemical specificities end into one and the same glomerulus. Un-
fortunately, the above related investigation did not involve enough different
stimuli in order to assess eventual glomerular similitudes of responsiveness.
On the basis of our present fragmentary knowledge, it is therefore apparent
that, owing to a dramatic 25,000 to 1 reduction in the number of input channels at
the expense of their intensity range but without significant loss in their individual
selectivity, the peripheral half-tone pattern is transcoded into a schematic binary
diagram, involving about 2,000 parallel channels, which is the actual input to the
subsequent sensory processing.
3. Discrimination at Mitral Cells Level

The second order olfactory neuron is the center of convergence of ascending,
descending and lateral influences and, as such, plays a major part in the elaboration
of the final message to be transmitted to the higher centers. Mitral cells were
historically the first structures in the olfactory system to be submitted to an
electrophysiological study. As early as 1942, in a pioneer work, ADRIAN recorded
unit activity in the mitral layer of the olfactory bulb of the hedgehog. He found
spontaneous discharges at each inspiration, with increased frequency when a
distinct odor was added. More recently, MOZELL and PFAFFMANN (1954), WALSH
(1956) and MANOIA et al. (1962) confirmed that bulbar units react differently to
different odors.
A new insight on the question was provided by a series of studies from D0VING
(1964, 1966b, 1966c) analyzing single unit activity in the olfactory bulb of the
frog stimulated by different chemicals. The bulbar units usually display a slow,
irregular spontaneous activity. On chemical stimulation, three possible types of
responses are observable: excitation, inhibition, or no response. Inhibition is the
more common, about 80 % of the whole sample. The remaining 20 % is equally
distributed between activation and no response. When the stimulus is changed, the
response of a given unit mayor may not change. The same unit always gives the
same response to the same substance and does not vary with stimulation intensity.
When two units appeared to respond identically to a series of stimuli, it was alway!>
possible to find another substance which was discriminated. Some units appeared as
"specific" in that they were inhibited by all but one stimulus tested. Only the
frequency of firing was significantly related to the stimulation; pattern of firing.
on the opposite, appeared rigidly characteristic of units and independant of stimu-
lation (D0VING and HYVARINEN, 1969). By computer analysis of the pattern of
responses of over 500 units to 33 different substances, it was possible to determine
the degree of electrophysiological similarity between all possible pairs of stimuli.
Each odour was then represented by a point in a diagram. The distance between the
points was inversely rated to their degree of similarity (SHEPARD, 1962a, b). It was
by no way possible to enter all the points into the same, even three dimensional,
chart without "stress". The psychophysical measure of subjective odour relatedness
performed on a sample of 10 substances fell in fair agreement with electrophy-
siology. An even better agreement was found for the series of homologous
aliphatic alcohols. It is rather satisfactory to find such an agreement between frog
and man!
A striking difference arises when comparing odour similarities at the glomerular
or the mitralleveI. Only the latter coincides with subjective similarities whereas
the former is rather akin to physico-chemical similarities. This means that central
processing of the olfactory message under the influence of behavioral or other
Odor Discrimination 199
relevant informations is achieved in the deutoneurons whose efferent pattern of

activation is therefore deeply reshaped with respect to the glomerular input.
4. Discrimination at Cortical Level

Very little is known about unit activity elicited by odour stimulation in the
olfactory cortex. HABERLY (1969) found in the prepiriform cortex of rat about
20 % of the units unequivocally responding to odours; among these units a great
majority were inhibited, less than 20 % were activated. An off-response was often
present, and the latency of the activated cell was several seconds long. A slight
amount of odour specificity was found. As BIEDENBACH and STEVENS (1969a, b)
have shown that electrical stimulation of lateral olfactory tract in the cat is
followed first by brief activation in the superficial units and then by long lasting
inhibition of the same cells under the influence of deep cells, it is likely that the
inhibited units found by HABERLY were superficial cells whereas activated ones
were deep cells. More experimental data are needed before further understanding
the olfactory cortex function.
5. Behavioural Experiments
Olfactory discrimination reaches maximal theoretical performances since it is
possible to ascribe a distinct odour to any distinct molecule provided it reaches the
olfactory epithelium in sufficient amount. It is therefore not surprising to find
how specific olfactory cues are to animal behaviour.
The interference of olfaction in reproductive mechanisms has long been
studied: Le MAGNEN (1952a), later confirmed by KOSTER (1965), showed that
human threshold for some steroid substances is highly dependent on the menstrual
cycle and more generally on the endocrine status of the subject; analogue phe-
nomena related to the olfactory identification of the sexual partner in the rat were
simultaneously reported (LE MAGNEN, 1952b); WHITTEN (1958) found that female
mice kept in a group had their oestrous cycles shortened and regularized under the
influence of male odour; BRUCE (1963) discovered, also in mice, the pregnancy
block induced by the odour of a strange male. In a different context, olfactory
cues are known to playa role in interindividual relations: ROPARTZ (1967) has
carefully analyzed the incidence of distinct cutaneous, genital or urinary odours in
social behaviour of mice; quite precise odorant territory landmarks are used by
pangolins resulting from special and diverse anogenital secretions (PAGES, 1968).
All of these examples involve critical identification of highly significant
olfactory patterns and their discrimination against a background which contains
sometimes very close non significant components. It is not surprising in this
respect to hear of a true "language of odour" (BRUCE, 1964).
The preceding experiments concerned spontaneous olfactory discrimination. It
is also quite feasible to achieve animal learning of olfactory detection and/or
discrimination in order to measure thresholds, adaptation or habituation. MOULTON
(1963) has shown in rabbit with chronically implanted electrodes in the olfactory
bulbs that spike activity is largely dependent upon efferent centrifugal influences:
unilateral destruction of ipsilateral connections between olfactory bulb and cortex,
including the anterior limb of the anterior commissure results, on the operated side,
in a great reduction of background activity and a parallel increase in response to

olfactory stimulation without any reduction during repeated stimulation while the
unoperated side remains unchanged with high fluctuating background, irregular
amplitude of responses to odours and a strong tendency to habituation. BENNETT
(1968) has demonstrated that unilateral removal of olfactory bulb does not impair
previously learned performances of the rat in odour detection and discrimination.
It is only when more than 50 % of both anterior limbs of anterior commissure are
severed that performances begin to be reduced. A complete loss of learned behavior
occurs after complete bilateral transsection of anterior commissure, but post-
operative deficit is reversible by a new learning. The decussating efferent fibers in
the anterior commissure appear thus to be functionnally predominant upon the
efferent fibers of the lateral olfactory tract but the latter alone can eventually
support learning mechanisms.
Olfactory Sources Localization

This ultimate step, in the absence of which olfaction would remain almost
useless, involves head orientation and locomotor activity driven by sniffing and
smelling any significant scent signalling some source of interest. This is indeed the
final integration of a complex sensory input into an oriented behaviour.
1. Elementary Mechanisms
In the past, human experimentation had led to the conclusion that it was
impossible, in a purely olfactory situation, to assess the side of an unilateral
stimulation (VON SKRAMLIK, 1925). An apparently contrary experiment was
reported by VON BEKESY (1964) who stated that it was possible to perceive the
first or stronger stimulated side during birhinal olfactory stimulation with as little
as 1 msec time interval or 10 % difference in concentration between the two
channels. The pendulum swung once again when SCHNEIDER and SCHMIDT (1967)
clearly demonstrated, still in human experiments, that slight differences in con-
centration (0.5 to 3 %) or a time interval of 100 ms between bilateral stimuli were
regularly mistaken unless the subject was allowed to move his head, or the stimuli
were trigeminal.
Recent animal experiments (LEVETEAU and MAC LEOD, 1969b, 1969c) reduced
these apparent contradictions. By recording glomerular potentials elicited by
bilateral olfactory stimulation in the rabbit, it became apparent that temporal
inhibition could reach a maximum of 50 %provided the onset of the two stimula-
tions were kept within 10 msec from an optimal value of 3 msec. Out of this
narrow range, no interbulbarinhibition was noted. Concentration differences yielded
a corresponding maximal inhibition when precisely matched; however, the concen-
tration effect was less sensitive. These data provide an objective correlate for VON
BEKESY'S observations as well as a clear explanation of SCHNEIDER'S contradictory
finding since he used too large time intervals and too small concentration differ-
ences. As for VON SKRAMLIK'S report, it appears that a dissymetrical bilateral
stimulation is perceived more unilateral than a true unilateral one (which never
occurs normally) obviously because bulbar activity projections to piriform lobe as
well as piriform projections to olfactory bulbs are bilateral (CALLENS, 1967).
References 201
Accordingly, when stimulation is bilateral the same projections ensure fine inter-
actions resulting in precise evaluation of birhinal differences.
Another fruitful finding of SCHNEIDER and SCHMIDT is that other sensory clues
may help side discrimination when olfactory clues alone become inadequate.
2. Behavioural Experiments
The preceding experimental evidence afford an explanation of how paired
olfactory organs can work to help the animal placing its head axis along the con-
centration gradient related to an odorant source. KLEEREKOPER (1967) reports
that a young shark approaches a prey source following a logarithmic spiral which
is exactly the pathway predicted if bilateral equalization of the two nasal inputs is
the relevant implication. In another experiment, he reported that various fresh
water and marine fishes failed to reach an olfactory attractant source unless a very
small water stream was associated. In this case, olfactory arousal was simply
shown to elicit an inborn rheotaxis.
The latter observation clearly applies to homing salmon finding its way through
successive river branchings back to its parental spawning grounds (HASLER, 1966).
Although specific odour has been shown to be the actual stimulus (HARA et al.,
1965) it is clear that no gradient exists so far from the source. It is therefore the
inborn tendency of the animal to swim upstream that is triggered by the olfactory
perception, realizing a refined model of sensory cooperation.
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Chapter 9
Spatial and Temporal Patterning*

By
MAXWELL M. MOZELL, Syracuse, New York (USA)
With 5 Figures
When the available information is incomplete, the mechanisms proposed to

explain the quality discriminations mediated by a sensory system generally involve
the specific tuning or the selective sensitivity of the receptors themselves. That is,
it is generally suggested that the receptors are themselves differentially responsive
to different sub-ranges of the total range of stimuli to which that sensory system
as a whole is responsive. These stimulus sub-ranges to which the receptors are
particularly sensitive may be mutually exclusive, as suggested by the most
stringent interpretation of the "Doctrine of Specific Nerve Fiber Energies."
However, as in color vision, they may overlap so completely that the receptors
differ only relatively in their responsiveness to the different stimuli. In either case,
the receptor signals how well a given stimulus matches the sub-range of stimuli to
which it is most sensitive and a code for quality is thus generated.
However, it is not always true that the selective sensitivity of the receptors
themselves is responsible for the analysis of qualities. For example, the frequencies
of the changes in air pressure impinging upon the auditory system are analyzed by
the cochlea in accordance with physical principles not at all indigenous to the
receptors per se. Because of the physical properties of the basilar membrane, each
frequency produces a membrane displacement which travels as a wave along its
length for a characteristic distance with a characteristic change in amplitude. This
allows different frequencies to be spatially separated and differentiated from each
other along the length of the membrane. The receptors of the Organ of Corti, also
arranged along the length of the basilar membrane, signal the course of these
travelling waves. This means that the receptors need not have any selective sensi-
tivity of their own; all they need do is signal whether, and by how much, their
positions along this analyzer have been affected. Such signals apparently form the
basis for the encoding of frequency in the auditory pathways.
In olfaction most attention has been focussed upon the sensitivity of the single
receptor as a basis for the discrimination of different odorants. However, evidence
has been accumulating to suggests that a spatial analysis of odorants may likewise
be made across the extent of the olfactory epithelial sheet. This mechanism, in
* The preparation of this manuscript and much of the author's data reported in it have
been supported by National Institutes of Health Grant NB 03904.
206 M. M. MOZELL:
analogy to audition, would require the receptors to simply signal the activity at
their respective positions along the analyzing epithelium rather than signalling
their selective sensitivities. As early as 1950 ADRIAN (1950, 1951) proposed such a
regional analysis of odorants by the olfactory mucosa. Since the activity of the
primary olfactory units at the level of the mucosa had not yet been successfully
recorded, Adrian based this proposal upon multiunit recordings from the secondary
units of the rabbit olfactory bulb. He found that some chemicals would, at low
concentrations, excite the units of the anterior bulb more readily than the units of
the posterior bulb, whereas other chemicals excited the posterior units more
readily than the anterior units. Several esters were included in the former group of
chemicals; several hydrocarbons in the latter. In addition to this spatial differ-
entiation of odorants Adrian also reported a temporal differentiation, viz., the
multiunit discharges in response to esters had shorter durations and were more
abrupt in their growth and decay than were responses to hydrocarbons. Thus, it
appeared that the discharge of centripetal impulses leaving the bulb could perhaps
represent different odorants by a code possessing both a spatial component (the
region of the bulb giving rise to the discharge) and a temporal component (the
duration, rise time and decay time of the discharge). However, at first it appeared
that this spatial representation could not adequately encode different odorants
because, as noted above, it was only at the lowest concentrations that the different
odorants excited distinctly different regions of the bulb. At suprathreshold con-
centrations the excitation produced by any given chemical very often spread
throughout the entire bulb. Thus, over most of the range of environmental con-
centrations normally encountered, the spatial code appeared to break down. If,
however, instead of only a qualitative observation, the actual magnitudes of the
discharges elicited in different regions of the bulb were quantified with electronic
summators and then compared, it became clear that odorants were still spatially
differentiated even at suprathreshold concentrations (MOZELL and PFAFFMANN,
1954). This spatial differentiation, however, appeared to be based upon the relative
responsiveness of each of the different regions rather than upon an absolute
responsiveness of one region coupled with an absolute non-responsiveness of the
other region. For example, as shown in Fig. 1, the response produced by heptane,
a hydrocarbon, when compared to the response produced by amyl acetate, an
ester, was relatively greater in the posterior bulb than in the anterior bulb. In
other words, from the anterior bulb to the posterior bulb amyl acetate lost
effectiveness relative to heptane as a stimulus. Thus even at higher concentrations
a basis for a bulbar spatial differentiation did appear to exist.
Fig. 1 also gives graphic representation of the temporal differentiations pre-
viously observed by ADRIAN (1950). In each animal the amyl acetate response is
shorter than that of heptane. In addition, the response comes to a sharper peak
indicating a faster rise time and a faster decay time.
This apparent spatiotemporal code could serve as a major basis for olfactory
discrimination only if each different odorant could produce its own peculiar
combination of spatial and temporal characteristics. It is obvious that the data so
far discussed, comparing as it does merely the anterior and posterior regions, give
evidence of only a gross spatial differentiation. Thus, the spatial aspect of the
spatiotemporal code may appear somewhat inadequate when one considers the
Spatial and Temporal Patterning 207
large number of odorants that can be discriminated. However, when MOULTON

(1965, 1967) later sampled the simultaneous discharges from many regions of the
unanesthetized rabbit's olfactory bulb with an array of implanted electrodes, he
was able to confirm a finer grain of spatial differentiations for different chemicals.
Anter ior bulb Pos er i or bulb
A nimal A
til A ir
3 '-_______A_m_Y'"'----1A_c_e_a_t_e
I~ Amyl Aceta te H ~p t<;lne
:::-
~
Animal 0
jAlr ~
AmY I Ace a e Heptane
~~~ _________ ~ ________ ~ I I I
Fig. 1. Summator records of the discharges from the anterior and posterior regions of the
olfactory bulb following stimulation by heptane and amyl acetate, each of which is maintained
at a constant supra threshold concentration for each of the animals (MOZELL and PFAFFMANN ,
1954)
c:F=ill
n- Propo 01 10. 3
0
n-Amyl Ace ate 10- 2
I 'lll
n - Oc one 10. 2
~
er -Bu yl Aleo 0110 2
~
a' -Ionone 10- 2
~
2-Undeeonone 10- 2
n-Hep ono llO- 2 5 atole 10- 3 n-Volenc ACid 10. 1
Eugenol 10- 2 Butyl E her 10- 2

~
d-llmonene 10- 1 ~
Fig. 2. Histograms for 12 different odorants showing the sizes of the summated responses
recorded from six sites in the olfactory bulb. Numbers refer to concentrations expressed as
dilutions of the saturated vapor at 22 C (MOULTON, 1965)
208 M. M. MOZELL:
As shown in Fig. 2, when only six of these regions are scanned, it is quite apparent
that different odorants produce differently patterned profiles of discharge magni-
tude. As the author suggested, it is probably safe to assume that the central
nervous system can discern finer subtleties in the spatiotemporal discharge patterns
than can the available electrophysiological recording techniques, and since the
latter has shown a large variety of such patterns, the actual variety occurring in the
Fig. 3. A frog's right olfactory nerve as it branches over the dorsal aspect of the olfactory sac.
L lateral aspect; M medial aspect. The calibration line represents 1 mm (MOZELL, 1967)
bulb may be quite imposing. It should be pointed out, however, that not only the
odorant itself but its concentration as well can playa role in determining the exact
discharge pattern. As Moulton noted, when the concentration is increased, the
discharge magnitude at any given region for any given chemical may increase
disproportionately relative to the discharge at the other regions. Although this
finding complicates the deciphering of the bulbar pattern of activity by showing
the code to be more complex than at first thought, it does not negate the possibility
of a spatiotemporal encoding of odorants at the level of the olfactory bulb.
It was because of this apparent spatiotemporalrepresentation of odorants in the
olfactory bulb that ADRIAN (1950, 1951), in attempting to account for it, suggested
a spatiotemporal analysis of odorants at the level of the olfactory mucosa. ADRIAN
reasoned that the spatial and temporal differentiations recorded from the bulb
must have been impressed upon it by precursory spatial and temporal analyzing
mechanisms operating at the level of the mucosa. There was a sound anatomical
basis for this contention since the olfactory epithelium is, to some degree at least,
topographically represented in the bulb. Although its preciseness varies from region
to region, LE GROS CLARK (1951) nevertheless demonstrated histologically in
rabbits a regional isomorphism in the projection of the mucosa upon the bulb. For
instance, retrograde degeneration studies showed a very precise projection of the
upper and back parts of the epithelium into the glomeruli on the upper surface of
the bulb. Several other epithelial regions were demonstrated to also project to
localizable regions of the bulb although they do so more diffusely.
It is evident that thus far the most potent argument favoring a spatiotemporal
analysis of odorants at the mucosa has been the extrapolation of olfactory bulb
data to the more peripheral level. It was necessary, therefore, to obtain evidence
of such a spatiotemporal pattern of activity from the level of the olfactory mucosa
itself. In order to sample the activity in different regions of the mucosa MOZELL
(1967) took advantage of the prolific branching of the olfactory nerve at its
peripheral end (Fig. 3). After first determining that these different branches
actually supply different regions of the mucosa (MOZELL, 1964a), he recorded
simultaneously the multiunit responses to odorants from the two most widely
separated nerve branches on the dorsal aspect of the frog's olfactory sac (MOZELL,
1964 b, 1966). One nerve branch, the most medial, subserved a region near the
external naris which, of course, was among the first regions contacted by the
incoming odorized air. The other nerve branch, the most lateral, subserved a region
nearer the internal naris, thus subserving a region farther along the flow path. The
neural discharge recorded from each nerve branch was quantified with an elec-
tronic summa tor and the ratio of the discharge magnitude recorded from the
lateral nerve branch to that recorded from the medial nerve branch (the LB/MB
ratio) was used as one of the measures with which to compare the relative activity
of the two mucosal regions in response to odorous stimuli. For example, an odorant
yielding a ratio of 0.1 was interpreted as having produced a steeper gradient of
decreasing activity along the mucosa (from the external naris towards the internal
naris) than was an odorant yielding a ratio of 0.5. A second measure comparing the
relative activity of the two mucosal regions was the time lapse (or latency dif-
ference) between the onset of the responses recorded from the nerve branches
supplying each of them. The magnitude of each of these measures depended upon
the chemical presented, each of the four chemicals producing a characteristic ratio
and latency difference (MOZELL, 1966). An example of but one of the response
arrays taken from but one of the frogs is given in Fig. 4. Note that if at any con-
centration one were to rank each chemical in accordance with the ratio it produced,
octane would rank highest (since it had the largest ratio) followed in order by
d-limonene, citral and geraniol. Note also that this rank order of chemicals would
be the same no matter which concentration were chosen. A variety of statistical
tests were used to determine how closely this order corresponded from animal to
animal and a very high degree of consistency was found. Indeed, at all concentra-
tions in all animals octane most often gave the highest ranking ratios, and d-
limonene most often gave the next highest. Neither the octane nor the d-limonene
14 Rb. Sensory Physiology, Vol. IV
210 M. M. MOZELL:
ratios ever fell into the two lowest ranks. In contrast, geraniol and citral shared the
two lowest ranks but neither of them ever occupied either of the two highest ranks.
Likewise, although showing considerably more variability, especially at the lower
w
z
w
~ -../'- ~. /.,- Y.,-
2 .
:i '--'" '- ~ '- "-" '-
I
"
Fig. 4. Summated neural discharges showing the response of one frog to a single presentation
of every stimulus. Partial pressures are given along the top in terms of 10-2 mm Hg. The upper
response of each pair is recorded from the lateral nerve branch and the lower is from the medial
nerve branch. The stimulus marker shows only the onset of the stimulus. Vertical time lines
occur once every 10 seconds. The stimulus volume is 0.4 cc (MOZELL, 1966)
concentrations, the different odorants could be ranked according to the size of the
latency differences they produced on the two nerve branches. D-Iimonene and
octane gave consistently shorter latency differences than did citral and geraniol
with octane tending to give the shortest. It should be noted that the chemicals
which gave the shortest latency differences were the very same ones which gave the
smallest ratios. As will be discussed below, this suggested that these two measures
were actually measuring "two sides of the same coin." Thus, although based upon
a rather small number of chemicals, there was now evidence from the periphery of
the olfactory system (rather than by extrapolation from the bulb) that the mucosal
analysis of odorants may very well involve some spatial and temporal mechanisms
of differentiation.
Octane ...Heptaldehyde
o Nonane d-Limonene
o Butyl Acetate Benzaldehyde
+ Butanol o Furfurol
Amyl Acetate 6. Methyl Benzoate
4- Heptanone * Carvone
1.2
r=-.68
1.0 o 0--- P <.01
o
0.8
..
~
d 0.6
n::
m
~ 0.4
co
....J
0.2 - 06.
o +______ L -_ _L -_ _~
*
0.1 05 1.0 25
Relative retention time
Fig. 5. The LB(MB ratios produced by each of twelve different chemicals. All the odorants are
presented at the same concentration (a partial pressure of 56 x 10- 2 mm Hg) and at the
same volume flow rate (20.6 cc(min). The correlation coefficient (r) and its probability (P) are
noted. When the symbols for a particular chemical are connected by dashed lines, that chemical
has more than one peak in its chromatogram; the symbols then represent the peaks with the
shortest and longest retention times (MOZELL, unpublished but similar to figures in MOZELL,
1970)
MOZELL (1970), in order to determine the generality of this possible spa-

tiotemporal analysis, pursued this line of investigation with a larger sample of
odorants. At the same time he tried to determine more precisely what role con-
centration and flow rate might play in the development of the spatiotemporal
activity across the mucosa. As seen in Fig. 5, each chemical did elicit a character-
istic gradient of activity across the mucosa as measured by the LBjMB ratios.
However, the ratios produced by several of the chemicals appeared to increase
somewhat as a function of the concentration and flow rate at which they are
presented. This could be embarrassing to the concept of a spatial analysis of
odorants for if the ratios do not predominantly carry information concerning the
odorant identity per se but are also considerably confounded with information con-
cerning concentration and flow rate, the ensuing code might be of little use to the
14*
212 M. M. MOZELL:
central nervous system in the identification of odorants. However, quantitative

tests, including the analysis of variance, showed that although both concentration
and flow rate do indeed playa role in the determination of the ratios, the magnitude
of their effects are actually quite small especially when compared to the effects
produced by varying the chemicals themselves as illustrated inFig. 5. In comparison,
each ten-fold increase in concentration increased the ratio by only 0.09; a ten-fold
increase in flow rate was calculated to increase the ratio by only 0.16. Therefore,
although an odorant's concentration and flow rate may modify to some degree the
gradient of activity which it elicits across the mucosa, the general level of this
gradient of activity is, in the first instance, determined by the odorant itself.
Indeed, it is interesting to speculate that the slight change in ratio that did occur
with variations in concentration may be just enough to explain the common
observation that the quality of an odorant often changes slightly as its concen-
tration is altered.
It is apparent from the foregoing that enough evidence has by now been
accumulated to give at least some credence to the suggestion that the analysis and
coding of odorants at the level of the mucosa involves a spatiotemporal organization.
This suggestion, however, makes it almost imperative that at least some attention
be given to the mechanism by which different odorants can give rise to the different
gradients of activity which have been observed across the mucosa.
In an attempt to explain the spatiotemporal differentiations he observed in the
olfactory bulb, ADRIAN (1950), extrapolating to the periphery, suggested that the
molecules of different odorants might spread across the olfactory mucosa in dif-
ferent spatial and temporal patterns in accordance with those molecular properties
that are able to affect their progress. Such properties included their diffusion rates
in air and their solubilities in mucus. Later BEIDLER (1957), being more specific,
suggested that the progress made by the molecules of different chemicals might be
determined by the binding strengths with which they are adsorbed to the recep-
tors; those adsorbed least strongly would move farther and more rapidly across
the mucosa than those adsorbed most strongly. MONCRIEFF (1955) had already
shown for the nasal epithelium in general, if not for the olfactory epithelium in
particular, that chemicals do indeed differ in the facility with which their molecules
can migrate across it. He prepared the nasal passageways in the head of a freshly
sacrificed sheep such that odorized air pumped into one nostril emerge from the
opposite nostril. He could then measure the elapsed time between the introduction
of a particular odorant into the first nostril and its detection by human subjects as
it emerged from the second nostril. These times varied considerably from chemical
to chemical, thus indicating differential adsorption of chemicals by the nasal
epithelium. However, as will be discussed below and has been demonstrated by
MONCRIEFF (1955), such a differential adsorption phenomenon can occur on a
variety of non-biological materials as well as on a variety of biological, but non-
olfactory, tissues. In order to maintain that this phenomenon may have some
biological significance, such as in olfactory discrimination, it would be necessary
to demonstrate its reflection in the neural discharges transmitted to the central
nervous system.
Such a demonstration is given by MOZELL'S data (1964a, 1966, 1970) which also
gives evidence from the level of the olfactory mucosa that the molecules of dif-
ferent odorants differ in their abilities to migrate across it. He recognized two very
plausible mechanisms which could explain his previously described observation
that each chemical produces a characteristic gradient of activity across the mucosa.
First, it was possible that from one region of the mucosa to another there may be
a disproportionate change in the relative number of receptors particularly sen-
sitive to each of the chemicals. Secondly, the mechanism may not at all depend
upon the selective sensitivities of the receptors per se but rather, as suggested here,
upon the facility with which the molecules of different chemicals migrate across
the mucosa. To determine which of these two alternatives was the more likely,
MOZELL (1964a) reversed the flow direction of the odorized air across the mucosa,
i.e., the odorized air was made to flow from the internal naris to the external naris.
For each chemical the resulting pattern of relative discharge magnitude recorded
from the two nerve branches were the reverse of the pattern produced when the air
travelled from the external naris to the internal naris. That is, the nerve branch
yielding the larger response when the odorant was introduced through the external
naris became the one yielding the smaller response when the odorant was intro-
duced through the internal naris. Unless one were to assume some unlikely con-
trivance such as one side of a receptor being more sensitive to a given chemical than
the other side, this reversal of pattern as a result of the reversal of flow direction
would have to favor the second explanation (molecular migration) over the first
explanation (topographically distributed selectively sensitive receptors) as a basis
for the observed gradients of activity across the mucosa.
If, as this data suggests, the olfactory mucosa separates and identifies odorants
on the basis of the facility with which the molecules are able to migrate across it,
it would seem warranted to make an analogy, as suggested by BEIDLER, between
olfaction and a non-biological system which, also in order to analyze and separate
chemicals, uses the same basic phenomenon of differential molecular migration.
That is, the analogy was made between olfaction and chromatography. It should
be emphasized that this analogy is made only in regard to the basic principle (viz.
the analyzing of chemicals by the propensity of their molecules to migrate along a
medium) and not to anyone particular set of operational details used in any
current laboratory application of this principle. One would expect that if the nose
did operate chromatographically, the parameters necessary for its operation would
develop to reflect its own requirements and would not necessarily strictly mimic
those of anyone currently established method. For example, it may simultaneously
combine some of the characteristics both of gas chromatography and of thin-layer
chromatography. In any case, the basic principle of chromatography is the same
and it is this basic principle that is suggested as a model for olfactory differen-
tiations.
In this regard it is interesting to note in Fig. 5 that not only are the LB/MB
ratios, as emphasized before, characteristic of the different chemicals but their
sizes are related to the facility with which the molecules of the different chemicals
pass through a gas chromatograph fitted with a carbowax column. With only one
exception, those chemicals taking the longest time to pass through the carbowax
column (i.e., those with the longest retention times) also appear to have the least
facility to migrate along the mucosa (i.e., give the smallest LB/MB ratios). As can
be seen, the correlation between the ability of a chemical's molecules to migrate
214 M. M. MOZELL: Spatial and Temporal Patterning
along the carbowax column and its ability to migrate along the olfactory mucosa
is high enough and significant enough to strongly suggest a relationship. Thus, it
might be argued that similar processes determine the migration along both these
media. That the two media are not identical in their ability to separate these
odorants is shown most dramatically by butanol, but some non-conformity is not
unexpected since the olfactory mucosa is not, after all, made of carbowax. To
determine why butanol differentiates between the olfactory mucosa and carbowax
in this regard may ultimately be extremely informative in understanding some
of the details of chemical separation at the mucosa. In the meantime, the behavior
of all the other chemicals supports the concept that an analysis by chromato-
graphic principles, similar to those operating On a carbowax column, occurs at the
level of the olfactory mucosa.
The point should be made that the proposal of a spatiotemporal analysis of
odorants based upon a chromatographic effect across the mucosa does not at all
preclude the possibility that a selective sensitivity of the receptors themselves
might also playa significant role in olfactory discrimination. Indeed, it is known
(O'CONNELL and MOZELL, 1969; GESTELAND, et al. 1965) that the individual
olfactory receptor possesses different sensitivities for different odorants and that
the spectra of sensitivities differ between receptors, albeit with much overlap.
These two possible mechanisms for discrimination, selective receptor sensitivity
and analysis across regions, need not be mutually exclusive, and by operating
together they can generate a combined code with many more permutations than
could either alone. Such a code would appear much more able to represent the
apparently vast number of discriminable odorants. It should be pointed out,
however, that One would expect some interaction between a chromatographic
process which operates On chemicals across the mucosa and the response of at least
some of the units selectively sensitive to those chemicals. Due to the chromato-
graphic effect, there would be at any given moment a gradient of chemical con-
centrations along the mucosa. This would cause the receptors positioned at various
points to respond differently to the same chemical not because they differ in their
selective sensitivities but rather because they are stimulated with different COn-
centrations. Thus, the concentration entering the nares does not necessarily specify
the concentration reaching many of the receptors. Indeed, if, as suggested in an
earlier paper (MOZELL, 1966), the initial chromatographic effect occurs in the non-
olfactory nasal passageways On the surface of non-olfactory epithelium (see
MONCRIEFF, 1955), the concentration entering the nares might not even specify the
concentration reaching any part of the olfactory mucosa. To make matters even
worse, the amount of the discrepancy could very well differ from chemical to
chemical depending upon its attraction to the surface.
Finally, it would be well to emphasize that the separation of chemicals by a
chromatographic process is not at all UnCommOn and indeed it is a naturally
occurring physical phenomenon. It occurs whenever and wherever, as in the nose,
the molecules of a substance are moved along a liquid or solid surface. Of course,
some surfaces are better at separating some substances than are others. The data
reviewed here suggest that the olfactory system in its evolution has simply come
to take advantage of this chromatographic phenomenon - a phenomenon which,
because of the inevitability of the physical principles involved, would occur quite
References 215
commonly in nature whether or not there were a living organism able to appre-
ciate it.
References
ADRIAN, E. D.: Sensory discrimination with some recent evidence from the olfactory organ.
Brit. med. Bul!. 6, 330-331 (1950).
- Olfactory discrimination. Ann. Psycho!. 50, 107-113 (1951).
BEIDLER, L. M.: Facts and theory on the mechanism of taste and odor perception. In: MITCHELL,
J. H., JR. (Ed.): Chemistry of natural food flavors, pp. 7-47. Chicago: Quartermaster
Food Container Institute for the Armed Forces 1957.
GESTELAND, R. C., LETTVIN, J. Y., PITTS, W. H.: Chemical transmission in the nose of the frog.
J. Physio!. (Lond.) 181, 525-559 (1965).
LE GROS CLARK, W. E.: Projection of the olfactory epithelium on the olfactory bulb in the
rabbit. J. Neuro!' Neurosurg. Psychiat. 14, 1-10 (1951).
MONCRIEFF, R. W.: The sorptive properties of the olfactory membrane. J. Physio!. (Lond.)
130, 543-558 (1955).
MOULTON, D. G.: Differential sensitivity to odors. In: Cold Spr. Harb. Symp. quant. Bio!.
Sensory Receptors 30, 201-206 (1965).
- Spatiotemporal patterning of response in the olfactory syst3m. In: HAYAsm, T. (Ed.):
Proceedings of the second international symposium of olfaction and taste, pp. 109-116.
New York: Pergamon Press 1967.
MOZELL, M. M.: Evidence for sorption as a mechanism of the olfactory analysis of vapors.
Nature (Lond.) 203, 1181-1182 (1964a).
- Olfactory discrimination: Electrophysiological spatiotemporal basis. Science 143,1336 -
1337 (1964b).
- The spatiotemporal analysis of odorants at the level of the olfactory receptor sheet. J. gen.
Physio!. 50, 25-41 (1966).
- The effect of concentration upon the spatiotemporal coding of odorants. In: HAYASHI, T.
(Ed.): Proceedings of the second international symposium of olfaction and taste, pp. 117 -
124. New York: Pergamon Press 1967.
- Evidence for a chromatographic model of olfaction. J. gen. Physio!. 56, 46-63 (1970).
- PFAFFMANN, C.: The afferent neural process in odor perception. Ann. N. Y. Acad. Sci. 58,
96-108 (1954).
O'CONNELL, R. J., MOZELL, M. M.: Quantitative stimulation of frog olfactory receptors. J.
Neurophysio!. 32, 51-63 (1969).
Chapter 10
Olfactory Psychophysics *
By
TRYGG ENGEN, Providence, Rhode Island (USA)
With 12 Figures
Contents
Psychophysical Scaling . . . . . . . . . . . . . 217
Adaptation . . . . . . . . . . . . . . . . . 219
Effect of the Intensity of the Adapting Stimulus. 219
Effect of the Duration of Presentation of the Adapting Stimulus 220
Recovery from Adaptation 222
Cross-Adaptation. 224
Detection of Odors . . . . . 226
Classical Theory . . . . . 226
Contemporary Detection Theory 227
The Effect on d' by Odorant Concentration 232
Individual Differences in Detection . 234
Absolute and Differential Threshold 235
Odor Quality and Classification . 236
Multidimensional Scaling 237
The Distance Model . . 237
The Content Model. . . 238
Interpretation of Factors 242
Conclusion 242
References 242
Psychophysics is the quantitative study of the relationship between stimuli

and sensations obtained in experiments with human and animal subjects. It has
always been an interdisciplinary field and is as much part of physiology as of
psychology. Without physiology, psychophysics is the study of a "black box,"
and without psychophysics, sensory physiology would only have subjective notions
about the behavioral operating characteristics of sensory systems. The limitation
of psychophysics in this respect is that it represents only a molar approach which
cannot distinguish between the effects of odorants on the fifth, ninth, and tenth
cranial nerves. Animal psychophysics is able to provide greater physiological pre-
cision by surgical removal of other sensory systems, but the present chapter must
be limited to recent data and concepts developed on human observers.
* This chapter was written while the author was supported by a Public Health Service
Fellowship (I.F3MH-39, 236-01) from the Behavioral Sciences Institute of Mental Health.
Psychophysical Scaling 217
Three basic psychophysical topics will be discussed: 1) perceived intensity as

a function of concentration, 2) odor detection and threshold, and 3) odor quality
and classification.
Psychophysical Scaling
The Power Function may be the most basic problem in psychophysics in that it
attempts to determine the mathematical form of the function relating perceived
intensity as a function of stimulus intensity for the whole effective stimulus con-
tinuum, from the weakest detectable odors to the strongest the subject can tolerate.
The most common methods for scaling is by direct estimation, for example, the
method of magnitude estimation, which requires the subject to match numbers to
the perceived effects of stimuli (STEVENS, 1966). The numerical judgments are
usually averaged for groups of 10 to 20 subjects, although individual functions
have also been studied, and the averages are plotted as a function of the con-
centration of the stimuli. These methods are very direct and only assume that the
subjects are able to quantify perceived intensity at a simple arithmetic level (see
ENGEN, in press, for a general discussion of this methodology). Data obtained
with such estimation methods have shown for a variety of odorants, including both
pure compounds and coffee odor, that perceived odor intensity conforms to the
power function (STEVENS, 1957) which states that
(1)
where R is perceived intensity, S stimulus intensity, and k and b are constants,

which refer to the intercept and slope respectively when the averages are plotted
in double logarithmic coordinates. Expressed in logarithmic terms the function
becomes
Log R = b (Log S) + Log k . (2)
The exponent b is the most interesting parameter in that it seems to distinguish

between different sense modalities. For example, its value is about 0.33 for visual
brightness compared with about 0.60 for odor intensity.
CAIN (1968) has performed an extensive study of the psychophysical scale in
olfaction with a series of homologous alcohols. He presented these odorants diluted
in air by an olfactometer. Concentration was controlled by flowmeters and was
delivered to the subject through Teflon tubing at a flowrate of 4 or 6 11m. A series
of relays and variable timers operated solenoid valves for precise timing of stimuli,
and the subject's breathing through the nose was paced by a metronome set at
30 c/m. The subjects (students) were instructed to estimate the intensity of the
smell of each of seven different concentrations presented twice each and in an
irregular order. Each subject was instructed to describe each stimulus with a
number he judged to be proportional to its subjective intensity and to make the
ntios of the numbers assigned to different stimuli correspond to the ratios of
subjective intensities. The judgments were transferred to a common reference
number of 10; for example, if the subject said, "100," this estimate was multiplied
by 0.1, if 5 by 2, etc. Fig. 1 presents the data obtained for pentanol which is
representative. The linear fit in logarithmic coordinates indicates that the data
conform to a power function.
218 T. ENGEN: Olfactory Psychophysics
Since the value of the exponent of this function (0.58) is less than 1, perceived
intensity increases as a negatively accelerated function of concentration in linear
coordinates. As the concentration of pentanol is doubled, perceived intensity is
increased only by a factor of approximately 1.7, and this ratio of 2/1.7 between
concentration and odor intensity appears to be constant throughout the range of
values. In other words, the olfactory system compresses the stimulus input.
In addition, the overall subjective range of odor intensity appears to be short
both when measured electrophysiologically (MOZELL, in press) and psycho-
physically (ENGEN and McBURNEY, 1964). It is also noteworthy that the exponent
2.0..-------------.
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0. 1
-0.84 -0.54 -0.24 0.0.6 0.36 0.66 0.96

Log concentration (mg / I)
Fig. 1 (from CAIN, 1968). Perceived odor intensity as a function of the concentration of pentano]
has been shown to be related to the water solubility of the odorant. CAIN (1968)
obtained an almost perfect rank-order correlation between the size of the exponent
and the water solubility of his own odorants and those scaled by other investigators
(JONES, 1958a and b; REESE and STEVENS, 1960; ENGEN and LINDSTROM, 1963;
ENGEN, CAIN, and ROVEE, 1968). The exponents range approximately from 0.3 to
0.7 with air dilution of the odorants.
If concentration is varied by liquid dilution in comparable units, the value of
the exponent is approximately halved. The reason for this important observation
is not entirely clear. STONE (1963) obtained more reliable threshold data with an
olfactometer than with sniff-bottles, where concentration depends on the ideality
of the solution, and this provides at least part of the answer.
It should be mentioned that the exact form of the psychophysical function
depends to some extent on the method by which judgments were obtained. FECH-
NER (1860) proposed a logarithmic psychophysical law such that a straight line
should be obtained for the data plotted in Fig. 1, if average judgments were plotted
as a function of the logarithm of concentration in a semi-logarithmic plot. Obvi-
ously, that function is not supported by the present data, or any other obtained
with direct methods in olfaction, but it is always possible that a logarithmic or
some other function will describe the data obtained in another judgmental task.
FECHNER based his theory on the subjects' abilities to discriminate very small
stimulus differences, a problem which will be considered below in connection with
detection. There is no independent, external criterion for deciding the problem
of validity of the subject's judgment and, hence, the nature of the "true" psycho-
physical function. However, the power function has fared very well in numerous
experimental tests, and on that basis it may be concluded that it is the best
available description of how human subjects describe the growth of intensity with
increase in concentration. It also agrees well with comparable electrophysiological
data (VON SYDOW, 1968).
Adaptation
Adaptation is perhaps the most important problem in psychophysics. Olfaction
is often used as a textbook example to illustrate sensory adaptation. It is often
claimed that while some senses (hearing, for example) are very stable, olfactory
sensitivity decreases very readily when exposed to a constant stimulus. The
typical anecdote used as illustration of this involves a person entering a room with
a noticeable or even strong odor, which decreases and then disappears after only a
short time in the room. The most common work cited in support of this strong
adaptation hypothesis is ZWAARDEMAKER'S work on threshold in 1895 (cf. PFAFF-
MANN, 1951). More recent work on suprathreshold stimuli seems to be in general
agreement with this view (e.g., STONE, 1966). One may not have come to this
conclusion on the basis of physiological evidence, for it has been shown in various
animal preparations that the olfactory system responds with good consistency
even after prolonged stimulation (e.g., OTTOSON, 1956). BEIDLER (1957) has sug-
gested that adaptation may be mediated by a central mechanism.
Effect of the Intensity of the Adapting Stimulus

After having established the psychophysical functions under conditions design-
ed to prevent adaptation with sufficient time between stimulations (60 sec), CAIN
and ENGEN (1969) observed the effect of adaptation by exposing the subjects to
three inhalations of a concentration of the odorant immediately prior to judging
the same test concentrations (Fig. 1). The subject would inhale the adapting con-
centration from one Teflon tube and upon signal would move his head over
6 inches while exhaling. He would then inhale the test concentration from another
Teflon tube, and then make his judgment. For pentanol the relatively low, medium,
and high adapting concentrations were 0.5,2.1, and 9.2 mg/I. The subjective ratio
between adjacent pairs of these adapting concentrations is 2.25 as determined
from the psychophysical function in Fig. 1.
The adaptation results are presented in Fig. 2. As a result of adaptation, the
psychophysical function becomes steeper, and the magnitude of the adaptation
effect is related to the concentration of the adapting stimulus. Based on work in
vision and audition (STEVENS and STEVENS, 1963) one would expect adaptation to
produce a deviation from a straight line in a log-log plot by an increase in the
steepness of this function for low concentrations. This effect is illustrated in Fig. 3.
This adaptation effect has suggested reformulation of the power function by
adding a constant representing the threshold (or "physiological zero"). The
modified power function is

(3)
where So refers to an estimated threshold and (S - So) indicate effective stimulus
values. If one corrects for threshold, the curvature in Fig. 3 will be eliminated and
the power function restored. The essential effect of adaptation then is to increase
threshold and the exponent of the function. The present adaptation data cannot
be described that simply. Instead of becoming progressively steeper near threshold,
lOr------------------------, 2.0 , - - - - - - -- - - - - - ,
:::iii 15
- - -
o
unadapted
low adaptation ~ 15
c:
2!
c:
c medium adaptalion
high adaptation
'"
C
~ 1.0
~
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o ~~~~~~~~~~~~ O~~~~-L __~L-_ _~_ _ _ _~

-084 -054 -024 006 036 066 096 -10 05 10
Log concentration (mg t I) Log concen rat ion ( mg t I)
Fig. 2 Fig. 3
Fig. 2 (from CAIN, 1968). Effect of adaptation with concentrations of 0.5 (0), 2.1 (0) and
9.2 (x) mgjl on the psychophysical function for pentanol shown in Fig. 1. Each adapting
stimulus was inhaled three times before presentation of each of the seven test concentrations.
When less than seven points are plotted it means that medium judgments of zero were obtained
for the missing points, that is, under adaption the lowest concentrations cannot always be
detected
Fig. 3. Expected effect of adaptation on perceived odor intensity plotted against the concen-
tration of the test stimulus as the adapting concentration increases from a relatively low to a
high value
the present function (Fig. 2) has a tendency to flatten, although other adaptation
functions obtained by CAIN (1968) were like those in Fig. 3. The reason for this
may be that as the odor becomes harder to detect, the subject takes deeper breaths
and thereby increases the flowrate, and both flowrate and concentration have been
shown in electrophysiological studies to determine the summated neural discharge
(TUCKER, 1963). Further research with human subjects should be able to control
this variable by monitoring breathing pneumographically, but it is not unlikely
that the deviation from the power function is caused by changes in the subject's
responses to weak stimuli (see discussion of threshold below).
Effect of the Duration of Presentation of the Adapting Stimulus

The effect of duration was studied by exposing the subject to 5, 8, and 15
inhalations of either the low (0.5 mg/l) or medium (2.1 mg/I) adapting concentra-
tions compared with 3 inhalations used in observing the effect of intensity on

adaptation. (One cycle of inhalation and exhalation corresponds to 2 sec.) Although
there possibly was a tendency for the exponent to increase as a function of the
number of inhalations, it could not be concluded that the effect was reliable.
Graphically, these adaptation functions did not deviate appreciably from the
adaptation functions shown in Fig. 2 which were obtained with 3 inhalations.
The variability of the data may conceal potentially significant differences, but,
in any case, relatively large differences in adapting time seem to produce much
smaller decreases in perceived intensity than moderate differences in the concen-
tration of the adapting stimulus. (It should be noted that all the effects described
here were replicated by CAIN on other alcohols.) Presumably, much longer durations
of constant stimulation would be required to show the decline in sensitivity which
is believed to be characteristic of olfaction.
This kind of experiment has been reported by EKMAN, BERGLUND, BERGLUND,
and LINDVALL (1967) and in some pilot experiments by STONE (1966). EKMAN et al.
used a direct scaling method similar to CAIN'S in order to assess the intensity of
the perceived odor of hydrogen sulphide (in consideration of the possible application
of these results to environmental hygiene). The subjects (students) breathed at a
normal rate of 12 to 20 inhalations per min. The odorant was controlled by an
olfactometer and presented to the subject in a Teflon hood at 90 11m and evacuated
by a fan, which produced a slight negative pressure in the hood. The hood was
fitted with an oval opening, which made a snug fit against the subject's face. The
concentrations of the odorant were varied by adding various amounts of the
odorant in a constant flow of nitrogen which in turn was diluted by purified air.
The results were essentially the same for all concentrations. and only the data
obtained for the medium adapting concentration of 2.6 ppm will be presented here.
This odorant was presented to the subject for a period of 12 min which was followed
by a recovery period of 12 min during which fresh air was presented in the hood.
The recovery period was interrupted by intervals of 4 sec during which the odorant
was presented. The stimulus was always the same, but the subject was not informed
about the variables studied in the experiment. During the adaptation period the
subject was requested to estimate perceived intensity after 1/4, 1/2, 3/4, 1.0, and
2.0 min and every minute thereafter for a total of 12 min, and then at 1/4, 1/2, 3/4,
1.0, 4.0, 8.0, and 12.0 min during the recovery period. (Since the recovery data
were based on fewer observations and are therefore less reliable the judgments for
1/4, 1/2, and 3/4 min were combined and plotted at 1/2 min in the final analysis.)
Four naive subjects participated, and each subject was tested 10 times with a
minimum of 24 hours between tests. All subjects indicated the same functions,
and their data have been combined in Fig. 4.
The longer the exposure to the odorant the lower is the perceived intensity of
that odorant. The decrease in perceived intensity is relatively rapid at first but
then more gradual. It is interesting, and perhaps important, that perceived
intensity for the average subject did not reach zero and does not appear likely to
do so with this concentration. Perceived intensity approached zero only in case of
one subject, but to the other three it retained its stimulating effectiveness at a
distinctly higher level. Very similar results were obtained for 0.7, 0.9, and 6.4 ppm
which sample adequately the whole effective stimulus range for this compound.
Threshold was estimated at 1.60 X 10-2 ppm, and 10.0 ppm was estimated to be
the useable maximum for the test situation (personal communication by the
authors).
The adaptation function is well described by the exponential function
R = a + bjcT , (4)
where R is perceived intensity, T the duration of the stimulus, a the asymptote of

the adaptation function, and a + b the initial value of the function at T = 0
(EKMAN et al., pp. 181-182).
I
I
I
I
I
?: I
III 50 I
: .,9.... -----6"-----0-
C
2 : 1''-
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I I
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41
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15
'>-"-<>..a.....:1'"'O'".o..-Ci.
u
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0 4 8 12
Time (min)
Fig. 4 (from EKMAN et al., 1967). Adaption and recovery of the perceived intensity of hydrogen
sulphide at a concentration of 6.4 parts per million as a function of duration. The horizontal,
dotted line indicates the cessation of hydrogen sulphide and presentation of fresh air. The
results are the mean of four subjects
In general, these data seem consistent with the physiological data referred to
above. They do not seem to support the hypothesis that odor sensitivity is likely
to disappear all together under constant stimulation. Tests with other odorants
would be valuable.
Recovery from Adaptation

Fig. 4 also shows that recovery of sensitivity is relatively rapid. Complete
recovery to the preadaptation level is reached after 4 min and possibly earlier in
an apparently negatively accelerated growth function. The necessity of introduc-
ing the odorant for recovery tests precludes obtaining exactly comparable data.
The stimulation during those intervals would tend to retard the growth of per-
ceived intensity. KOSTER (1968) has proposed that the rate of recovery also depends
on the nature of the odorant.
CAIN'S procedure allowed for more convenient observation of recovery after
short intervals. In his situation it was measured in terms of the number of inhala-
tions of room air following a defined number of inhalations of the stimulus prior to
the inhalation of the test concentration. Fig. 5 presents the results which show that
after only 3 inhalations (or approximately 6 sec) there is a noticeable change in the
psychophysical function brought about mainly by increase in the perceived
intensity of the lower concentrations which are most influenced by adaptation.
This recovery seems rapid; for example, VON BEKESY (1964) observed that per-
ceived odor intensity immediately following the presentation of a stimulus grows
relatively slowly and might require several seconds to reach maximum intensity.
On the basis of studies in a number of other sense modalities, EKMAN and his
colleagues (e.g., EKMAN, FROBERG, and FRANKENHAEUSER, 1968) have shown that
2.0.
-
>-
Ul
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2
1.5
8 beaths of adaptation
o 3 beaths of recovery
"C
ClI
1.0.
.>
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ClI
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0.5
-l

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- 0..84 -0..54 -0.24
Log concentration (mg III
Fig. 5 (from CAIN, 1968). Perceived odor intensity of pentanol after eight inhalations of a
2.1 mgjl concentration (.) and after eight inhalations of this concentration followed by three
inhalations of fresh air (0). The straight line is the nonadapted function in Fig. 1
perceived intensity of suprathreshold stimuli is a simple logarithmic function of

duration
R = a + blog T, (5)
where R is perceived intensity and T is duration of stimulation. Knowledge of this
relation should be an interesting supplement to the data presented in Fig. 4.
Temporal relations would be useful in olfaction as they have been in other sense
modalities, e.g., the dark adaptation function in vision.
One note of caution may be worthwhile regarding time-course experiments.
It is well known in psychology that a subject's expectation of what will happen
will introduce a response bias for him to "perceive" it. (This topic will also be
discussed below in connection with detection.) For example, in the adaptation
experiment by WOODROW and KARPMAN (1917) the subjects were instructed to
report when the odor of a constant stimulus disappeared. Such instructions will
instill in the subject the experimenter's expectation that the odor will indeed
disappear. Even without specific instructions which may introduce a certain per-
ceptual bias, the subject may have his own conception of the situation or develop
one during the course of the experiment and respond accordingly. STUIVER (1958),
for example, served as his own subject. Psychologically, the problem is not that
seeing - is-believing but the other way around. The earlier studies (cf. PFAFFMANN,
1951) tended to show change in sensitivity as a linear function of time, but the
linearity may be at least partly the result of response bias. The work of both
EKMAN et al. (1967) and CAIN (1968) represents great improvement in this aspect
of the methodology. Their instructions were designed to prevent such response
biases from occuring, and in addition the direct estimation methodology provides
the opportunity for maximum response variation. Nevertheless, further work on
the problem of response bias in the time-course experiments would be useful. One
must make sure that the psychophysiological relation entails the proper psycho-
logical variable, and this is especially important in the difficult task of detection
which will be considered later.
Cross-Adaptation
The adaptation effect discussed so far may be more precisely described as self-
adaptation in that perceived intensity of an odorant is affected by prior exposure
to the same odorant. Cross-adaptation involves prior exposure to an adapting
odorant before presenting a different test odorant. Both procedures are based on
the same assumption about the effect. In self-adaptation it presumably results
from some form of fatigue to continued or repeated stimulation of the same receptors.
Cross-adaptation has therefore been considered evidence that different odorants
stimulate the same receptors (LEMAGNEN, 1948; MONCRIEFF, 1956; CHEESMAN and
TOWNSEND, 1956). It has been hoped that the stimulus classification resulting
from cross-adaptation may lead to the discovery of a common molecular attribute
for each class which presumably stimulate specific receptors. Lack of cross-
adaptation indicates the functioning of different receptors. "Selective adaptation"
is another term for the effect.
This apparently logical approach has not fulfilled its promise. Although definite
cross-adaptation effects have been observed, the degree of cross-adaptation does
not appear to vary systematically with, for example, the degree of physical
similarity of the compounds as defined by a series of linear homologous alcohols.
Several properties of the molecule, e.g., water solubility, vapor pressure, etc. might
operate simultaneously but at different levels for different odorants (ENGEN, 1963).
In addition, it was mentioned earlier that adaptation may depend upon both
central as well as peripheral factors. The most general weakness of the reported
experiments on cross-adaptation has been the lack of control of the perceived
intensity of the odorants tested. Most of the work has been limited to the so-called
absolute threshold, but one cannot use threshold concentration as a unit with
which to measure perceived intensity, unless one is willing to make the question-
able assumption that the exponent of psychophysical functions is constant for
different odorants (see above). Another procedure is to work with constant concen-
trations, but this clearly does not guarantee the equivalence of perceived intensities
required in order to observe the effect of quality (MONCRIEFF, 1957). The concen-
tration of the adapting and test odorants, respectively, should be selected from
comparable psychophysical functions (CAIN and ENGEN, 1969). One can then
determine if (1) cross-adaptation has the same kind of effect as self-adaptation,
and if (2) two odorants cross-adapt each other to the same extent as one would
expect from a simple, unidimensional hypothesis of selective adaptation.
CAIN and ENGEN (1969) performed cross-adaptation experiments which
were identical to the self-adaptation experiment except that a different alcohol
was used as the adapting stimulus. Propanol was used to adapt pentanol and vice
versa. The same relatively low, medium, and high concentrations (0.5, 2.1, and
9.2 mg/l) were used as pentanol-adapting stimuli in scaling seven concentrations of
propanol with the method of magnitude estimation. Three adapting concen-
trations of propanol were also selected to be used in scaling seven concentrations
of pentanol under cross-adaptation. These relatively low, medium and high pro-
panol concentrations were 2.0, 6.3, and 21.6 mg/l, and the perceived ratios between
adjacent pairs were 2.25. That is, they were matched to the three pentanol adapt-
ing concentrations in perceived intensity such that 0.5 and 2.0, 2.1 and 6.3, and
9.2 and 21.6 mg/l of pentanol and propanol respectively are of equal perceived
intensity but of different quality.
20.
15 - - - unadapted
o low cross-adaptation
>.
o medium
iii
c ~ h'9h
III .0.
S
-0
>
III
Q;
c3- cs
t 0.5
III
a.
m
0
--'
0.
- 0..5 L........::-':-:----::-::-:---::c':-:---::-::-::----::-::-::----="::-::--::-:
-0.64 -054 -0.24 0.06 0.36 0.66 0..96 -0.06 016 0.48 0.76 lOS 138 l68
Log concentration (mg I II
Fig. 6. The effect of cross-adaptation of propanol (C a) and pentanol (C5 ). Perceived intensity is
based on the median judgment of 18 subjects (not the same as in Fig. 1). The high (x) adapt-
ing concentration was inhaled three times and the low (0) and medium ( 0) eight times before
each of the test concentrations. The straight lines show the nonadapted functions for these
odorants. See text for details
After some inhalations of one of these adapting concentrations of pentanol, for

example, through one Teflon tube, the subject would exhale, and in the next
breath inhale one breath of one of seven different concentrations of propanol from
the other Teflon delivery tube and make his judgment of its perceived intensity.
The subject was not aware of the nature of the experimental conditions. The order
of presentations of the stimuli were irregular as before. The various combinations
of adapting and test stimuli were presented in different sessions with at least
90 sec between any pair of adapting and test stimulus in order to allow recovery of
adaptation between trials.
The results are presented in Fig. 6 together with the psychophysical functions
obtained for both pentanol (Os) and propanol (0 3) without any prior adaptation.
The graph shows that the effect of cross-adaptation seems to be the same as the
15 Hb. Sensory Physiology, Vol. IV /1
226 T. ENGEN: OUactory Psychophysics
effect of self-adaptation; that is, the psychophysical function becomes steeper as a

function of increase in the concentration of the adapting stimulus. On the other
hand, the effect of cross-adaptation is found to be smaller than the effect of self-
adaptation, as MONCRIEFF (1956) has observed. This can be seen by comparing the
self-adaptation data for pentanol (Fig. 2) with the cross-adaptation data for pro-
panol (Fig. 6) for matched adapting concentrations. This difference between self-
and cross-adaptation holds also for propanol which was more affected by cross-
adaptation than pentanol. Cross-adaptation of propanol by pentanol at medium
concentration (Fig. 6) is approximately the same as self-adaptation of pentanol at
the low adapting concentration (see CAIN and ENGEN, 1969).
While some aspects of the data are consistent with the rationale of the cross-
adaptation method, the fact that the effect of cross-adaptation is asymmetric
demands a more complex multidimensional approach in interpreting the infor-
mation obtained from cross-adaptation into fundamental classes of odorants.
Multidimensional scaling, which will be considered below, represents a new
approach to classification where a direct approach to odor similarity replaces the
indirect approach through cross-adaptation. What may even be more difficult to
subsume under the rationale of the cross-adaptation method are recent findings
that, while inhibition or adaptation of one stimulus by another is the usual finding
in cross-adaptation experiments, cases exist in which prior exposure of one odorant
will facilitate or increase the preceived intensity of the other odorants (ENGEN and
BOSACK, 1969; CORBIT, 1969).
A potentionally important generalization is indicated by the present findings
that self-adaptation and cross-adaptation affect the psychophysical function in
the same way, and that different odorants show equivalent self-adapting effects
for adapting concentrations matched in perceived intensity. That is, the degree of
adaptation may be predicted by knowing the psychophysical intensity of the
odorant independently of its perceived quality. A study of more odorants to test
this generalization would be constructive, and a more precise quantitative descrip-
tion of the extent and course of adaptation should be possible in terms of the
parameters of the psychophysical function. According to formula (3) the effect of
adaptation would be expected to decrease constant k and increase constant So
andb.
Detection of Odors
Classical Theory
From the beginning the concept of absolute threshold played a major role in
psychophysics and psychophysiology (PFAFFMANN, 1951; MONCRIEFF, 1954). There
is presumably a minimum stimulus value required to initiate a response from a
subject. The methods for determining this value were developed in order to
evaluate stimulus-response relations according to the theory that the lower the
threshold, the more efficient the stimulus. Such a program has never been suc-
cessful in olfaction. Thresholds do not predict responses to suprathreshold stimuli
(e.g., ENGEN, 1965), and thresholds for the same compounds have been found to
vary greatly for both different psychophysical methods and techniques of dilution
(cf. MONCRIEFF, 1954).
Detection of Odors 227
According to classical threshold theory, a stimulus reaching the receptor

initiates a neural response which affects a center in the brain. The magnitude of
this effect will vary with the intensity of the stimulus, the sensitivity of the
receptor, the efficiency of the neural connections, and the background activity of
the center. If the stimulus effect is of a certain magnitude on a trial, the center will
respond and lead to a conscious preception of the stimulus. The minimum stimulus
intensity which produces this preception represents the so-called momentary
threshold. The various factors listed above will produce random variation in the
momentary threshold over trials, and this variation can be described by the normal
distributions. When the probability of detection is plotted against stimulus
intensity, a function will be obtained which corresponds to the normal cumulative
distribution, which in olfaction sometimes is described as the "frequency of smell-
ing" distribution. The mean of the distribution of the momentary thresholds is the
statistical definition of the absolute threshold. In classic theory it was assumed
that the important independent variable was the stimulus intensity, and that the
use of carefully trained observers eliminated the effect of extraneous, non-
stimulus variables.
Contemporary Detection Theory

This classic theory now seems too simple, because it has been shown that in
order to evaluate the effect of the stimulus it is necessary to measure and control
certain psychological variables (GREEN and SWETS, 1966). The two most important
variables are the subject's expectation that a stimulus will be presented and the
consequences of his detection performance. This approach may be especially useful
in olfaction where the lack of reliability is well-known and where the likelihood of
a false alarm (false positive) is great. SLOSSON (1899) demonstrated long ago the
ease with which olfactory hallucination can be induced by showing that his
subjects readily agreed that distilled water had a very disagreeable odor. This
"trick" works so well that it has been used in television audience-participation
shows. It is obvious to anyone who has served as a subject in a threshold experi-
ment that it is not usually clear whether or not a stimulus was perceived. When it
is known that a stimulus was presented, as in typical classical procedures, the
subject is inclined to expect and thus to agree that some perceptual effect took
place. Threshold is not normally experienced as a sharp point but appears to be a
region where stimulus effects vary vaguely between the perceptible and the
imperceptible. For this reason the subject himself must decide where in that region
to establish a criterion whereby he can categorize his perceptions into "Yes, I
smelled it," and "No, I did not smell anything." Detection theory rejects the
threshold theory of classical psychophysics and proposes that weak as well as
strong stimulation produce a continuous perceptual effect. Classic theory and
detection theory agree that the detection task is influenced by variability or
"noise," but their accounts of variability are basically different.
Detection theory assumes that the stimulus always has an effect, but its effect
must be assessed against the effect of noise and background factors which also
have perceptual effects. Noise is always present in the form of extraneous olfactory
stimulation. The design of a Camera Inodorata has always been somewhat of a
high but unrealistic ideal. Noise may be contributed by the subject, the environ-
ment, or spontaneous activity of the system; it may be introduced on purpose by

the experimenter in a masking or cross-adaptation experiment, and it may result
from variability in the stimulus in that a different number of molecules actually
reach the epithelium at each trial even though a constant stimulus is presented.
Noise (N) is assumed to stimulate the sensory system and produce a normally
distributed perceptual effect which can be described by its mean effect as shown
on the left in Fig. 7.
?:
'iii
c
<11
"0
~
:ac
.0
o
!l:
Hypothetical perceptual dimension
Fig. 7. Hypothetical sensory effects of noise and stimulus in detection theory
The effect of the stimulus (8) is always added to the noise (8 N) level present
at the moment and causes a displacement of the noise distribution as shown on the
right in Fig. 7. The weaker the stimulus the smaller will be the displacement. The
perceptual effect of the stimulus then should be measured as the difference between
the two means:
(6)
where d' is a measure of stimulus effect, MSNis the mean of the stimulus-plus-noise
distribution, M N the mean of the noise distribution, and aN the standard deviation
of the noise distribution which is assumed to be equal to the standard deviation for
the stimulus-plus-noise distribution (aSN). Finally, it is assumed that in an actual
experiment the subject uses conditional probabilities associated with the per-
ceptual effects in deciding whether or not a stimulus was presented at a given trial.
Table 1 illustrates a simple experimental situation and the results of a simple
Yes-No detection experiment. Assume that a weak concentration is presented on
series of trials and randomly mixed with so-called blanks, consisting of the diluent
used in the experiment, which defines the meaning of noise (N) in this situation.
A trial is indicated by the experimenter, and the subject must decide by smelling
alone whether or not a stimulus was presented. Since there are only two alternative
responses and stimulus conditions, all the information of the matrix is contained in
the cells containing the proportion of hits and false alarms. These represent the
conditional probability that the subject would correctly respond "Yes" when the
odorant was presented, p(y/8N), and the conditional probability that the subject
would respond "Yes" when the diluent was presented, p(yJN). The proportion of
false alarms indicates the likelihood of the subject responding that he per-
ceived the odorant when noise alone was presented. Fig. 8 illustrates the location
Table 1. Stimulus-response matrix in a Yes-No detection experiment
Response alternatives
Yes No
SN p (yjSN) p (njSN)
Stimulus Hit Miss
alternatives
N p (yjN) p (njN)
False alarm Correct rejection
p (yjSN) + p (njSN) = 1.0
p (yjN) + p (nfN) = 1.0
of these proportions in the model. In part A the subject responded "Yes" on

approximately half of the noise trials, but in part B, presumably under other
circumstances, he hardly ever responded "Yes" on noise trials. It should be
stressed that in the latter case the subject also responded "Yes" less often than the
odorant was actually presented. The difference between part A and B illustrate a
relatively low versus high criterion in deciding whether or not an odorant has been
presented. It is important to keep in mind that the effect of the criterion is independ-
ent of the stimulus effect on detection; the same stimulus was used in part A and
B, and the separation between the two distributions is constant. For each criterion
False alarm
.
<II
C
"C
.D
o
.D
o
ct
Criterion B
Hypothellcal perceptual dimension
Fig.8. Schematic representation of the predicted effects of different judgmental criteria..

The proportions of hits and false alarms vary but the separation between the distribution is
unaffected
230 T. ENGEN: OHactory Psychophysics
adopted by the subject for saying "Yes," he will produce a different pair of pro-
portions of hits and false alarms. According to the theory a plot of these proportions
of hits against the proportions of false alarms should generate the curved function
shown in Fig. 9. The parameter of this function is d' which can be determined by
use of a table relating proportions to standard scores (z-scores) under the normal
curve, by subtracting false alarms from hits, and by using the standard deviation
1.0r------------=~
0.8
r-::l
Z
lo.6
VI
en
~ 0.4
0.2 0.4
False alarms [~YI N)]
1.0
Fig. 9. Schematic representation of the predicted relationships between the proportions of hits
and false alarms according to classical theory and modern detection theory
(0") of formula (6) as the unit of measurement on the hypothetical perceptual

dimension according to the formula
d'= z(y/SN) - z(y/N) , (7)
for example, if p(y/SN) , the proportion of hits, is 0.83 and p(y/N) , the proportion
of false alarms, is 0.31, d' = 0.95 - (- 0.50) = l.45. The function shown in Fig. 9
represents a single stimulus intensity and thus the same intensity and one value of
d'. For that reason it is called an isosensititivity function or ROC curve (for
receiver operating characteristic). Different points on the curve represent different
criteria: In the lower left corner the subject is very reluctant but in the upper right
corner he is very willing to respond "Yes." Which criterion the subject will use is
partly determined by the consequences of his judgment, which may be analyzed
with a so-called payoff matrix. If the subject is penalized for making a false alarm,
he will tend to say "Yes" relatively rarely, but if he is rewarded for each hit
without regard to false alarm, he will be more likely to respond "Yes," etc.
SEMB (1968) has shown support for this assumption about the criterion in the
detection of odor by demonstrating the ease with which subjects can change their
performance to conform to criteria which were arbitrarily selected by the experi-
menter to correspond to threshold. Threshold was first determined conventionally
by a method of limits, and the subjects (students) were paid the usual hourly rate
for their work. In the next experiment the same subjects were paid for their per-
formance according to a payoff matrix with criteria which were sometimes higher
and sometimes lower than the previously measured threshold. The subject was
given no other information about the experiment but was rewarded with 2 cents
for saying "Yes" when the concentration presented was higher than the criterion
point for that session and "No" when it was lower. For either of the two possible
errors he was fined two cents. The results left no doubt that the subjects could
adopt the experimenter's criterion and, by implication, can establish their own
criteria and respond accordingly. The well-known difference between detection and
recognition thresholds (ENGEN, 1970) reflect changes in criterion rather than a
change in the olfactory system.
Table 2. Payoff matrix'
Response alternatives
Yes No
Odorant higher than criterion + 2 cents - 2 cents

concentration lower than criterion - 2 cents + 2 cents
a These payoff matrices are usually arranged so that the person will on the average earn
at least the student hourly rate of 1.50 dollars per hour, but often a little more.
With payoff constant, it is possible to generate a similar isosensitivity function

by influencing the subject's expectation with variation in the stimulus-presen-
tation probability. If a weak stimulus is presented all the time, the subject will
come to expect it on each trial and will therefore have a stronger tendency to
respond "Yes" whether or not it is presented. Likewise, if the likelihood that a
stimulus is presented is very low, the subject will be more likely to respond "No"
whether or not a stimulus was presented on a given trial. Manipulation of stimulus
presentation probability has produced a relationship between conditional proba-
bilities of hits and false alarms like the one shown in Fig. 9. No such experiment
has been reported in olfaction.
According to classical theory, response bias represents a constant value with
an effect proportional to the hit rate. As a result the proportion of hits should be a
linear function of the proportion of false alarms as illustrated by the dotted
straight line in Fig. 9. According to classical theory, proportions of hits are there-
fore corrected by the following formula:
o = p(yjSN) -p(yjN)
(8)
p 1 - p(yjN)
where cp is the proportion of hits corrected for false alarms. However, a great
deal of data, especially in vision and hearing, have failed to support this formu-
lation, and instead indicate that the concave downward isosensitivity function
represents the correct basis for correcting detection data. In Fig. 9 the effect of
stimulus intensity is to displace the curve away from the diagonal, which represents
chance performance, toward the top-left corner, which represents perfect detection.
This dimension d' is continuous and, theoretically, varies as a function of the
displacement of S N from N (Fig. 7).
The Effect on d' by Odorant Concentration

SEMB (1968) has obtained such isosensitivity curves in olfaction for various
values of d' which are consistent with the theory. The main purpose of the experi-
ment was to test the hypothesis that odor detection may be considered a decision
based on two normal distributions. His experimental procedure was more complex
than the basic Yes-No experiment described above. He obtained observations on
both concentration and criterion in the same experiment. The odorant (8), n- butanol,
was diluted in diethyl phthalate which also was used as the "blank" (n). Both 8
and n were sniffed from cotton wrapped around a glass rod, which was kept in a
test tube partially immersed in the liquid when not in use. Seven concentrations of
n-butanol were selected to cover an estimated range of perceived intensity from
roughly chance performance to almost perfect detection. A trial consisted of four
steps: 1) Diethyl phthalate (undiluted diluent) was always presented first to
provide a stable reference point for each trial; 2) a two second pause; 3) the pre-
sentation of either n-butanol (8) or diethyl phthalate (n) again; 4) the subject was
then required to judge whether or not 8 had been presented. Instead of responding
by either one of two categories ("Yes" or "No"), he was free to use anyone of six
response categories representing various degrees of confidence that the response
should be "Yes" or "No":
1. Very certain that n was presented.
2. Quite certain that n was presented.
3. Uncertain, but only n was presented.
4. Uncertain, but 8 was presented.
5. Quite certain that 8 was presented.
6. Very certain that 8 was presented.
Different categories of the rating scale represent different criteria and should
provide data conforming to the isosensitivity function (GREEN and SWETS, 1966).
8 was presented on half of the trials and n on the other half in random sequence
with PIs) = Pin) = 0.50. Each of the eight concentrations were presented a total of
600 times in five sessions of 120 trials. Only the last 400 trials with each concen-
tration were used in the final analysis of the data in order to eliminate the more
variable performance, which characterizes early detection performance. The two
subjects who participated in this experiment were students who did it as a part-
time job. They were paid for their performance, one cent for each hit and correct
rejection and a fine of one cent for each false alarm and miss. The results for the
two subjects were the same, and one set of results will be presented here.
With six response categories there are five category boundaries, and it is
assumed that the subject will use a certain response category when the perceptual
effect of a stimulus exceeds that category boundary. For example, any trials on
which the subject responded with 6 were treated as if he responded "Yes," and all
other trials with that concentration for which he responded 1, 2, 3, 4, or 5 were
treated as a response of "No." This provides, for a certain criterion, a definition of
hit rate and a conditional probability computed by dividing the numbers of trials
rated 6 by the total number of trials on which that concentration was presented.
The same procedure is followed for the false alarm rate for trials on which n was
presented. For the next criterion the response categories are divided into 5 and
6 versus 1, 2, 3, and 4 etc. for each of the five category boundaries. In this manner
an isosensitivity function was obtained for each of the seven concentrations.
The hit rate, p(yjSN), is plotted against the false alarm rate, p(yjN), for the
six criteria in Fig. 10. The obtained isosensitivity curves are consistent with the
detection theory; that is, each concentration produces a function describing six
different criteria under constant sensitivity as measured by d'. There is a d' value
for each concentration with a range of less than 0.1 to more than 3.0 for 0.109 to
109.282 millimoles respectively.
o 02 ot. 06 08 10
FalsI' alarms [Ply! N)1
Fig. 10. The effect of concentration on d'. The proportions of hits are plotted as functions of the
proportions of false alarm for various concentrations of n-butanol: .0.109 millimoles with d'
about 0.05, 0 0.546 millimoles with d' about 0.75, 0 5.464 millimoles with d' about 1.20,
l'> 10.928 millimoles with d' about 1.65, x 27.320 millimoles with d' about 2.00, V 54.641 milli-
moles with d' about 2.65, 109. 282 millimoles with d' about 3.30
This psychological analysis of the detection task seems especially valuable for
sensory physiology. Presentation of a detection rate as a threshold value without
the specification of the effect of non-stimulus factors is not adequate and would
have indicated a different sensitivity for each criterion as indicated by the values
of the hit rate on the ordinate of Fig. 10. It suggested that d' or a similar index
replace "frequency of smelling" as a measure of olfactory sensitivity. Plotting the
values of d' in Fig. 10 as a function of their respective concentrations indicate that
odor sensitivity increases as a power function of concentration
d'= cSn (9)
where c is a constant determined by the choice of units; S is concentration, and n
is the slope of the function. The value of n in SEMB'S experiment was 0.30, compa-
rable to the value obtained in direct scaling of suprathreshold concentrations
(ENGEN, CAIN, and ROVEE, 1968). This indicates that even for weak concentrations
the olfactory system compresses stimulus input. More precise specification of this
psychophysical function should be useful. In addition d' has a specific meaning
related to the "ideal observer" and the meaning of noise. Further research on
noise in the olfactory system is needed, and for the present d' is used only as an
index of detectability.
Individual Differences in Detection

One other advantage of this index should be noted. CORBIT (1968) measured the
detectability of several homologous alcohols with an air-dilution olfactometer,
according to the simple Yes-No detection situation of Table 1. Presentation of the
stimulus was controlled by flowmeters, a series of relays, and variable timers. On
a trial either air (n) or air plus odorant (8) was presented to the subject through a
Teflon tube and was indicated by a signal light. The subject indicated by pressing
one of two buttons whether n or 8 had occurred. The response was recorded on a
counter at the experimenter's panel, and the subject was simultaneously informed
by light signals whether or not his response was correct. The order of presentation
of conditions was random with Pn = Ps = 0.50. Each of the three college students
who served as subjects was given three sessions of 100 trials of training with each
of four alcohols in order to stabilize performance and to determine approximate
concentrations for the subsequent detection analysis. During training the subjects
were paid 1.50 dollars per hour. Three additional training sessions were given under
a payoff matrix, which rewarded the subject with three cents for each correct
response and fined him three cents for each error. Then followed five sessions of
100 trials for each of the odorants for which d' was determined.
Table 3. Concentrations of stimuli in rug/l in air and corresponding values of d' for three sUbjects
(from CORBIT, 1969)
Odorant Subject 1 Subject 2 Subject 3

concentration d' concentration d' concentration d'
n-propanol 0.046 1.44 0.051 1.38 0.051 1.44

n-butanol 0.013 1.56 0.013 1.38 0.020 1.38
n-heptanol 0.001 1.40 0.001 1.16 0.001 1.24
The results are shown in Table 3. In agreement with earlier studies (e.g.,
MOULTON and EAYRS, 1960; ENGEN, 1965), detectability as indicated by d'
decreases as the homologous series of alcohols is ascended. However, the inter-
esting aspect of Corbit's data is, first of all, the extremely small variability in both
concentration and d' for different subjects compared with thresholds, for example,
obtained with the conventional method of limits (e.g., compare present data with
JONES, 1955, and SEMB, 1968; versus ENGEN, CAIN, and ROVEE, 1968). The reason
for expecting both lower detection values, in the same unit of concentration
(STONE, 1963), and smaller individual differences is that the detection procedure
provides both a standard of noise and continuous feedback of information about
performance, which make it possible for subjects to form and maintain a lower
criterion. The difficulty of comparing detection values from different laboratories,
species, and subjects will not be resolved as long as the comparison is made in
terms of one selected concentration value per odorant without control of non-
stimulus variables. The threshold is not absolute, for any minute concentration
can probably be detected depending on the time (or trials) available for the pre-
cision of the estimate of response probabilities. More reliable generalization about
odor sensitivity should result from a quantitative comparison of sensitivity as
measured by an index like d' plotted as a function of comparable values, as in
formula (10).
Absolute and Differential Threshold

The difference threshold or limen can be measured by the same methods used
to measure the so-called absolute threshold, and the same theory has been applied
to account for the data. The subject's task is also similar in the two situations.
Differential sensitivity is indicated by the small change in stimulus intensity that
a subject will discriminate as a difference in perceived intensity. (Such discrimi-
nation should not be confused with psychophysical scaling, because the differential
threshold is only an index of resolving power at different points on the scale). If the
assumption is correct that absolute threshold indicates the smallest intensity that
can be detected above the noise level, then the usual distinction between absolute
and differential sensitivity is arbitrary.
L1 S is usually obtained by the method of constant stimuli. The subject is
presented a standard stimulus, which is a constant value, and a comparison
stimulus of a different value with instruction to judge whether or not the perceived
intensities of the two stimuli are the same or different. The value of the comparison
stimulus is varied over trials, and L1 S is defined as the difference in intensity a
subject can detect 50 % of the time. Likewise, absolute threshold presumably is
the stimulus intensity a subject can detect 50 % of the time without a standard of
comparison, but as has been indicated the subject will adopt a standard (criterion)
of his own in that case. Absolute sensitivity is thus a special case of differential
sensitivity (cf. also EKMAN, 1959).
Tests of differential sensitivity have usually been related to Weber's Law
which was originally written
L1S = kS, (10)
where L1 S refers to an increment of S, the value of the standard stimulus, and k is

a constant. Weber's fraction, or k, tends to be constant for moderate values of S
but usually increases greatly as S is decreased and detection becomes a problem
for the subject. For that reason Weber's Law has been modified to
L1S=k(s+a), (11)
where a is a small value on the stimulus dimension and may be considered the
minimal value that will be reported as different from the effect of noise or the
absolute threshold in a conventional sense.
STONE and co-workers have demonstrated with a variety of odorants controlled
by an air-dilution olfactometer that Weber's Law (11) provides a good description
of olfactory discrimination (cf. STONE and BOSLEY, 1965). L1 S was determined by
the method of constant stimuli. Several different standard values were used, and
there were six different comparison stimuli placed evenly above and below each
standard. The subject judged each pair of standard and comparison stimuli six
times in a random order. Each stimulus was presented in a plexiglass hood, which
fitted over the subject's head for 1 sec at 10 sec intervals. Using pure air as the
standard, 50 % detection values (So) were estimated at the end of the discrimi-
nation work. Data were collected from volunteer subjects following a practice
session. The results of nine subjects were pooled, and Weber's Law tested by
regression analysis.
0
12
.....
'"E 8
('1')52
~ 4
(/)
"l

0
s + So (xl0-3 mg II)
Fig. 11 (from STONE and BOSLEY, 1965). Weber fractions plotted against concentration
adjusted to threshold
In Fig. 11 S is plotted against S + 8 0 according to formula (11). In addition to

the representative odorants shown, the function fitted to the data is based on data
obtained earlier on 2-heptanone, 2-octanone, n-heptyl alcohol, and ethyl valerate.
The function clearly seems to describe both trigeminal and olfactory stimulation.
Acetic acid provides the widest range of values, and those obtained for the odorants
not shown here are close to those plotted for propionic acid.
L1 S is 0.281 and indicates that a change of approximately 28 %in concentration
is required before the average subject will detect it at the 50 %level. By this index
the sense of smell is the dullest of the senses, but for reasons indicated above
evaluations on the basis of classical threshold are subject to biases. With reference
to this STONE and BOSLEY (1965) suggest possible problems with motivation and
attention in their discussion of individual differences. Further refinement of the
basic information now available can be expected from the application of detection
procedures in controlling such non-stimulus variables in discrimination as in
detection tasks.
Odor Quality and Classification

The study of smell has to a large extent stressed the classification of odors based
on subjective description of the quality of the odor, and it has been expected that
analysis of such classes of similar odors will reveal chemical and physical correlates
of the perceived similarity. The most recent and prominent system has been
developed by AMOORE (1965) and is based on the hypothesis that similar odors
have similar stereochemical properties. Perhaps the most important aspect of this
approach is the development of a glossary of odor qualities, but no system has as
Odor Quality and Classification 237
yet been successful as shown by the comprehensive review by HARPER et al. (1968).
The problem is semantic. For example, in the American work of SCHUTZ (1964)
"sulpherous" was found to be similar in meaning to "goaty," but these are dif-
ferent according to a related English study (HARPER et al., 1968). Even if researchers
could agree on the terms to be used to describe odors in perfumery and food
technology, the critical question is still whether or not those terms are relevant
when a subject discriminates one odor from another.
Multidimensional Scaling
The recent psychophysical approach to the quality of odors rejects verbal
labels as arbitrary, and it has instead attempted to increase the generality of the
information obtained by simplifying the responses and testing them against
multidimensional mathematical models. It is proposed that the psychophysics of
qualitative perceptual differences must be extracted from perceived similarity
(proximity or distance) of stimuli or from the extent to which different stimuli
elicit the same perceptual quality (content). To accomplish this the subject's
responses must be related to a spatial or geometric model of the perceptual space.
Both the evaluation of the responses and the model can be tested with empirical
data. The ideal kind of solution suggested by BIENFANG (1941) is that odors be
represented by a three dimensional Euclidean space with "an axis of clarity," "a
radius of strength," and "a circumference of note" analogous to brightness,
saturation, and hue of the color solid.
The Distance Model

WOSKOW (1968) has reported the first comprehensive study of multidimensional
similarity analysis. In his experiment each of 20 inexperienced volunteer subjects
rated all the 300 pairs of 25 odorants in nine different sessions following one session
devoted to practice. The subject judged the qualitative similarity of each pair of
odors on a nine-point scale where 1 indicated "most alike" and 9 indicated "as
dissimilar as possible." The odorants were presented in 30 ml wide-mouth bottles
with stoppers removed only for sniffing. The experiment was performed in a well-
ventilated stainless steel room. The order of presentation of pairs was systematic,
and statistical analysis indicated no effect of order of presentation and cross-
adaptation of members within a pair. The mean rating of each pair defines the
distance between the members of each pair, and the results of the experiment are a
matrix of these distances for all 300 pairs. The problem is to reduce this matrix to
a relatively small number of psychological dimensions, which requires a judgment
about the model which might best represent the odor space. WOSKOW employed a
Euclidean model for the advantage of availability of computational methods,
because he considered it an advantage which allows transformation of distances
and orthogonal rotation of the derived axes. Following a detailed procedure
developed by TORGERSON (1958), the distance matrix was converted to a matrix
of scalar products, and this matrix was in turn factored by COMREY'S (1962)
minimum residual method.
The factor analysis produced the nine factors, three of which are shown in
Table 4. The columns of this factor matrix represent the orthogonal axes or dimen-
sions and the elements in the rows values for different odorants in a Euclidean
"odor space." For example, the position of methyl salicylate is -2.63 on the first
axis, 1.08 on the second, 1.13 on the third, etc. The reliability of the present results
was confirmed by various statistical methodological procedures and by comparison
with earlier smaller studies by WOSKOW and his colleagues.
Table 4. Dimensions of odor q'/.U1l,ity from factor analysis of multidimensional similarity

(Distance) judgments (from WOSKOW, 1968)
Odorant Factor
I II III
Methyl salicylate -2.63 1.08 1.13

Eugenol -2.31 1.61 1.40
Pyridine 3.82 -1.00 1.76
Safrol -2.89 1.24 0.23
Benzaldehyde -2.50 0.12 1.36
Guaiacol 0.84 2.98 0.39
Citral -2.35 -0.05 0.44
n-Butanol 0.55 -1.27 -1.20
Toluene 1.19 0.17 -1.99
Anisole 2.23 0.05 -1.94
n-Propanol -0.06 -1.36 -0.33
Acetic acid 3.56 -0.11 2.57
n-Pentanol -0.30 -1.08 -1.75
Seatol 3.64 1.60 0.18
Ethanol -0.70 -1.57 -0.61
n-Butyric acid 4.32 -0.66 1.94
n-Nonanol 0.01 -1.36 -0.55
Phenylethyl alcohol -1.67 -1.15 0.24
Vanillin -3.12 -1.27 2.62
l-Menthol -0.64 2.16 -0.54
d-camphor 0.03 3.03 -1.08
n-Hexanol -0.77 -1.34 -0.58
Pinene 0.17 1.59 -1.49
n-Octanol -0.27 -1.46 -1.54
n-Heptanol -0.22 -1.31 -0.74
Proportion of Variance: 0.631 0.126 0.104
The proportion of variance removed from the matrix of scalar products by

each factor is shown in the row below the factor matrix in Table 4. The first factor
accounted for 63 %, the seond 12 %, the third 10 %, etc. of the total variance in
the matrix of scalar products. In other words, three of the nine factors account for
most of the variance of similarity judgments. The other six factors add little
potential information about odor quality, for each accounted for a maximum of
4 % of the variance. The dimensionality obtained by WOSKOW is to some extent
determined by the assumption demanded by the Euclidean model that his simi-
larity judgments are metric data, that is, distances with a fixed origin. Recent con-
tributions to multidimensional scaling make it possible to approach a metric
representation with non-metric data, that is, without assuming absolute distances
and in both Euclidean and non-Euclidean spaces (SHEPARD, 1962a and b; KRUS-
lUL, 1964a and b; COOMBS, 1964). An interval scale without a fixed origin is then
obtained which permits any linear transformation of the data, which in turn
makes it possible to reduce the dimensionality of the obtained data. It is not
unlikely that these newer methods could reduce the number of factors extracted
from WOSKOW'S data. However, interpretation of the remaining factors may then
be more difficult, for the reduction is made possible primarily through bipolar
dimensions (see EKMAN, in press).
Inspection of Factor I in Table 4 suggested a hedonic dimension, and this was
verified by comparing the elements with judgments of pleasantness obtained
by the same subjects at the completion of their judgments of similarity. It seems
that the similarity of odors is determined largely by pleasantness, and WOSKOW
concludes his very informative paper by suggesting that the lack of success
of odor classification may have been partly due to the general neglect of this
variable. Perception of pleasantness has also emerged in other multidimensional
studies (ENGEN, 1964). Direct scaling with the method of magnitude estimation
has also shown that the perceived range of pleasantness of odors is unusually
large; for example, safrol is roughly 100 to 150 times as pleasant as pyridine
(ENGEN and McBuRNEY, 1964). Pleasantness is perhaps not a quality in the usual
meaning of the term in odor classification, but it undoubtedly plays a dominant
role in responses to odors. The connection of the olfactory system with the limbic
system may be the reason.
A straightforward interpretation was not possible with the other factors.
Factor II was tentatively described in terms of "coolness" and "woodiness"
because of its high values for menthol, camphor, pinene, and guaiacol; and no
interpretation was suggested for Factor III. In any case, the end result of multi-
dimensional scaling ought to be more than a further search for general labels. For
example, the data may be used to test hypothesis about physiological correlates or
physical determinants.
The Content Model

EKMAN (1963) has proposed an approach to multidimensional analysis which
promises to be valuable in olfaction where interpretation of factors is the primary
concern. The approach differs from the distance approach both in the measurement
of perceptual responses and in the model of the odor space. The judgments are
direct, as in magnitude estimation, and theoretically provide ratio scales with a
fixed origin. Using one odorant of a pair as the standard or unit, the subject is
asked to judge in per cent the extent to which the comparison odorant elicits the
same quality of odor as the standard. EKMAN'S model of the perceptual space is a
coordinate system where vectors represent different qualities. The length of a
vector corresponds to the perceived intensities of a particular quality as in Fig. 1.
The angular separation or cosine of two vectors represents the extent to which two
odorants produce the same quality of odor, or in the general case, share the same
"content." If the subject compares odors of different quality which have pre-
viously been matched in perceived intensity first by using one of the two as stand-
ard and then the other, the angular separation between the corresponding vectors
may be described by the geometric mean of the two estimates in the general
equation
(12)
240 T. ENGEN: OHactory Psychophysics
where cos "Pij is the angular separation between the vectors for the odors of
odorants i and j; qij is the numerical judgment of the extent to which odorant i
elicits the same odor quality as odorant j, and qji is the numerical judgment
obtained when i is the standard and j the comparison odorant. If the qualities of
the two odorants are the same, the situation is unidimensional and qijqji= 1, but
when there is a qualitative difference then qijqji < 1.
EKMAN (in press) concludes that the distance approach may be preferred when
it is desirable to describe a stimulus configuration in as few dimensions as possible,
and when interpretation ofthe dimensions is not a primary problem, as for example
in the analysis of the perception of geometric forms. The main explanation
for the preference involves the lack of a fixed origin of the distance scale which
permits reduction of the data to fewer dimensions but results in greater difficulty of
interpretation. By contrast, the content approach has fixed the origin of the
judgmental scale and permits only rotation of the axes obtained in factor analysis,
and the proper rotation and interpretation are usually obvious. This is an advantage
when the purpose of the analysis is to isolate and define qualities in homogeneous
or perceptually "non-analyzable complex stimuli" as in the case of colors and
odors.
This implies a solution to an old problem: Is olfaction a "synthetic" sense
modality like color vision, where a mixture of wavelengths yields a hue different
from the hues of the component wavelengths of the mixture, or "analytic" like
audition where a cord may be perceived as several individual notes? MOZELL (in
press) has suggested a crucial test of this problem. He points out that in vision, for
example, subjects cannot discriminate an orange hue resulting from a mixture of
different wavelengths from the orange hue of a single intermediate wavelength.
Olfaction may be considered synthetic if subjects are found unable to discriminate
the quality of odorants containing only one molecule from odorants containing
more than one type of molecule. Such a situation would be unidimensional with the
implication that qijqji = 1. According to MOZELL the idea that olfaction is analytic
refers mainly to the ability of trained chemists to identify known components of
mixtures, but information based on identification by smell alone is needed. In
general man's ability to perceive odors tends to be exaggerated by anecdotes about
perfumers and chemists (see ENGEN, 1970). The important but still untested con-
sideration for multidimensional analysis of odor is that the subject compares
the odors in overall similarity in order that previously unknown and basic dimen-
sions be extracted (see SHEPARD, 1964).
A set of odorants will provide a matrix of n(n - 1) pairs of odorants from
which values of cos "Pij in formula (12) may be computed. Analysis of these values
by the method of principle factors (HARMAN, 1960) will result in factor loadings
representing direction numbers regarding quality when the odorants are equal in
perceived intensity. Such an experiment, which is performed with n-heptanal and
amyl acetate defining two different odor qualities (EKMAN and ENGEN, 1962),
illustrates both the potential of the content method and a strategy for multi-
dimensional scaling in olfaction. Two of the odorants were undiluted n-heptanal
and amyl acetate and four were mixtures of the two consisting of 12.5, 25.0, 50.0,
and 75.0 % n-heptanal in amyl acetate. The odorants were sniffed from cotton
which was always immersed in the liquid odorant when not in use. Judgments were
obtained from 10 subjects (students) who were naive about the purpose of the
experiment and the nature of the odorants. All pairs of the six odorants were pre-
sented to individual subjects in an irregular order, and each member of the pair
was in turn the standard against which the subject judged the quality of the other.
Ta.ble 5a Analysis of results of multidimensional judgments according to content model

(from EKMAN and ENGEN, 1962)
Principle rotated
gl; cos 'PI; factors factors
1 2 3 4 5 6 1 2 3 4 5 6 I II I II
1 47 37 27 23 19 63 43 35 30 19 57 -45 72 09
2 84 74 60 38 37 71 58 41 34 77 -38 82 28
3 50 68 58 45 42 63 56 30 78 -22 70 40
4 45 57 69 81 56 70 52 81 12 48 66
5 38 45 71 61 80 71 78 37 29 82
6 20 32 22 49 62 64 51 09 81
Decimal point has been omitted
From the mean judgments of qiJ and qii' a matrix of cos "Pi; was determined with
formula (12). This matrix was factor analyzed, and the principle factors obtained
were rotated. The factor values are shown in Table 5. Fig. 12 shows the function
obtained when loadings of the odorants on Factor II are plotted against the loadings
\
1.0
6 5
0.8 r-
-Ci 4
c
0.. ".,
I
0.6 r-
I
-
..c
3
:::: 0.4 l-
... 2
oS
u
tf 0.2 r-
1

I I I I
0 0.2 0.4 0.6 0.8 10.
Factor I ("Amyl acetate")
Fig. 12 (from EKMAN and ENGEN, 1962). A plot of rotated factors from multidimensional
scaling of mixtures of amyl acetate and n-heptanal
for the same odorants on Factor 1. It is clear that Factor I corresponds to amyl
acetate and Factor II to n-heptanal. Similar successful tests of the content approach
have been made with judgment of color in which case wavelength was manipulated.
16 lib. Sensory Physiology, Vol. IV/I
242 T. ENGEN: Oliactory Psychophysics
Interpretation of Factors
The usual strategy followed in multidimensional investigations is to attempt to
sample the whole odor space with as many and diverse odorants as possible in
order to increase reliability of the description of the odor space. This is bound to
lead to problems of interpretation of a relatively large number of factors, or at
least to the necessity of considering hypotheses related to a large number of
potential correlates of odor. A better strategy therefore may be to select odorants
systematically according to established hypotheses based, for example, on mole-
cular (BEETS,1964), stereochemical (AMOORE and VENSTROM, 1966), or gas chromato-
graphic (MOZELL, 1966) considerations. The potential contribution of this approach
has been demonstrated by D0VING who compared electrophysiological data from
the frog with multidimensional judgments from human subjects. The odorants
were classified as exciting, inhibiting, or having no effect on single neurons in the
frog's olfactory bulb. The similarity of these responses to different odorants was
expressed in chi square values. The data on man were obtained by both content
and distance methods, and comparisons were made with previously published (e.g.,
WOSKOW, 1968) as well as original data. The rank-order correlations between the
data from man and frog were generally high and reliable at the 1 % level. This
agreement indicates that a psychophysics of quality based on multidimensional
scaling can produce perceptual qualities related to physiological processes and in
turn to parameters of the odorants (D0V1NG, 1966a and b; D0VING and LANGE,
1967).
Conclusion
It seems reasonable to conclude that the psychophysics of olfaction is capable
of a level quantification close to that in vision and hearing, thanks largely to
improved technology in stimulus presentation and application of new mathematical
methods. Even in the study of quality one is no longer limited to introspective
accounts of odor perception, although these may be of interest and value in certain
applied work.
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BEETS, M. G. J.: A molecular approach to olfaction. In: AmEN's, E. J. (Ed.): Molecular
pharmacology. New York: Academic Press 1964.
BEIDLER, L. M.: Facts and theory on the mechanism of taste and odor perception. In: MITCHELL,
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B1ENFAN'G, R.: Dimensional characterization of odors. Chronica Botanica 8, 249-250 (1941).
CAIN, W. S.: Olfactory adaptation and direct scaling of odor intensity. Unpublished Ph. D.
Thesis, Brown University 1968.
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(Ed.). Oliaction and Taste. New York: The Rockefeller University Press 1969.
CHEESMAN, G. R., TOWNSEND, M. J.: Further experiments on the olfactory thresholds of pure
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COOMBS, C. H.: A theory of data. New York: Wiley 1964.
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- Unpublished data, Brown University 1968.
DOV1NG, K. B.: Analysis of odour similarities from electrophysiological data. Acta physiol.
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- Psychophysical analysis of the odor intensity of homologous alcohols. J. expo Psychol. 70,
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JONES, F.: Olfactory absolute thresholds and their implication for the nature of the receptor
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JONES, F. N.: Scales of subjective intensity of odors of diverse chemical nature. Amer. J.
Psycho!. 191'i8a, 305-310.
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KRUSKAL, J. B.: Multidimensional scaling by optimizing goodness of fit to a nonmetric hypo-
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Chapter 11
Olfactory Genetics and Anosmia

By
JOHN E. AMOORE, Albany, California (USA)
With 1 Figure
Contents
General Survey of Olfactory Abnormalities. 245
Test Methods for Olfactory Function . . . . . . . 247
Possible Origin of Specific Anosmia . . . . . . . 248
Evidence about Inheritability of Specific Anosmia. 249
The Isovaleric Acid Anosmia and the Sweaty Primary Odor 251
Tentative Odor Classification Based on Specific Anosmias. 255
References . . . . . . . . . . . . . . . . . . . . . . 255
General Survey of Olfactory Abnormalities

There is an enormous variation among the human population as regards
olfactory acuity. Even persons with no obvious abnormality may range in sensi-
tivity as much as 1,000-fold between the least and the most sensitive observers
(AMOORE, 1968b). More precisely, the standard deviation (SD) from the mean
olfactory sensitivity in a sample of 443 subjects toward isobutyl isobutyrate was
1.71 binary dilution steps in a geometric concentration scale. Hence, taking the
normal mean detection threshold as unity, approximately 95 % of the population
should have personal thresholds between 1/10 tho and 10 times the mean threshold
concentration for a given odorant.
Individuals with sensitivities outside this range may be suspected of having an
olfactory abnormality, of which there are numerous types (Table 1). An extremely
high sensitivity of smell (and taste) has been described by HENKIN and POWELL
(1962) as an accompaniment of cystic fibrosis of the pancreas and in adrenal
insufficiency. Smell sensitivities averaging 10,000 times normal were reported,
with a rather general increase in responsiveness toward all odorous materials
tested. The condition may therefore be described as general hyperosmia. An inter-
mediate condition of specific hyperosmia is a theoretical possibility which seems
never to have been clearly established. It is to be anticipated that some persons
might, through inheritance or mutation, possess a specific semitivity for some
particular substance which the majority of people cannot smell. Proof would be
246 J. E. AMOORE: OHactory Genetics and Anosmia
required that the subject concerned had merely a normal sensitivity toward other
unrelated odorants.
Table 1. Range of abnormal olfactory sensitivities
Condition Relativl'! sensitivity
General hyperosmia (in cystic fibrosis) 10.000/1

Specific hyperosmia (not established)
High normal range (+ 2 SD) 10/1
Normal mean detection threshold..... ....... . . .. . . .... ... . ... ... . ..... 1
Low normal range (- 2 SD) 1/10
Specific anosmia (one primary loss) 1/100
General anosmia (1st. nerve loss) 1/100.000
Complete anosmia (1st. and Vth. nerve losses) 1/100.000.000
The commonest olfactory abnormality is a lowered sensitivity towards a single

odorant or a few related compounds, while most other odors are perceived normally.
This condition was first described by BLAKESLEE (1918) in respect of Verbena
flower scents. His allusion to persons "blind" to odors in a certain pedigree of
Verbenas gave rise to the popular term "odor-blindness" for this condition. GUIL-
LOT (1948) listed 8 chemical compounds exhibiting what he called "partial an-
osmia." He ascribed the diminished sensitivity to a deficiency in one of the funda-
mental or primary odors of which the sense of smell may be composed. The present
author prefers to emphasize the chemical specificity of the phenomenon by using
the term specific anosmia, for example, in respect to the isovaleric acid smelling
deficiency (AMOORE, 1967). The relative sensitivity of persons defective towards
this particular odorant averaged about one-hundredth of normal. The incidence of
specific anosmia in the population has been reported to vary from 0.1 % to 32 %,
depending upon the type of deficiency and the method of measurement (AMOORE,
1969).
In medical literature the name "hyposmia" is favored for milder olfactory
defects which are comparatively general in extent, such as occur during the common
head-cold. Perhaps the "uncomplicated anosmia" described by DUNCAN and
BRIGGS (1962) should be placed in this class. Most often suffered as a persistent
aftermath of a severe bout with influenza, this type of anosmia responded well and
often permanently to massive injections of vitamin A. The perception of an uncon-
ventional odor-sensation, different in quality from the usual reaction to a given
substance, is called "parosmia." Sometimes this is simply a manifestation of a
specific anosmia to the major component in a mixture of dissimilar odorants
(DOUEK, 1967), but it could also be due to a peculiarity of the nervous connections.
Total loss or absence of the sense of smell is known as general anosmia or
literally "anosmia." The olfactory or 1st. cranial nerve is utterly inoperative or
ineffective for one or other of various reasons listed by SCHNEIDER (1967). These
include mechanical blockage of the airway; infectious or chemical interference with
the olfactory receptors; tumors, trauma, or infection near the olfactory nerve and
bulb, and certain congenital and hysterical conditions. In the fatty acid series of
odorants, the normal observers averaged very roughly lOO,OOO times the sensi-
Test Methods for OUactory Function 247
tivity of a person "\\ith permanent general anosmia caused by head injury (.!MOORE,
VENSTROM, and DAVIS, 1968). The incidence in the population of general anosmia
due to accident or disease may be about 0.2 % (PATTERSON and LAUDER, 1948).
A new syndrome of complete anosmia was described by HENKIN (1966). Such
patients lack not only 1st. nerve sensitivity but also fail to show any responses to
irritant chemical vapors which normally excite other areas of the nasopharynx
served by the V tho (trigeminal) and also the IX tho and X tho cranial nerves. These
non-olfactory responses to inhaled vapors are loosely included under the older
expression "common chemical sensitivity" (PARKER, 1922). Toward pyridine,
which the complete anosmics could not detect even undiluted, the sensitivity of
normal observers is at least 100 million times greater (HENKIN, 1967).
The more severe olfactory abnormalities are often merely secondary symptoms
of an un-related primary condition, which is usually not genetically controlled or
even predisposed. There are some interesting exceptions, however, HENKIN and
KOPIN (1964) reported that two out of six patients with familial dysautonomia
were hyposmic in addition to the tasting deficiency from which they all suffer.
SPARKES et al. (1968) described a family exhibiting hypogonadotrophic hypo-
gonadism associated with anosmia. An X-linked inheritance seemed likely; the
genetic defect probably being manifested through the hypothalamus which is
associated with both olfactory and pituitary function.
A general anosmia of indeterminate origin was noted by MAINLAND (1945) who
traced the defect through three generations and ascribed it to a single autosomal
dominant gene. PATTERSON and LAUDER (1948) contributed further information
on a curious "delayed anosmia" which occasionally comes on in middle age, affect-
ing about one person in a thousand. Again a dominant pattern of inheritance was
suggested.
Test Methods for Olfactory Function
Many specialized types of olfactometer have been developed, but reasonably
satisfactory demonstrations of olfactory abnormalities can usually be achieved
with simple "sniff-tests." These are based on sets of stoppered bottles containing
appropriate dilutions of test odorants in water or other non-odorous solvent.
AMOORE et al. (1968) gave detailed instructions for individual threshold measure-
ments and statistical evaluation of the results. A test odor series with successive
halving of the concentration is recommended. At each concentration step a sorting
test should be applied to prove that the subject really can distinguish the test odor
flask from a similar flask merely containing pure solvent. A set of five flasks (two of
which contain odor) affords confidence at the 90 % level when the selection is
correct (2/5 test). For clinical purposes a set of three flasks, among which one has
odor, would be adequate (1/3 test, 66 % confidence), especially if the test is
repeated in doubtful cases.
A "general smell test" was used by AMOORE et al. (1968) to eliminate subjects
with hyposmia or general anosmia. It consists of seven 2/5 tests, containing "match-
ing" concentrations of seven standard odorants representing the stereochemical
theory classes (AMOORE and VENSTROM, 1966). The test odor concentrations (for
example, 300 ppm v/v phenylethylmethylethyl carbinol in water) are roughly
30 times the mean threshold concentrations. Persons with general anosmia fail all
248 J. E. AMOORE: Olfactory Genetics and Anosmia
these odors except the 5 % formic acid standard, which is a strong trigeminal
stimulant in addition to its rather feeble true odor. Before testing for specific
anosmia, it is recommended that any persons failing more than one of these test
odors should be rejected. Such persons may have a hyposmia (or specific anosmias
to more than one of the general test odorants).
In testing for complete anosmia, HENKIN (1966) used 30 % formalin, whose
vapor these patients entirely failed to distinguish in a 1/3 test from water. Nor
could they tell pure pyridine from water. They could sometimes, but still not
reliably, distinguish concentrated hydrochloric acid or concentrated ammonium
hydroxide from water.
The tests for general hyperosmia are described by HENKIN and POWELL (1962).
From the data there given, it would appear that a solution of 0.1 mM hydrochloric
acid would be readily distinguished by odor from plain water in a 1/3 test by most
ofthese patients. No normal observer could detect anything less than 1,000 times
stronger.
The experimental definition of specific anosmia is a little more subtle and
somewhat arbitrary, because the abnormality is generally much less dramatic
quantitatively than in the extreme conditions just described. AMOORE et al. (1968)
recommend that any general anosmics or even hyposmics in the population sample
to be tested should first be eliminated by the general smell test. The test odorant,
towards which specifically anosmic persons are sought, should be carefully purified.
The mean threshold for the test odorant is determined on a large group of normal
observers, preferably at least 25. As an arbitrary definition of specific anosmia, the
- 2 SD point on the Gaussian distribution of individual olfactory thresholds was
selected. By this criterion any people who are not general anosmics, but fail to
perceive the test odorant in a "screening test" at say 10 times the mean normal
threshold concentration, would be classified as specific anosmics to the chosen
compound. The actual thresholds of the specifically anosmic subjects can then be
determined using a concentration series of 2/5 tests.
It is important that testing for suspected olfactory abnormalities should be
carried out in a systematic and quantitative manner with statistical control. Other
factors to be borne in mind include the following: There is a deterioration in sensi-
tivity with age at the average rate of doubling the threshold concentration each
22 years (VENSTROM and AMOORE, 1968). The same authors reported negligible
influence from the sex of the subject or from the use of tobacco. However, smoking
or candy-eating immediately prior to the test causes about a four-fold increase in
threshold concentration, as does a head-cold or nasal allergy (AMOORE et al., 1968).
A surprisingly large (10-fold) decrease in sensitivity between morning and evening
was noted by STONE and PRYOR (1967) in respect to the isonitrile odor; if confirmed
for other odorants, this is a matter which should be controlled. These various
factors ought to be guarded against, because each could be large enough in certain
circumstances to give a spurious impression of specific anosmia.
Possible Origin of Specific Anosmia

The phenomenon of specific anosmia is a strictly olfactory defect, in contrast
with the more severe kinds of abnormality which are usually non-olfactory in
Possible Origin of Specific Anosmia 249
origin. Hence a particular interest attaches to the specific aberrations. Smell and
taste are chemical senses, and it is worth recalling that virtually all chemical
defects in living organisms can be traced to a defective protein; for example, the
inborn errors of human metabolism or the metabolic mutants induced in micro-
organisms by radiation. It has recently been established that the ultimate cellular
receptor substances for sapid and odorous molecules are in fact specialized proteins.
DASTOLI and PRICE (1966) isolated a sweet-sensitive protein from taste-buds, and
later a bitter-sensitive protein was reported (DASTOLI, LOPIEKES, and DOIG, 1968).
Following promptly on these successes, ASH (1968) has just announced pre-
liminary evidence for an odor-sensing protein (responding to linalool) in extracts
from rabbit olfactory epithelium. The idea of a different selective receptor protein
for each of the primary modalities of taste and smell is implicit in these findings.
Hence the most obvious explanation for a given type of specific anosmia would
be that the deficiency is due to absence or defect (variation) in the corresponding
olfactory receptor protein (AMOORE, 1968a). (Conversely the predicted specific
hyperosmia would be due to the presence of an abnormal but effective protein).
Other explanations could apply in special cases such as an absent receptor cell
type, an interrupted nervous pathway, or faulty cerebral association. Nevertheless,
the hypothesis of a defective receptor protein seems most concordant with current
thinking in biochemical genetics.
Evidence about Inheritability of Specific Anosmia

To date just three types of specific anosmia seem to have been seriously
investigated from the standpoint of inheritance. These are for the odors of n- butyl
mercaptan (skunk), hydrogen cyanide, and Freesia scent.
PATTERSON and LAUDER (1948) tested 4,030 persons with a 0.0075 % solution
of n-butyl mercaptan in 90 % methyl alcohol. They found 17 persons who failed
to perceive the odor, which even at this dilution was decidedly obnoxious to the
average person. Additional tests were applied with other characteristic odors, to
eliminate any general anosmics. There remained six typically "smell-blind" per-
sons, four of whom could not even detect an odor in a ten-times stronger solution
of n-butyl mercaptan. Two of the defective persons were from families in which
both parents were normal. The authors concluded that if the smell blindness is
inherited, it would appear to be as a recessive character, because children of normal
parents have been affected. The information is however too meagre to be con-
clusive.
The hydrogen cyanide anosmia has received much more attention, but the
situation remains exceedingly confused. At the present time no agreed conclusion
has emerged. The first systematic study was by K.mK and STENHOUSE (1953) who
tested 244 persons for the ability to distinguish a 20 % solution of potassium
cyanide from plain water. They found that 18.2 % of males but only 4.4 % of
females were unable to discriminate between the samples. These proportions sug-
gested that the inability to smell HON is a sex-linked recessive. The data were
further analyzed in 61 families, but this lent only partial support to the hypothesis.
FuKUMOTO et al. (1957) presented data on 433 persons, obtaining results in very
close agreement with K.mK and STENHOUSE.
A similar study by HUSER et al. (1958) on 50 persons from a hemophilic pedigree

reached a completely different conclusion. The non-smeller trait appeared to be
neither a recessive nor sex-linked. Instead they concluded that it is inherited as a
dominant, probably autosomal. The whole question was re-opened recently by
BROWN and ROBINETTE (1967) who made the important advance of testing their
subjects with a series of dilutions of potassium cyanide and not with just a single
high concentration. They showed first of all that cyanide behaves quite unlike any
other of the eight odorants they studied in that there is a far wider quantitative
range of thresholds in the population, and the distribution is trimodal rather than
unimodal. When applied to 2,885 schoolchildren, the modes appeared at dilutions
of 10-6, 10-3 and 10-1 g cyanide/l water. There was no overall difference in the
distribution of boys and girls, suggesting that sex linkage was not involved.
However, 86 pairs of parents were also tested, and the fathers in particular pre-
sented a somewhat different picture. Among these adult men there was a decrease
in the frequency of "good smellers" and a large increase in the "non-smellers"
group, so that they greatly outnumbered the non-smeller women. Although this
result is reminiscent of the unequal division between the sexes noted by some of
the earlier investigators, the distributions among the children within the families
examined by BROWN and ROBINETTE (1967) provided no support whatsoever for
any simple sex-linked segregation pattern. In fact they felt impelled to conclude
from their studies that the genetic proportion of the control of hydrogen cyanide
threshold seems to be small compared with the total variation.
The third systematic investigation of specific anosmia, although not yet
published, is yielding some very interesting results. I am grateful to Dr. K. G.
MCWHIRTER of the Genetics Laboratory at Oxford University for allowing me to
place on record some of his observations on the ability to smell Freesia flowers. His
results are reassuring as they tend to support the belief that there is an important
genetic control in at least this type of anosmia. Here are some quotations from his
letter of August 29, 1968:
"The specific anosmia to the scent of Freesia is very striking as this is to the
majority of subjects one of the most powerful of all flower smells. Tests on succes-
sive groups of undergraduates show that about 5-8 % are wholly or virtually
completely anosmic. Familial data show that homozygous recessives for a single
autosomal Mendelian gene are anosmic. The notation Fr for the dominant gene
permitting the perception of Freesia scent and fr for the recessive is being pro-
posed."
"The scents of very many other plants (and also a number of odoriferous
animals) show striking variations in their power to excite human subjects of
varying genotype. The few familial data so far collected indicate that these are
also genetic polymorphisms with a dominant gene permitting perception of the
scent. The first three chosen for study on a population basis suggest dominant
gene-frequencies in the useful 25-75 % range. There is no evidence of interaction
so far. Hence the assumption is that each variation in phenotype is due to a
distinct genetic control."
"Since all these specific anosmias seem to reflect gene-frequencies which fall
within the range of genetic polymorphism, as defined by Ford, i.e., they are too
frequent to be explained as being maintained by recurrent mutation alone, there
The Isovaleric Acid Anosmia and the Sweaty Primary Odor 251
must be a large series of selection pressures operating on the genes controlling

them. One may expect the new loci to be useful in human gene-mapping."
A few cautious conclusions may be permissible from these pioneering investiga-
tions of olfactory genetics. The work is difficult enough from the olfactometric
standpoint, on top of which the genetic phenomenon appears to be far more
complicated than anybody anticipated. The balance of opinion would seem to
favor the non-smelling trait to be a recessive character. If so, the result would at
least be in agreement with the idea of a selective receptor protein representing a
primary odor. Only if an individual failed to inherit the gene for producing the
particular receptor protein from either parent, would he experience the specific
anosmia. The heterozygous individual would probably be unrecognizable as a
phenotype, because the expected halving of sensitivity is much the same as the
e:xperimental error in measuring a person's threshold (AMooRE et al., 1968).
Olfactory genetics deserve intensified research effort partly to assist in unravel-
ling the hypothetical primary odors of the sense of smell and also as a contributor
to the fund of accessible traits for studies in population genetics.
The Isovaleric Acid Anosmia and the Sweaty Primary Odor

So far only one type of specific anosmia has been thoroughly studied with
regard to the chemical extent of the smelling deficiency (AMOORE, 1967). By
systematic application of the olfactory tests mentioned above and fully described
elsewhere (AMOORE et al., 1968), ten persons were found exhibiting specific anosmia
to isobutyric acid. These ten individuals were thereafter treated as a test group, for
purposes of measuring their average olfactory deficiency compared with a large
control group of 97 normal observers. The group threshold deficiency towards
isobutyric acid was 5.6 binary steps in the odorant concentration scale.
Subsequently the group of specific anosmics was tested with 17 other odorants
which are chemically related to isobutyric acid. The results are shown in Fig. l.
The higher the points on the graph the greater the deficiency of the anosmic group
to that odor. Among the straight chain (normal) fatty acids, the deficiency
exhibits a broad plateau between 4 and 7 carbon atoms. The maximum deficiency
of 6.6 binary steps was observed with the branched-chain isovaleric acid, which is
thus identified as the best representative for this variety of specific anosmia. Alter-
ing the functional group (from the carboxylic acid) to the aldehyde or alcohol
changed the odor type and largely removed the smelling deficiency.
According to the concept of GUILLOT (1948) the phenomenon of specific anosmia
may be due to a defect in one of the primary odors of the sense of smell. If this
idea is accepted, it follows that the above "map" of the anosmic deficiency is
equivalent to a profile of the corresponding primary odor specificity (A:~moRE,
1967). The most a ppropriate subj ective description ofthis primary odoris "sweaty. "
This would appear to be the first systematic experimental delineation of any
primary odor.
Purified isovaleric acid is the best example known for this odor primary.
Evidently its molecular size and shape in conjunction with the appropriate
carboxylic acid functional group approximates to the ideal stimulant for the cor-
responding olfactory receptor protein. The molecular size and shape parameters
~
Table 2. Olue8 to the olfactory code& ~
general odor classifications specific anosmia analyses

line ZWllR- LINNAlIiUS HENNING CROOKER AlIOORlIi SOHUTZ WRIGHT HARPER MISOEL- reputed specific anosmlas estab- probable
no. DEHAKER & & 6t. al. LANEOUS lIshed primary
HENDER- MIOHELS primary odor
SON odorant
(1895) (1756) (1915) (1927) (1952) (1964) (1964) (1968)
30 (sub)
classes 7 classes 6 classes 4 claBBeS 7 classes 9 claBBes 8 classes 44 classes additional ;-.
1 fruity hexyl fruity y-undeca- ~
acetate lactone
2 Waxy soapy
3 ethereal ethereal etherish etherish ethylene- trlchloro- benzene methyl ~
solvent dichloride ethylene cyclo-
propyl ~
ketone
4 Camphor camphor camphor 1.8-cIneole naphtha- p-dichloro adaman-
mothballs lene benzene tane
5 clove aromatic aromatic eugenol benzyl- anlsole
alcohol
6 cinnamon spicy spice spicy cinnam- salicyl-
aldehyde aldehyde
7 aniseed benzo-
thlazole
J
8 minty minty minty menthone menthol tert-butyl
carbinol I
9 thyme thymol ~
10 rosy geraniol phenyl-
ethanol !lIJ,.
11 citrous fruity cltral cltrous geranlal
12 Almond spicy almond hydrogen Isobutyr-
cyanide aldehyde
13 jasmine flowery floral floral PEllE
carbinol g;.
i
14 orange- fragrant fragrant fragrant fragrant
blossom
15 lily
16 violet lonone famesol
17 vanilla sweet vanilla vanillin benzyl anisic cyclotene
sweet salicylate aldehyde
18 amber animal
111 musky ambrosial musky musk macro- androste- musk versallde
cyclic nol xylol
musks (4)
20 leek alllaceous garlic allyl iso- phenyliso- allicin propenyl-
thlocy- thlocy- sulfenic
anate anate acid
21 fishy ammonia hexyl-
fishy amine
22 bromine iodoform
23 burnt burnt burnt burnt affective burnt
24 phenolic carbolic
25 caproic hirclne caprylic sweaty isobutyric phenyl- caproic Isovaleric sweaty
* acid acetic acid acid
acid
26 - cat-urine = 251
27 narcotic repulsive phenyl-
Isocyanide
28 bed-bug
29 carrion nauseous sickly putrescine ~
30 fecal fecal skatole indole
31 resinous resinous resinous
paint
io
32 foul putrid sulfurous unpleas- putrid mercap- dimethyl thiophane g
ant sulfurous tans (3) disulfide
33 acid acid formic acetic
acid acid
34 oily oily
35 - rancid rancid = 251 [
36 metallic metallic <:>
37 meaty
38 moldy 2-heptan-
one t
39 grassy
40 bloody
41 cooked- methional [
vegetable
42 sandal cedryl-
acetate 8
43 watery
44 urinous androsta- f
dienone <:>
45 S;
<:>
46
47
48
49
50
(non-
olfactory) I I I (pungent) (trigemi-
nal)
(pungent
& 5 others)
r
a Citations for the general odor classifications are included in the References to this chapter. The sources of the reputed specific anosmias
are listed in the original publication (AMOORE, 1969); from which this Table is reprinted by courtesy of Rockefeller University Press. N)
at
<:.0:>
are amenable to quantitative assessment using silhouette photographs of scale

molecular models. These may be conveniently appraised by a scanning computer
(AMOORE, PALMIERI, and WANKE, 1967). A correlation coefficient as high as 0.80
was obtained between molecular shape and anosmic deficiency. Hence there is a
good prospect for eventual quantitative treatment of odor qualities by computer
methods. Refining the procedures could yield detailed inferences about the con-
figuration of the molecular receptor site on the olfactory receptor protein.
formic 'celie PrGp iCinlC Bul,oC Vmlulc

7
...
6
1 : : ' \
r__I
<II
~S Tert , . '. o
r
,b" lO "'!~i l,,\o'?!
VI
l'
'<l
0"
~4
(>'-
T \ T
i\ I
~ 6 \. I~l
~3~ 2~~11{ 0 Al dehyde

~ 1 1
1t__~____ ~ ~
0 l '
L-__
o Alcohol
__
1
____L-__
I
~
!
__-L_E5_t_er____ ~
2 3 5 6 7 8 9 10
Number of carbon atoms
Fig. 1. Influence of molecular structure on degree of specific anosmia to fatty acids and related
compounds. Vertical lines indicate standard error, SED. The aldehyde, alcohol and ester are
the isobutyl derivatives. Reprinted from AMOORE (1967) by courtesy of Macmillan Journals
The isovaleric acid smelling defect is occasionally quite remarkable in its

magnitude. Among 443 otherwise normal observers, AMOORE (1968b) reported
finding two subjects with far-and-away greater deficiency than even the other
specific anosmics. These two persons were at least 15,000 times less sensitive than
the average normal observer. As discussed previously (AMOORE, 1968a) there could
be more than one explanation for this result. Possibly the extreme defectives
completely lack the appropriate receptor protein, while persons with intermediate
deficiency simply have an abnormal protein with lowered sensitivity. Alterna-
tively the extreme defectives may lack more than one receptor protein (multiple
specific anosmia) with some overlap in their specificity profiles. A number of
possible variations are to be anticipated, because in vivo protein defects can range
from single amino acid deletion or substitution through increasingly drastic
abnormalities up to complete absence of the specific protein. These considerations
could lead to qualitative as well as quantitative changes in the chemical specificity
and sensitivity pattern. A thorough genetic analysis of the specific anosmias may
be indispensable for disentangling the various possibilities.
References 255
Tentative Odor Classification Based on Specific Anosmias

It should be possible in time to extend the special principles enunciated in the
preceding section into a general classification of odors as originally envisaged by
GUILLOT (1948). Up to now 62 instances of specific anosmia to different named
chemicals have been described (AMOORE, 1969). These compounds are listed on
the right side of Table 2. By comparison with existing classifications of odors (left
side of Table 2) and from considerations of chemical and odorous relatedness,
these known anosmias may be reduced to 27 through the grouping of likely re-
dundancies. Hence somewhere between twenty and thirty seems to be a reasonable
estimate for the likely final number of primary odors in man. The problem of
identifying the primaries may be regarded as the problem of the "olfactory code."
Table 2 represents the initial plan for a very large experimental survey which
has hardly begun. Evidently the classification of odors is a much more complex
problem than most people had suspected. Nevertheless here is a rich and largely
un-tapped mine of information which could be exploited in at least two meaningful
ways. Systematic investigation of all specific anosmias should eventually reveal
all the primary odors, hence providing an objective classification of odors. Further-
more the wealth of corresponding genetic defects and variants could supply ample
material for research in population genetics.
References
AMOORE, J. E.: The stereochemical specificities of human olfactory receptors. Perfum. essent.
Oil Rec. 43, 321-323, 330 (1952).
- Specific anosmia: a clue to the olfactory code. Nature (Lond.) 214, 1095-1098 (1967).
- Specific anosmias and primary odors. In: Theories of odor and odor measurement, 71-85
(TANYOLAc, N. ed.). Istanbul: Robert College 1968a.
- Odor-blindness as a problem in odorization. Proc. Operating Sect., Amer. Gas Assoc.,
Distribution Conf. 242-247 (1968b).
- A plan to identify most of the primary odors. In: Olfaction and taste III, 158-171 (PFAFF-
MANN, C. ed.). New York: Rockefeller University Press 1969.
- PALMIERI, G., WANKE, E.: Molecular shape and odour: pattern analysis by PAPA. Nature
(Lond.) 216, 1084-1087 (1967).
- VENSTROM, D.: Sensory analysis of odor qualities in terms of the stereochemical theory. J.
Food Sci. 31, 118-128 (1966).
- - DAVIS, A. R. : Measurement of specific anosmia. Percept. Mot. Skills 26, 143-164 (1968).
ASH, K. 0.: Chemical sensing: an approach to biological molecular mechanisms using difference
spectroscopy. Science 162, 452-454 (1968).
BLAKESLEE, A. F.: Unlike reaction of different individuals to fragrance in Verbena flowers.
Science 48, 298-299 (1918).
BROWN, K. S., ROBINETTE, R. R.: No simple pattern of inheritance in ability to smell solutions
of cyanide. Nature (Lond.) 216,406-408 (1967).
CROCKER, E. C., HENDERSON, L. F.: Analysis and classification of odors: an effort to develop
a workable method. Amer. Perfumer ess. Oil Rev. 22, 325-327, 356 (1927).
DASTOLI, F. R., LOPIEKES, D. V., DOIG, A. R.: Bitter-sensitive protein from porcine taste
buds. Nature (Lond.) 218, 884-885 (1968).
- PRICE, S.: Sweet sensitive protein from bovine taste buds: isolation and assay. Science 1M,
905-907 (1966).
DOUEK, E. E.: Smell: recent theories and their clinical application. J. Laryng. 81,431-439
(1967).
DUNCAN, R. B., BRIGGS, M.: Treatment of uncomplicated anosmia by vitamin A. Arch.
Otolaryng. 76, 116-124 (1962).
FUKUMOTO, Y., NAKAJIMA, H., UETAKE, M., MATSUYAMA, A., YOSHIDA, T.: Smell ability to
solutions of potassium cyanide and its inheritance. (In Japanese, with English summary).
Japan. J. Human Genet. 2 (1), 7-16 (1957).
GUILLOT, M.: Anosmies partielles et odeurs fondamentales. C. R. Acad. Sci. 226, 1307-1309
(1948).
HARPER, R., BATE-SMITH, E. C., LAND, D. G., GRIFFITHS, N. M.: A glossary of odour stimuli
and their qualities. Perfum. essent. Oil Rec. 69,22-37 (1968).
HENKIN, R. 1.: Complete anosmia: the absence of olfaction at primary and accessory olfactory
areas. Life Sci. 6, 1031-1040 (1966).
- The definition of primary and accessory areas of olfaction as the basis for a classification of
decreased olfactory acuity. In: Olfaction and taste II, 235-252 (HAYASill, T. ed.). Oxford:
Pergamon Press 1967.
- KOPIN, I. J.: Abnormalities of taste and smell thresholds in familial dysautonomia:
improvement with methacholine. Life Sci. 3, 1319-1325 (1964).
- POWELL, G. F.: Increased sensitivity of taste and smell in cystic fibrosis. Science 138,
1107-1108 (1962).
HENNING, H.: Del' Geruch, I. Z. Psycho!. 73, 161-257 (1915).
HUSER, H. J., MOOR-JANKOWSKI, J. K., TRUOG, G., GEIGER, M.: Klinische, genetische und
gerinnungsphysiologische Aspekte der Hamophilie B bei den Blutern von Tenna, mit
einem Beitrag zur Genetik der Gerinnungsfaktoren. Acta Genet. 8, 25-50 (1958).
KIRK, R. L., STENHOUSE, N. S.: Ability to smell solutions of potassium cyanide. Nature (Lond.)
171, 698-699 (1953).
LnrnAEUS, C.: Odores medicamentorum. Amoenitates Academicae 3, 183-201 (1756).
MArnuND, R. C.: Absence of olfactory sensation. J. Heredity 36,143-144 (1945).
PARKER, G. H.: Smell, taste and allied senses in the vertebrates. Philadelphia: J. B. Lippincott
1922.
PATTERSON, P. M., LAUDER, B. A.: The incidence and probable inheritance of smell blindness.
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SCHNEIDER, R. A.: The sense of smell in man: its physiologic basis. New Eng!. J. Med. 277,
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SCHUTZ, R. G.: A matching-standards method for characterizing odor qualities. Ann. N. Y.
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SPARKES, R. S., SIMPSON, R. W., PAULSEN, C. A.: Familial hypogonadotrophic hypogonadism
with anosmia. Arch. intern. Med. 121, 534-538 (1968).
STONE, R., PRYOR, G.: Some properties of the olfactory system of man. Percept. Pyschophys.
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VENSTROM, D., AMOORE, J. E.: Olfactory threshold in relation to age, sex or smoking. J. Food
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WRIGHT, R. H., MICHELS, K. M.: Evaluation of far infrared relations to odor by a standards
similarity method. Ann. N. Y. Acad. Sci. 116,535-551 (1964).
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Chapter 12
Olfactory Response and Molecular Structure

By
M. G. J. BEETS, Hilversum (Holland)
With 10 Figures
Contents
A. Introduction. . . . 257
B. Basic Concepts. . . 260
C. Response Intensity. 265
D. Electrophysiology . 268
1. Recordings from the Receptor Membrane 268
2. Recordings from Nerve Fibres . . . . 278
3. Recordings from the Olfactory Bulb . . 280
E. The Verbal Expression of Odor Sensation . 285
1. Odor Classification. 286
2. Odor Similarity . 287
3. Odor Transition . 297
F. Optical Antipodes 300
G. Adaptation . . . 304
H. Specific Anosmia. 308
I. Indirect Relations 311
K. Conclusions . . 313
References . . . . . 316
A. Introduction
All properties of a compound, chemical, physical and physiological, are
unambiguously determined by the complete definition of its molecular structure
(BEETS, 1957).
However, most of these properties are the observable and measurable effects
of an interaction between the molecules of the compound and a more or less
complex system consisting of the interaction partner or partners and external
conditions. They can only be observed and measured as reproducible consequences
of the molecular structure if the parameters of this system are explicitly defined
and do not vary beyond narrow limits between observations of the same property.
In the case of chemical and physical properties the system is generally simple
and well-defined. It may consist of a reaction partner in a chemical reaction, or of
17 Rb. Sensory Physiology, Vol. IV/I
258 M. G. J. BEETS: Olfactory Response and Molecular Structure
light of a discrete wave length in absorption spectroscopy, each combined with 11

set of external conditions. In all such cases the effect is observed and measured as
the direct consequence of the interaction and the interaction mechanism is
frequently well understood.
The system involved in physiological properties is invariably a highly complex
part of a living or freshly killed organism. Its structure, function and scope are
imperfectly understood. The information resulting from the interaction can be
tapped at various parts of the system but only after it has been subjected to one
or more cryptic transcoding and processing operations.
The relationship between the original and the measured information is rarely a
simple one. Ingenuity as well as speculation are often required for its reconstruc-
tion. This is superlatively the case in olfaction and a discussion of the relationship
between structure and odor can only be meaningful if these complications are
taken into account.
The process of olfaction in terrestrial animals starts when the odorant molecules
which have found their way through the devious air passages of the nasal cavity,
are absorbed on to the mucosae containing the olfactory, the trigeminal and the
vomeronasal nerve endings. For most classes of odorants the trigeminal and the
vomeronasal nerve endings seem to playa part in the formation of the olfactory
code message (BEIDLER and TUCKER, 1955; MOULTON and TUCKER, 1964).
Although we know very little of the relative contributions of the three nerve
types to the message, it seems reasonable to assume that for most odorant mole-
cules the impulses generated in the olfactory membrane carry an important part
of the total information and consequently are largely responsible for the dis-
crimination of olfactory quality.
The primary effects of the interaction between molecules and receptor sites are
summated in the receptor cells and translated into nerve impulses carrying the
information elements originated in the receptor cells. The total information
pattern consisting of these elements travels up the olfactory nerve into the olfac-
tory bulb where it undergoes new processing steps leading to strong convergence
which does not necessarily decrease the amount of pertinent information in the
message.
In its new, condensed or perhaps sorted out form the message is delivered to
the information processing center of the brain where it is compared with memory
contents. Irrespective of the result of this comparison, the message is translated
into the observed odor sensation but in case memory elements are found compar-
able with elements of the message presented, the resulting odor sensation contains
a recognition pattern which may be expressed verbally.
This crude outline of the olfactory process suffices to define the scope of our
subject.
Information on the effect of stimulation can be obtained at various points in
the process; by means of electrophysiological techniques at the olfactory mem-
brane, in the olfactory and trigeminal nerves and in the olfactory bulb and, by
analysis of the verbal odor expression, at the terminus of the process.
Electrophysiological techniques provide us with objective information tapped
from sites which are damaged or at least strongly influenced by the instrument
used and which are part of an organism which is injured or, at best, functioning
Introduction 259
under abnormal conditions. The verbal expression gives us subjective and often
psychologically distorted information obtained from the intact, normally function-
ing system. Its quality requires upgrading by means of statistical techniques, of
comparative analysis of the semantics involved in a series of observations and of
methods which influence the olfactory system in a way which the subject cannot
comprehend and, consequently, which he cannot subjectively distort.
Both types of information are causally although differently related to the
information generated in the interaction and, through this, to the molecular
structure. Consequently both types will be discussed in this chapter in so far as
odorants of defined structure are involved in the experiments.
Unfortunately the amount of information available is only a fraction of what
it could have been if the multi-disciplinary nature of the subject had been realized
in an earlier stage and especially if there had been closer coopcration between
physiologists and chemists. Although the situation is rapidly improving, much
excellent work, especially in the electrophysiological field, has been carried out
with ill-defined and unreproducible mixtures of odorants such as coffee, decaying
fish, lilac, cedar and orange. Work of this type may have enabled its authors to
draw important conclusions on other aspects of the problem but for our knowledge
about the structure-odor relationship it has no value. Progress in this field will be
greatly accelerated by the exclusive use of painfully purified single chemicals.
Until more experimental information will be available regarding the role of
trigeminal and vomeronasal (TUCKER, 1963) effects in the olfactory process, the
use of compounds with molecular weights lower than 130-140 such as carbon
disulfide, diethyl ether, lower alcohols and acetone, should be avoided except when
there is a well-considered reason to use such materials.
A third reason why the available information shows deplorably largc gaps is
that materials synthesized in academic institutes are rarely evaluated on odor
whereas those originated in commercial laboratories are limited to structural
types of commercial interest such as musks and ambers. Consequently, systematic
synthetic work in other classes is virtually non-existent. We have detailed infor-
mation on the structural parameters of the musk odor but we know little of those
of the camphor odor. The important and simple problem of the odor of optical
antipodes has not yet been solved satisfactorily. This indicates the necessity of
incorporating systematic organic synthesis combined with organoleptic and
electrophysiological evaluation in academic research on olfaction.
Since the molecular structure is the common origin of all properties of a
compound, these properties are necessarily interrelated. Although the nature of
this interrelationship may be extremely intricate and cryptic, it does at least
present the theoretical possibility of discovering meaningful links between olfac-
tory information and one or more other characteristics of the odorant.
The direct relationship between olfactory information and molecular structure
as well as the indirect inter-relationship between such information and other
properties belong to the scope of this chapter.
This text does not present a complete review of the relevant literature. Much
of the earlier work has been rendered obsolete by the progress made in the last
decade. Many excellent modern papers have been omitted either because of the
17
doubtful nature of the odorants used or because their results and conclusions do
not contribute significantly to our knowledge of structure-odor relations.
Rather than presenting a complete compilation of vaguely or unconnected
structure-odor relationships, I have attempted to select those which may con-
tribute significantly to our understanding of the olfactory process and especially
of olfactory discrimination.
In the next paragraph some basic concepts will be discussed which, without
perhaps belonging to the realm of solid facts, are based on reasonable assumptions.
These concepts will serve as the starting point for a discussion of structure-response
relations which will enable us, in the concluding paragraph of this chapter, to
review what we have learned and in how far the concepts from which we started
can be expanded, confirmed or rejected.
In following this course, no mortal author can avoid a fair amount of specu-
lation and subjectivity. However, as long as we are aware of their existence, such
drawbacks are acceptable in view of the advantage that this approach enables us
to make a direct attempt to give the phenomena a meaningful place in the olfac-
tory process.
B. Basic Concepts
All theories of olfaction based on tele-activation of receptors by means of
molecular radiation have now been abandoned (OTTOSON, 1956). Concepts involv-
ing chemical or enzymatic interaction between receptor and odorant molecule, in
which a chemical change of the latter is the basis of olfactory interaction, have
perhaps not yet reached this final stage, but in view of the vast diversity of odorant
structures such theories are hardly tenable. This does not mean that the receptor
site may not have an enzymatic function which is triggered by absorption of a suit-
able molecule. A mechanism of this type, which does not involve a chemical con-
version of the odorant molecule, has recently been proposed by MARTIN (1970). An
excellent review, including a discussion of the older theories, has been published by
JONES and JONES (1953).
This brings us to a point at which we may consider a physical and reversible
contact between the odorant molecule and one or more molecular components of
the membrane structure, preceding olfactory interaction, as an established fact.
The simplest model of the interaction scene we can envisage is an interface
between two layers, which we may call the carrier phase and the receptor phase.
The carrier phase is a microlayer of mucus to which odorant molecules are
continuously supplied from both the mobile phase above and the receptor phase
underneath and from which such molecules are continuously removed towards
both phases. If the carrier phase is defined as being sufficiently thin it may be
considered to be stagnant and to represent a steady state with respect to odorant
concentration during the major part of the inspiration. The concentration of
odorant molecules in the carrier phase defines the non-intrinsic term of odor
intensity.
The interaction between odorant molecules and receptor components, the
source of the elementary bits of information from which the olfactory code message
is constructed, occurs in the framework of an adsorption-desorption process across
the interface. The criteria governing such processes in general may throw some
Basic Concepts 261
light on the question how odorant molecules are presented to the receptor com-
ponents and how the characteristics of this presentation influence olfactory inter-
action. For this reason we may consider the adsorption of a population of identical,
rigid and non-polar molecules at an interface between an aqueous and a non-
aqueous phase.
Adsorption processes are statistical phenomena. Although all molecules have
the same structure, they will enter the interface in an almost random pattern of
orientations which may, in this case, be represented by a shallow Gauss curve,
using frequency and angular deviation of the interface-perpendicular from the
longitudinal axis as coordinates.
In the case of rigid, polar molecules, possessing a sterically accessible functional
group, the orientation pattern can be expected to be much more homogeneous.
Practically all molecules will be adsorbed in the same orientation. The orientation
pattern can now be represented by a sharp, narrow Gauss curve.
As the polarity or the steric accessibility of the functional group decreases, the
position of the energetically preferred orientation and consequently, also the
orientation pattern degenerate and the Gauss curve representing the latter
becomes more and more shallow. The frequency distribution representing the
orientation pattern of molecules with two polar groups that are sufficiently far
apart in the structure may show two maxima.
Adsorption of flexible molecules at an interface is governed by variations of
orientation as well as of conformation, and presents a much more complex pattern.
We are now interested to know how this outline of the behavior of molecules in
a conventional interface between two layers changes when the adsorption process
involved in olfaction is considered. In this case, the non-aqueous phase has a
structure, consisting of or containing sites on the poly-molecular, mono-molecular
or sub-molecular level with a roughly specific tendency to accommodate specific
molecular profiles with varying efficiency in a reversible complex between mole-
cule and receptor site.
In this chapter we will abstain from discussing the nature of this accommodation
since theories of olfactory interaction are discussed by J. T. DAVIES in this volume.
It may be that a poly-molecular matrix is punctured (DAVIES, 1953), that the
membrane structure contains permanently available cavities of specific shape
(A~100RE, 1952) or that a molecular receptor component or part of such a com-
ponent can undergo a conformational change in order to accommodate the steric
requirements of an odorant molecule. The author favors the latter concept but
since we know little about the structure of the receptor membrane, all three con-
cepts are purely speculative. Also, at this stage of our knowledge, the choice is
arbitrary since a potential receptor site may have the same steric characteristics
as a permanently existing one.
The transition from an adsorption phenomenon at a conventional interface to
the process taking place at the receptor membrane does not necessarily change
much in the statistical patterns.
Unlike the unstructured non-aqueous phase of the conventional system, the
olfactory membrane has a structure containing sites not only with a certain
affinity for the arriving molecules but also with specific steric requirements and
polarity.
262 11. G. J. BEETS: Olfactory Response and Molecular Structure
A non-polar molecule will probably adapt its coniormation to the steric

requirements of the site to which it is presented, as far as its orientation permits
this. Since only one of the partners has a polar character, there is no driving force
towards reorientation and the molecule can be expected to participate more or less
efficiently in the complex in the orientation in which it arrives or to be released
without forming a complex in cases where the steric requirements of oriented
profile and site are incompatible.
Consequently, a population of non-polar molecules with identical structures
will be presented to the population of sites as having an almost infinite variety of
profiles. Interaction will lead in this case to a blurred code message which the
brain is unable to identify or analyse as it is unable to identify a blurred photo-
graph of a fast-moving object (BEETS, 1967). This is roughly in agreement with the
weak, uncharacteristic odors of pure, saturated hydrocarbons.
Rigid, polar molecules will participate more or less efficiently in the interaction
complex mostly in a single orientation dictated by their own polarity and by that
of the sites. Finally, flexible, polar molecules have an extra degree of freedom in
the adaptability of their coniormation to the steric requirements of the sites.
However, the latter are different for different sites. Consequently, flexible polar
molecules, even although their orientation pattern is homogeneous because it is
dictated by the polarity of the sites, may be able to complex and interact in various
coniormations with several site-types.
All this is not meant to become the basis of a new theory of olfaction. It is
merely an attempt to find out what we can learn from analogous, better-known
systems, and what to look for in the observed facts which we shall discuss in the
next paragraphs. In order to facilitate this we may draw the following conclusions
from the theoretical considerations given above.
The complexity of, i.e. the number of different types of information on the
molecular level in, the primary information pattern can be expected to decrease
with decreasing molecular flexibility, increasing polarity and increasing steric
accessibility of the functional group.
Identifiable odor types, characterized by easy recognition and large populations
of representatives, in short, the types named "primary odors" by AMOORE (1952),
can be expected to be related to primary information patterns of minimum com-
plexity (maximum homogeneity) and consequently to occur mostly in the classes
of rigid polar molecules (e.g. musks) and never in non-polar molecules (e.g.
saturated hydrocarbons). True "mono osmatic odors", (BEETS, 1968) i.e. odors
caused by activation of a single type of receptor site by a single type of molecule
probably do not exist and are only approximated in insects, e.g. the stimulation of
specialized receptor cells by the queen substance in the honey bee (KAISSLING and
RENNER, 1968). In man and in vertebrates, with an undoubtedly considerable
number of only roughly specific receptor site types, the code message can be
expected always to have a fairly complex (heterogeneous) character.
The preferred coniormations of flexible molecules in the olfactory interaction
complex are not necessarily identical with those of free molecules or molecules
absorbed at an unstructured interface, since we can expect the molecule to adapt
its conformation to the steric requirements of the site to which it is presented.
Since we know nothing of the latter, the practical consequence is that it seems
Basic Concepts 263
justified to compare molecular models of compounds with similar odors in con-

formations with maximum profile similarity.
The preferred orientation of a polar molecule on a specific receptor site can be
assumed to be related roughly in the same way to the direction of the dipole axis
for molecules with similar odors. The degree of profile similarity for such compounds
should be studied by comparing their molecular models in the same orientation
with respect to their dipole axis.
Of the principles discussed above, the central role of the molecular dimensions
(profile, shape, size) for odor has now been fairly generally accepted, perhaps the
more readily because of the firmly established relation between steric aspects and
other types of biological activity. Among the first to recognize the importance of
molecular shape in olfaction we find, apart from the much quoted LUCRETIUS,
TROLAND (1930) and PAULING (1946). The importance of orientation and con-
formation patterns in olfactory interaction seems to supplement that of the profile
criterion. In the course of this chapter we shall see in how far these principles are
corroborated by the observed structure-response relations.
The major aspect of the problem of olfaction on which we may hope to improve
our insight by studying olfactory response-structure relations is olfactory dis-
crimination. The olfactory code message containing the information which enables
the brain to record, to analyse and eventually to recognize an odor picture is the
end product of a series of processing operations in the olfactory system for which
the raw materials were generated mainly by the interaction following absorption
of odorant molecules on to the epithelium.
The anatomy of the olfactory system in general, and of the olfactory epithelium
in particular, have been reviewed by GEREBTZOFF (1953), GASSER (1956), DE Lo-
RENZO (1963) and FARBMAN (1967) and also in other chapters of this volume. Here
we shall merely mention a few facts which are of particular importance to our
subject.
The olfactory epithelium in many species is covered by a thin sheet of mucus
into which the ducts of the Bowman glands inject their pigmented secretion. This
mucous layer may very well be an efficient colloidal medium for absorption and
desorption of organic molecules of widely varying structure in both directions.
However, it is an unstructured aqueous colloidal system. Its thickness and its
composition probably vary considerably with time, location on the epithelium and
condition of the subject. Also its position in the nasal cavity and its limited size
make it hard to believe that it could playa stationary phase-type part in a sorption
mechanism of olfactory discrimination analogous to gas chromatography (MOZELL,
1964a, 1964b;MouLToN, 1965; DOLL and BOURNOT, 1949; JONES and JONES, 1953).
The epithelium itself (REESE, 1965), is a densely-packed structure of receptor
cells and supporting cells and its surface is a mosaic of the peripheral endings of
both cell types, each of which carries hairlike filaments which are embedded in the
mucus. For the olfactory cells these are called cilia, in general 1-20, in man 6 to
8 per cell. Their length is 10-200 micron and their thickness about 0.1 micron.
The terminal processes of the supporting cells, the microvilli, are much more
numerous (in man about 1000 per cell) and much smaller (about 1 micron) than
the cilia. According to HOPKINS (1926) more than half the length of the cilia lies at
the surface of the mucus.
The supporting cells probably playa role in the electrical mechanism of signal
formation (MAcLEOD, 1963; TUCKER and SHIBUYA, 1965) but they carry no
receptor sites capable of interaction with odorant molecules (TAKAGI and YAJIMA,
1965). Also no relation has been discovered between the pigment present in the
supporting cells and olfactory interaction or discrimination (BRIGGS and DUNCAN,
1961; MOULTON, 1962; TAKAGI and YAJIMA, 1965; MOULTON and BEIDLER, 1967).
At this stage of our knowledge there is no longer any serious doubt that
olfactory stimulation and the formation of the olfactory signals take place on and
in the receptor cell and, although conclusive evidence is still lacking, the limiting
membrane of the cilia is generally thought to be the scene of olfactory interaction
and to contain the receptor sites (MOULTON and BEIDLER, 1967). Assuming this to
be true, a rough calculation made for a small mammal and based on a number of
108 receptor cells, each carrying 13 cilia with a length of 100 micron and a diameter
of 0.15 micron, shows that about 600 cm2 is available for interaction between
odorant molecules and receptor sites. OTTOSON and SHEPHERD (1967) have recently
discussed the possible arrangements of receptor sites on the cilia and their ability
to transmit excitation from the sites to the dendrite and further to the axon
hillock where impulse initiation is presumed to take place.
Each receptor cell is linked by a single nerve fiber, without branching to the
glomeruli in the olfactory bulb. Of the 50 million receptor cells in the rabbit 25,000
are thus directly connected with a single glomerulus of which there are about
1,900 (ALLIsON and WARWICK, 1949). It has been observed that these axons enter
a glomerulus from many different directions and LE GROS CLARK (1957) suggests
that this may be explained as a re-sorting of fibers (and consequently of information
carried by impulses generated in the receptor cells) on a functional basis.
Apart from a topical localization in the sense that certain areas of the epi-
thelium are projected to local regions of the bulb, which has been demonstrated
experimentally by LE GROS CLARK (1950), he further suggests a second type of
localization; i.e., a redistribution of fibers derived from receptor cells which differ
in their sensitivity to different odorous substances so that impulses generated in
cells with identical or similar interaction patterns with a certain group of related
odorants are led predominantly to certain individual glomeruli.
Each glomerulus transmits information to about 24 mitral cells of which there
are about 48,000 in the rabbit. ADRIAN (1955, 1959) found, by recording from
intrabulbar electrodes, that the mitral cells can be divided into 5 or 6 different
types, each of which is particularly sensitive to one group of odors.
A detailed discussion of the physiological aspects and the construction of an
information theory (HAINER et al., 1954) are beyond the scope of this chapter.
However, we may formulate a few tentative principles of the generation of olfac-
tory information as a basis for critical comparison with the known facts on
structure-response relations. The following steps seem to agree roughly with our
present knowledge.
1. Generation of an information bit by interaction of a molecule in a specific orientation and
conformation with a specific receptor site. The energy effect produced varies with interaction
efficiency.
2. Summation of information bits produced by simultaneous or quasi-simultaneous inter-
actions in the membrane of a single receptor cell. We assume that the cell fires when the sum-
mated energy effects reach a critical value. Information produced by the receptor cell is called
Response Intensity 265
the information element. Firing frequency is determined by interaction efficiencies (represent-

ing the intrinsic term of response intensity) and by interaction frequency (odorant concen-
tration; non-intrinsic term of intensity). As we shall see, the position of the cell on the mem-
brane may be the major source of qualitative information in the information element.
3. On the level of the membrane, the assembly of information elements form the primary
information pattern. This is the precursor of the olfactory code message, the final product of
the olfactory system.
We can be fairly certain (GASSER, 1955, 1956; GESTELAND et al., 1963; DOVING,
1967) that the information elements retain their individuality through cell body
and nerve fiber up to the level of the glomeruli. In man there are about 10 million
olfactory receptors, each of which can conduct a maximum of about 10 nerve
impulses per second over its neural extension. Thus, the informational capacity of
the receptor membrane is about lOS elements per second. This is roughly equi-
valent to the informational capacity of the optic system (BEIDLER, 1966). ENGEN
and PFAFFMANN (1959, 1960) estimated the informational content required for
specific olfactory behavior determination.
It seems that the olfactory bulb is a processing center in which the information
available in the primary information pattern is sorted out and upgraded in two
successive processing steps taking place in the glomeruli and the mitral cells. It is
attractive to assume that the original, extremely complex primary information
pattern is reduced to a simpler form in which all the qualitative and quantitative
aspects of the primary information are retained, perhaps in the same way that all
essential information of a three-dimensional object is retained in a simple two-
dimensional hologram. This simple form is the olfactory code message which is
presented to the brain for analysis and identification.
In short, olfactory discrimination depends only partly on the informational
content of the pattern generated in the primary process at the receptor level, but
also on the ability of the whole olfactory system to process this information and
on that of the central nervous system to discriminate between two similar olfactory
patterns.
In this we may quote ADRIAN (1949-1950) who wrote: "If we had the dog's
much larger and more elaborate olfactory organ, it would not necessarily follow
that we should distinguish many more smells than we do at present; and if the
dog had ours he might still have a range of discrimination wider than we have
though not as wide as that of a normal dog."
It is the modest aim of this chapter to see what we can learn from the single
source of molecular structure about the primary step in the olfactory process in
which interaction between molecules and receptor sites generates the information
pattern which serves as the raw material for the various processing steps in the
olfactory system.
c. Response Intensity
Any olfactory response is characterized by its quality and its intensity. The
intensity consists of a non-intrinsic term, which is determined by the concentration
of the odorant in the carrier phase and an intrinsic term. Both the quality and the
intrinsic intensity are functions of the molecular structure and both are discussed
in this chapter. However, a few aspects of the intrinsic intensity require some
separate attention.
The obvious way to obtain information on intrinsic intensity is to measure the

minimum concentration at which a significant or, as some authors prefer, an
identifiable olfactory response can be observed, i.e. the threshold concentration.
We can safely assume that the primary information pattern of olfaction is a com-
plex assembly of contributions from a large number of types of interaction com-
plexes between odorant molecules and receptor sites. In this assembly one type
may quantitatively predominate and to the extent to which this is the case we
approach the non existent ideal of the mono-osmatic odorant. In some of the best
representatives of Amoore's primary classes we can expect this type of response
pattern. If this philosophy is correct, such odorants present particularly attractive
substrates for threshold determinations since the predominating type of inter-
action complex can be assumed to continue making its contribution to the odor
sensation or to the recorded response at concentrations at which the secondary
types have ceased to do so. In short, the predominating type is the last component
of the odor sensation to survive as the concentration decreases. This means that
for certain groups of odorants the threshold value could very well be a quantitative
criterion for a true mono-osmatic phenomenon. If this were the case we could say
that at threshold concentration Amoore's primary class odorants become truly
mono-osmatic in the sense defined by BEETS (1968), and we may even find that
they are characterized by some common criterion such as a narrow range of thresh-
old values for all odorants belonging to a single primary class or a particularly
transparent relationship between threshold value and physical properties or molec-
ular structure. This, of course, is pure speculation.
However, we should keep in mind that the absolute values of threshold con-
centrations reported in the literature have little or no meaning as far as they have
been determined in the conventional way with humans or, on the basis of behavior,
with other mammals. In such cases, the concentration of the odorant in the air
current entering the nose of the subject is known, but its relation to the con-
centration in the carrier phase, i.e., to the availability of odorant molecules for
interaction with the receptor sites is unknown since a large percentage can be
expected to be trapped by the mucous lining of the nasal cavity. STUIVER (1958)
calculated that approximately 2 % of the molecules in the air current entering the
nose reach the olfactory epithelium during normal breathing.
Many threshold values measured by conventional methods may show regu-
larities which are not or only partly related to olfaction and mainly to the relation
between the solubility of the odorant molecules in water and the degree in which
they are trapped before reaching the receptor sites. An example may be the
characteristic differences found by STUIVER (1958) in the thresholds of the three
nitro-phenols. The intra-molecularly chelated and consequently less water-soluble
ortho-isomer has a much lower threshold (5.0 x 109 ) than the two other isomers
(ca. 1013 ).
The unknown influence of trapping can only be avoided by establishing a direct
contact between the air current carrying the odorant and the receptors. Much of
the electrophysiological work reported satisfies this requirement and will be discuss-
ed in the next paragraph. The same holds for the study of behavioral thresholds
with insects such as the work of DETHIER et al. with blowflies. In most of their
papers, these authors described stimulation of the tarsal contact receptors with
Response Intensity 267
aqueous solutions of stimulants, using blowflies with amputated antennae and

labella to eliminate olfactory effects. This part of the work lies beyond the scope of
this chapter. DETHIER and YOST (1952) also determined for a series of alkanols
(CCC10) the concentration in the vapor phase rejected by 50% of a population of
blow flies with intact antennal receptors and found a linear relationship between the
logarithms of the rejection threshold and the number of carbon atoms. When
threshold concentrations were expressed in terms of thermodynamic activity, the
intermediate terms of the series roughly conform to the rule of equal effectiveness
at equal activity.
Although the absolute values of thresholds determined by conventional
methods may have a cryptic meaning, they can be applied with some confidence in
comparative studies of closely related odorants such as isomers or homologs for
which the effect of trapping can be assumed not to differ too much. For this reason
it is rather unfortunate that MULLINS (1955) chose for his study of thresholds a
series of C4 hydrocarbons which, in the pre-gas chromatography period, can hardly
have been free from minute traces of impurities with powerful odors and which, in
pure form, are probably odorless.
Several authors have studied the thresholds of n-alkanols by means of con-
ventional or behavioral methods. PASSY (1892), VON SKRAMLIK (1948), GAVAUDAN
et al. (1948) and MULLINS (1955) determined these thresholds in man, MOULTON
and EAYRS (1960) in rats. All this work indicates a linear relationship between the
logarithms of threshold and chainlength, the olfactive efficiency increasing with
increasing chainlength within the limits of the experiments (usually CC C12).
Since such a simple relationship is not found in some other homologous series
(PASSY, 1893; MULLINS, 1955), MOULTON and EAYRS (1960) mention a number of
reasons which could explain this discrepancy.
Recently, BEETS (1968) outlined a method, according to which intensity
estimates by a panel may bc used to assess the degree of relatedness between the
primary information patterns of odorants with similar odors such as musks, and
perhaps eyen to obtain an impression of the complexity of these patterns. In a
preliminary experiment, odor intensities were estimated by putting solutions of
various musks in odorless diluents in their correct position in a series of solutions
of a reference musk, e.g., pentadecanolide with concentrations increasing from
1-10 %. Intensity indices were computed based upon a value of 100 for the refer-
ence musk. This index was found to depend not only on the nature of the musk
but it also increases very significantly with dilution. The dilution effect is extremely
strong for the nitro musks and much weaker for polycyclic isochromane- and keto
musks. The variance between observers and between duplicates was found to be
high.
BEETS interpreted these results tentatively in the light of the concept that the
primary information pattern for all musk odors is a blend in which the pure musk
component predominates and in which secondary components are present of
which the nature, the number and the contribution vary with molecular structure.
The large variances observed may be explained by the inability of the observers to
distinguish between the total intensity of the sensation and the intensity of the
pure musk component in both the evaluated and the reference musk, resulting
in different and inconsistent interpretations of their impressions. The dilution
effect may indicate the degree of relatedness between the primary information
patterns of the musk evaluated and the reference musk or, in case the latter is
assumed to represent the closest possible approximation of the ideal mono osmatic
musk, the complexity of the primary information pattern of the evaluated musk.
The results, which clearly require extensive confirmation, suggest a low degree of
similarity between the information patterns of nitro musks and macro cyclic
musks.
D. Electrophysiology
Although most electrophysiological work has been undertaken in order to
obtain information on the functions and the structure of the olfactory system,
some systematic attempts have been made to find relations between odorant
structure and interaction pattern by recording from the receptor membrane, from
nerve fibres or from units in the olfactory bulb. In most of these cases the structural
chemist would have selected other odorants with more significant structural differ-
ences. Also, although he recognizes the technical difficulties involved, he would
have appreciated recording from a larger number of units, more representative of
the total response pattern.
1. Recordings from the Receptor Membrane

The activity in the olfactory membrane can be recorded in three different
forms, i.e. the slow potential or electro OlfactOgram (E.O.G.), the activity in a
small group of receptor units from an electrode in an arbitrary position in the
membrane and, finally, the activity in a single receptor unit.
OTTOSON (1956) studied the slow negative potential recorded from an electrode
in contact with the mucosa of the frog. This response is only obtained from the
area containing receptor cells. Consequently the E.O.G. must be considered as a
purely olfactory response.
The effect of chemical structure on the E.O.G. amplitude was studied for a
large series of substances (OTTOSON, 1958).
Increasing unsaturation (propyl-, allyl- and propargyl alcohol) results in a
fairly weak but progressive increase of the amplitude. The introduction of a
hydroxyl group in propyl alcohol (propanediol-l,2) and in propionic acid (lactic
acid) reduces the amplitude to nearly zero. Replacement of the hydroxyl group in
propyl alcohol by a primary amino group results in a roughly 4-fold increase of the
amplitude. Acetone, acetaldehyde and propyl alcohol have about the same stimu-
lating efficiency. The ratio between the E.O.G.-amplitude obtained with salicylic
aldehyde and 4-hydroxy-benzaldehyde is about 6.
The amplitudes obtained with the four isomeric butyl alcohols decrease in the
sequence secondary, normal, iso- and tertiary butanol. This order is different from
those obtained by ranging the four odorants according to decreasing vapor pres-
sure, decreasing volatility from an aqueous solution and increasing steric acces-
sibility of the functional group which are all tertiary, secondary iso- and normal
butanol.
In all this work, the stimulus was obtained from solutions of the odorants in
distilled water. Since the concentrations of the stimulants in aqueous solution are
not simply related to those in the stimulating air puff, the differences observed may
Recordings from the Receptor Membrane 269
reflect to an unknown extent concentration differences in the air puff and only for
the remainder differences between the intrinsic stimulating efficiencies.
OTTOSON attempted to reduce the non-intrinsic (concentration-) term as much
as possible by selecting low concentrations which did not surpass the linear part
of the S-shaped curve expressing the relationship between stimulus strength and
response amplitude. The "irregular" sequence found for the stimulating efficien-
cies of the four butyl alcohols suggests that the intrinsic term is represented in
OTTOSON'S results. However, the effects found in most of the other cases mentioned
are too closely related to volatility and concentration to be entirely convincing.
This is especially suggested by the large differences observed between propyl
alcohol and propanediol, between propionic and lactic acid and between the two
hydroxy benzaldehydes. In the latter case the functional groups in 4-hydroxy-
benzaldehyde are free for hydration in aqueous solution, whereas in salicylic
aldehyde hydration is suppressed in favor of intramolecular chelation. The result-
ing very large increase in volatility from an aqueous solution could easily explain
the effect observed by OTTOSON.
The possible superposition of intrinsic and concentration terms in the stimu-
lating efficiency is undoubtedly far less important for series of homologs and here,
(OTTOSON, 1958) some information on the intrinsic term can be obtained.
Fig. 1 shows some of the results.
In Fig. la the amplitudes obtained from primary alkanols with 1-8 carbon
atoms in two concentrations of the aqueous solutions are shown. The abnormally
low value found for pentanol is explained by the presence of isopentanol as an
impurity. In order to examine also the stimulating efficiency of the non-water
soluble alkanols the experiments were repeated with solutions of alkanols with
2-12 carbon atoms in mineral oil (Fig. lc). This demonstrates that the efficiency
reaches a maximum for heptanol whereas, in aqueous solution, octanol has a
higher efficiency. This may be explained by the difference between the curves
expressing the partial vapor pressures of the alkanols above solutions in oil and in
water (solvation effect).
OTTOSON suggests that the curves found for the stimulating effectiveness of the
alcohols in both solvents may be explained by DETHIER'S (1951) hypothesis that
water-soluble odorants stimulate via an aqueous phase and larger molecules via a
lipid phase. Also, CHADWICK and DETHIER (1949), in their work on tarsal reception
in blowflies found a break in the curve at 7 carbons for both the alkanols and
alkanals. DETmER (1952) also observed that the taste threshold in humans for
heptanol was lower than for octanol when both alcohols were dissolved in oil
whereas octanol has the lower threshold in aqueous solution. The similarity between
the curves found by DETmER and by OTTOSON indicate that excitation in contact
chemoreceptors and in olfactory receptors are based on similar mechanisms.
OTTOSON (1958) adjusted, by trial and error, the concentration of the alkanol
solutions in order to give them the same stimulating strength. The ratio between
the relative vapor pressures and the saturated vapor pressures which represents
the thermodynamic activity was found to be roughly linear except for the lowest
3 members of the series. This indicates that alcohols of equal thermodynamic
activity have equal stimulating effectiveness and, since the thermodynamic scale
can only be used for equilibrium processes, this might mean that for the alcohols
270 lV1. G. J. BEETS: Olfactory Response and Molecular Structure
with 1-3 carbons, olfactory stimulation does not involve an equilibrium. Thc
relationship between thermodynamic activitics and stimulating efficiency has also
been studied by DETHIER and YOST (1952), by GAVAUDAN et al. (1948) and by
MULLINS (1955). Some of this work, as far as it is of interest for the subject of this
chapter, is discussed in other paragraphs.
0;.
7
100 a
Prim. alkanols
80 in H2 O
'"
c-..
"0
::l 60
0/0
E 40
<!
20
0
_0------
I 2 3 4 6 7 8
100 b
Keto7
80

'"
"0
::l 60
.-
~ 40
<!
OL-__~__~__- L_ _~____L-~
1234 S 6 7
100/ ~o
~rim.alkanols
~
80
60 ',m', Oil/ \
\
E 40 /
<! 20 ~o_o
OL-~-L-L~__~~-L~__L-~~
I 2 3 4 S 6 7 8 9 10 II 12
From Ottoson (19S8)
Fig. 1. Relation of stimulatory effectiveness to chain length of primary aliphatic alcohols III
aqueous solution (a), in mineral oil (c) and of aldehydes and ketones (b). (From OTTOSON, 1958)
Fig. 1b shows that the stimulating efficiency of homologous aldehydes and

ketones increases with the chainlength within the limits of the experiment. For the
homologous fatty acids with 1-9 carbon atoms an irregular relationship was
observed with maxima for the acids with 3 and 6 carbons and a minimum for the
acid with 5 carbons. Impurities as well as concentration effects may be involved in
this result.
The characteristics of the E.O.G., recorded from the membrane of the frog,
especially the initial positive deflection, were studied by GESTELAND (1964;
GESTELAND et al., 1965), who classified a large number of stimulants into two
groups, one of which causes a strong and the other no or a weak initial positive
deflection. Both lists contain odorants with widely varying structures and no
correlation between either the structure or the properties of the stimulants and
the class to which they belong can be found.
In a later study, GESTELAND (1967) recorded the E.O.G. as well as the changes
in transfer impedance magnitude and in the transfer impedance phase angle for a
number of stimulants. All are consistently similar for the same stimulant anywhere
on the mucosa and from any frog and permit to distinguish a large number of
different odors. Unfortunately, the material does not comprise sufficient structur-
ally related stimulants to justify a search for similarity of the shapes of the curves.
GESTELAND et al. (1963) stimulated the receptor membrane of the frog with a
group of 25 odorants with varying structures, recording both slow potentials and
action spikes from low-impedance extra-cellular electrodes. The purpose of this
study was in the first place to collect some information on the specificity of the
receptors. The impression obtained from this work is that each of the receptors
responds to certain odorants and not to others. Most units respond strongly to at
least one odorant and weakly to many others. As a result of this study, the
authors attempted a rough classification of the receptor population into a number
of strongly overlapping types, each of which is characterized by the odorants to
which it responds.
Limonene, camphor, pinene and (weak response) carbon disulfide;
Coumarin and musk xylene;
Butyric, valeric and mercapto-acetic acid and cyclohexanol;
Benzaldehyde, nitrobenzene, benzonitrile, musk xylene and amyl alcohol;
Pyridine, musk xylol, cinnamic aldehyde and n-butanol;
Musk xylene and (weak response) benzaldehyde and nitrobenzene;
Pyridine;
Butanol, amyl alcohol, ethyl butyrate and geraniol.
Interesting as this classification may be, it involves small, arbitrarily chosen
fragments which are not representative of the total response pattern. Apart from
the fact that a single type in this classification seems to respond to a group of
odorants with somewhat related odors (such as benzaldehyde, nitrobenzene and
benzonitrile), this classification offers no clue to any correlation with odor type or
molecular structure.
On the basis of this work, GESTELAND formulates the tentative conclusion that
there are receptor sites belonging to different types on each cell, and that cells
differ by having sites of various types in different ratios. Even with a limited
number of site types distributed over the cells in varying ratios, this system seems
to be sufficient to generate a satisfactorily versatile olfactory code.
DRAKE et al. (1969) studied the correlations between the concentration of
odorants in air, measured by G.L.C., the E.O.G. peak amplitudes obtained by
stimulation of the epithelium of the frog with these odorants and intensity estimates
by human subjects. High correlations were found between the logarithms of any
two of these characteristics with the exception of hexanal in higher concentrations
in oil, since E.O.G. amplitudes pass through a maximum in this case. The slope of
272 M. G. J. BEETS: OHactory Response and Molecular Structure]
the linear relations found between electrophysiological and G.L.C. data and be-
tween psychophysical and G.L.C. data differs between odorants.
The number of odorants involved in this study is far too small to permit any
conclusion on the influence of molecular structure, but the method is mentioned
here since it may become useful for more extensive series of structurally related
odorants.
The olfactory receptors on insect antennae present an excellent and, up-to-now,
unique opportunity for electrophysiological recordings from single receptor units.
Also, the antennae permit the recording of slow potentials, representing the sum-
mated effect of olfactory receptor potentials of the whole antennae and named by
SCHNEIDER (1957) electro-antennogram or E.A.G. in analogy to the E.O.G.
All the units tested by SCHNEIDER and his co-workers have a reproducible
spectrum of odor stimuli to which they respond. On the basis of these spectra,
cells can be classified as "odor generalists" such as the basiconical cells of the
moth and the placoid sensilla of the honey bee which show unique although over-
lapping odorant spectra and "odor specialists" such as the sex-attractant sensitive
receptors of the male moth and the drone, which give stereo-typed responses to a
number of odorants (BOECKH et al., 1965).
In single unit recordings (LACHER, 1964), the response frequency often shows a
phasic-tonic course, i.e., it rises almost instantaneously to a maximum and drops
rapidly to a level which is either constant or which approaches asymptotically to
a constant value. Both the height of the maximum and that of the stationary level
can be used for response characterization. The receptor unit can respond to
olfactory stimulation either by an increase of the impulse frequency (excitation) or
by a decrease of the frequency (inhibition) or it does not respond. The meaning of
the inhibitory effect as a component of the primary olfactory information pattern
depends on the still undecided question whether the resting activity of the receptor
unit is due to traces of odorants in the air or the presence of the electrode (BOECKH,
1962) or whether it is an intrinsic activity of the receptors.
The shape of the E.A.G. varies with the structure of the odorant. Its amplitude
increases with odorant concentration and air velocity and can be used as a criterion
for the quantitative aspects of olfactory response (threshold determination).
BOECKH (1962) studied both the E.A.G. and the response frequency during
olfactory stimulation of the receptors of Necrophorus. These receptors are con-
sidered to be specialized cells; they respond to odorants which are possibly generat-
ed in meat in the first stages of decay, such as fatty acids, amines and benzyl
mercaptan but not to the lower molecular products generated in a later stage,
when the meat is no longer attractive to the insect, such as ammonia and hydrogen
sulfide. Receptor potentials recorded from a small number of units during stimu-
lation with a series of straight chain fatty acids show that the C6 to ClO-acids act
clearly as odorants, eliciting positive potentials in all cells with a maximum for
heptanoic acid. Propionic acid and butyric acid cause hyperpolarization without
impulse formation in the receptor cell. No response was obtained for the saturated
acids with 12-18 carbons but the unsaturated acids oleic- and erucic acid elicit
small positive responses.
SCHNEIDER (1963), recording E.A.G.'s from the sensilla of the silk worm moth
(Bombyx mori) , measured the electrophysiological threshold values for the four
stereoisomers of the sex attractant bombycol (hexadecadiene-1O-trans-12-cis-ol-l)

and compared these with the behavioral thresholds reported by other authors. The
results are summarized in Table 1. Both columns refer to different concentration
units and therefore can only be compared in a relative way.
Table 1
Hexadecadiene-l0-12-01-1 Behavioral Electrophysiological

threshold threshold
10 12
microgram/ml microgram
trans cis (bombycol) 10- 12 10- 10

cis trans 10- 3 10- 5
c~s c~s 1 10-"
trans trans 10- 5
10-I'on . 12-c ..
10-Clo-12-I,o"$
10-clI-12cl$
12-I,on5-12-
Iron.
Fig. 2. Molecular models of bombycol and its stereomers. (From SCHNEIDER, 1963)
18 lib. Sensory Physiology, Vol. lV!l
At higher concentrations (10- 3 to 101 fLg) of the four isomers, the E.A.G.
amplitude changes linearly with the logarithm of the stimulus concentration. In
this range bombycol produces significantly higher amplitudes than its stereomers.
At lower concentrations (10- 5 to 10-3 fLg) the amplitudes for all isomers are low
and none predominates clearly.
SCHNEIDER observes that the architecture of the molecules of the four stereo-
mers, the models of which are shown in Fig. 2, is important for the response.
Although the difference between the electrophysiological thresholds is less
characteristic than that between the behavioral thresholds, both indicate the high
stereospeeificity of the receptor popUlation involved in these E .A.G.'s and their
unique sensitivity for the bent profile of bombycol. The much lower preferential
sensitivity for the second bent molecule, the 1O-cis-12-trans-isomer, in which the
bend is closer to the functional group, is only obvious in the behavioral thresholds
which also show the low sensitivity for the two more linear stereomers. Only the
I,
01
i
';lIse a
Z!'"~ .~.-:---
100
'--------.
o... ....
I. f8C ] 5 7C
w,,--------~--------r_------~~
Imp/s':. h
o
60 1 ' . - ----,------,--,
:/~/.
.- .------'
2()
:0
o .- - - - Q ..... - - ....
.----. : ------
to fil 16 t8 C 5 6 7C
Fig. 3 Fig. 4
Fig. 3a and b. Response of 5 receptors of Apia mellifica to stimulation with aldehydesC1o-C1s
a Maxima of frequency curves, b Stationary levels. (From LACHER, 1964)
Fig. 4a and b. Response of 6 receptors of Apis mellifica to stimulation with fatty acids C3-C,.
All acids have straight chains except the C.acid for which isovaleric acid was used. a Maxima
of frequency curves, b Stationary levels. (From LACHER, 1964)
receptors of the male silk worm moth respond to bombycol but both the male and
female receptors to 2,4-hexadienol and cycloheptanone.
In a recent paper, SCHNEIDER et al. (1968) describe the threshold measurement
of bombycol, labelled with tritium.
LACHER (1964) recorded the nerve impulses from a series of single receptor units
(sensilla placodea) on the antenna of the honey bee during stimulation by a
number of odorants. The responses from 5 different units to stimulation with the
straight-chain fatty aldehydes with 10, 14, 16 and 18 carbons are shown in Fig. 3
and from 6 receptor units stimulated with fatty acids from 3 to 7 carbons in Fig. 4.
In each case the upper part (a) represents the maxima of the frequency curve
and the lower part (b) the stationary levels. It seems that for these homologous
series the influence of the structural relationship is expressed better in the stationary
levels, which are more homogeneous and vary more gradually with increasing
chain length than in the heights of the maximum which vary erratically with the
chain length.
Fig. 5 shows a typical representation of impulse frequency curves obtained
from 3 different receptor units by stimulation with a series of 4 structurally differ-
ent odorants.
IoWlI_t /~IJI
001 01 01
7~L:::::::::::::: =.I<.
7'
01 sec
B
A
PIlef1j4o/!JyIoce/ol Eugenol {opronsolJf't'
Fig. 5. 3-Dimensional representation of impulse frequency curves from 3 receptor units in the
honey-bee by stimulation with cinnamic aldehyde, phenyl ethyl acetate, eugenol and caproic
acid. (From LACHER, 1964)
The most interesting aspect of LACHER'S work for the subject of this chapter is
the recording of responses from 47 receptor units upon stimulation with 33 odor-
ants. Although the number of units from which responses were recorded (17 out of
3000 pro antenna for the worker bee and 30 out of 15,000 for the drone) hardly
suggests that the response spectra obtained are representative of the total spectra
of the antennae, it is tempting to look for correlations between these spectra and
the various odorant structures. An obvious way to do this is by combining the
odorants in pairs and to estimate the similarity between the oriented profiles of
the members of each pair. Comparison of these similarities for two pairs indicates
a trend (greater or smaller than; approximately equal), which can be compared
18*
with the trend in the similarities of the response spectra. The latter are obtained
by counting for each pair the number of excitatory-, inhibitory- and zero-responses
which the two odorants, combined in a pair, have in common, expressed as a per-
centage of the total number of units from which responses have been obtained for
both odorants. The author (BEETS, 1969) did this tentatively for the material
presented by LACHER for 30 receptor units of the drone. No correlation was found
when common inhibitory-, excitatory- and zero-responses were counted. However,
when only the excitatory responses were considered as contributing to the primary
olfactory information pattern, a more meaningful picture was obtained. Out of
46 trends of estimated profile similarity 32 showed the same direction as the corre-
sponding trends of spectrum similarity. In 6 cases the trends had opposite direc-
tions and in 8 cases the result was uncertain since profile similarities were difficult
to estimate by means of simple comparison of the molecular models.
There is little doubt that the available material is far too limited to draw a
conclusion. The attempt is only mentioned to indicate a method which may
become a useful tool as soon as recordings from larger numbers of units become
available. The t'3chnique described by AMOORE (1967 a) permits the quantitative
determination of profile similarities. Also, the odorants should be selected on the
basis of their structural relationships. In practically all work in this field, the con-
tribution of the structural chemist seems to be lacking.
SCHNEIDER et al. (1964) published similar material on the stimulation of
48 receptor cells in 27 sensilla basiconica of Antheraea pernyi (Asiatic silk moth)
with 13 different odorants. An attempt was made to characterize the response
pattern for each odorant by means of the ratio between the numbers of positively
and negatively responding units. There is no obvious relation between this criterion
and odorant structure.
The same authors recorded impulses obtained from 9 sensilla trichodea of
Antheraea by stimulation with 10 different odorants, including the sex attractant.
Each sensillum is served by 2-3 receptor cells of which only I or 2 are excited by
the attractant. The other odorants inhibit about half of these cells. The cells which
do not respond to the attractant are, in roughly equal numbers, excited, inhibited
or uninfluenced by the other 9 odorants. BOECKH et al. (1965) also working with
Antheraea, reported the reaction spectra of 27 single cells in the sensilla basiconica
for 10 odorants. In both cases the odorants have structures that are too widely
different and are too small in number to permit any search for correlations.
BOECKH (1968) recorded from single receptor units on the antenna of the
migratory locust. These cells had been found in previous experiments to be
sensitive to "grass" odors.
Of a series of odorants with varying functional groups possessing a straight,
saturated chain of 6 carbon atoms, only caproic acid was found to be effective.
Also in the C7 series, the alcohol and the aldehyde are ineffective and a response is
obtained only with heptanoic acid. This picture is changed by the introduction of
unsaturation; 2-hexenol, 2-hexenal and 2-hexenoic acid are all effective. Hexene-I
is ineffective but hexine-I elicits a weak response.
In the series of saturated fatty acids, formic and acetic acid are ineffective. In
the higher members the activity increases with the chain length to reach a maxi-
mum in caproic acid (C6 ) after which it decreases rapidly. Acids with 9 or more
carbons are inactive. For the same series electrophysiological thresholds were
determined. Caproic acid shows the lowest threshold of 109 molecules per cm 3 of
air. A different technique, using Cl4 -labelled caproic acid, resulted in a threshold
value of 1.1 x 1010 molecules/cm 3 (BOECKH, 1967b). A still lower threshold
(10 8 molecules/cm 3 ) was found for 2-trans-hexenal.
Hexen -2-trans-al Caproic acid
Hexen-2-cis-ol-l Hexanol-l
Fig. 6. Molecular profiles of trans-hexenal, caproic acid, cis-hexenol and hexanol
Replacement of the terminal methyl group in caproic acid by a carboxyl group

(pimelic acid), which completely changes the oriented profile, destroys the activity.
Also, and possibly for the same reason, 6-amino-caproic acid is inactive. The same
is true of the ester and the amide.
Introduction of unsaturation, e.g., hexenoic acid and hexadienoic acid, leads
to retention of the activity but it disappears in muconic acid (butadiene-l,4-
dicarboxylic acid). Also, the formate and other esters of the active hexenol, as
well as hexene-3-diol-2,5 and 4-ethyl-butanolide are ineffective.
Fig. 6 shows the molecular models of 4 of the odorants tested, viewed approxi-
mately in the direction of the dipole axis and in decreasing order of activity, i.e.,
trans-hexenal (+ + + ), caproic acid (++), cis-hexenol (+) and hexanol (-).
It seems that the active odorants show the "smallest" profiles whereas the
similarly oriented profiles of the inactive compounds of this series are bulkier.
In Fig. 7 (BOECKH, 1967 a) the response spectra for three widely different
types of receptor cells and a large number of odorants are summarized (hatched
areas: excitation; dotted areas: inhibition and white areas: no response).
Test
Corflon Carrion Gross Corflon Carrion Gross
slJbstoncfs
R~ceptor Receptor Receptor Recfptor Receptor
Colliphoro Thonotop/,ullJs L oClJslo
Fig. 7. Response patterns obtained by stimulation of 3 different types of receptor cells with
homologous series of acids, aldehydes, primary alcohols and various other stimulants. (From
BOECKH, 1967 a)
2. Recordings from Nerve Fibres

BEIDLER and TUCKER (1955) recorded from very small branches of the olfactory
nerve in the isolated epithelium of the opossum and measured the percentage
increase in the spike frequency above resting activity. The same was done for a
live rabbit preparation and the recordings were compared with the ones taken
from the trigeminal nerves of the same animal. Both nerve types responded to
most of the odorants tested. TUCKER (1963), by comparing responses from olfac-
tory and vomeronasal nerve twigs of the tortoise, found that the vomeronasal
response tends to predominate for the lower members of homologous series of
compounds (fatty acids and alkanols).
MOZELL (1964 b) took simultaneous recordings from two widely separated nerve
twigs of the frog. Various stimulants were found to elicit a different response
magnitude ratio between the two branches and also the time lapse between the
two nerve responses differed for different odorants. The ratio of the maximum
Recordings from Nerve Fibres 279
response of the lateral nerve to that of the medial nerve increases in the order:
geraniol, citral, d-limonene, octane, phenyl ether. The ratio found for naphthalene
was much like the one found for geraniol. The order across animals (2 groups of 10
and 4) was found to be statistically significant. Also, the average temporal differ-
ences between the odorants were significantly different except for geraniol and
citral. The only aspect of interest for the subject of this chapter is that two
structurally related chemicals like geraniol and citral are found closely together
according to both criteria.
Since the concentrations in this work were different for the various chemicals,
the spatio-temporal code could depend either on odorant concentration or on
molecular structure. This complication was eliminated by working with a flow-
dilution olfactometer (MOZELL, 1967). The same 4 chemicals were presented in
6 different concentrations and the number of animals (out of 10) in which the
lateral to medial nerve branch ratio produced by each chemical falls into a given
rank (1-4) at each concentration was determined. The same was done for the
latency difference. Although none of the chemicals showed ratios confined to a
single rank in all animals at a given concentration, a strong tendency for order-
liness was observed and the order found was the same at all concentrations.
d-Limonene and octane always ranked 3rd or 4th, geraniol and citrall st and 2nd.
The data found for the latency differences were more variable at low concen-
trations but their internal consistency increased with concentration. These results
strongly suggest that both the spatial code and the temporal code depend on the
structure of the odorant rather than on its concentration.
GESTELAND et al. (1965) recorded the activity in small bundles ofaxons im-
mediately underneath the basal membrane. By stimulation with an odorant, the
resting activity in the bundle, which is as chaotic as that in the receptor surface,
may be excited, inhibited or not affected. Stimulation with methanol, for example,
seems to affect three units of which the one associated with the largest spike is
inhibited, with the medium spike excited and with the smallest spike unaffected.
The few units of which the activity is recorded by the electrode in a single position
hardly permit any conclusion on structure-response relations. Two odorants which
cause very similar but by no means identical sensations in the human nose such as
menthol and menthone or benzonitrile and nitrobenzene, produce similar responses
in one electrode location but a short search suffices to discover another location
where the units discriminate between the two. This is exactly what we could
expect. On the basis of recordings with an odd selection of stimulants, comprising,
among others phenylethyl alcohol, tetraethyl tin, geraniol, methanol and diethyl
amino ethanol, the authors offer some interesting speCUlations on discrimination.
If every fibre and consequently every receptor cell would be sensitive to every
stimulant, i.e., if it would respond in a way anywhere between strong excitation
and strong inhibition, the universe of odors would be "seen" by every fibre from a
different point of view. The primary olfactory information pattern would then
have as many dimensions as there are fibres. This introduces the speculative con-
cept of the holographic nature of the primary information pattern.
Chaos in this context has two aspects, an infinite variety of receptor sites and
a random distribution over the epithelium. For biochemical reasons we must
reject the former; the number of receptor site types which can only be identified
with as many chemical (proteinaceous) individuals in the epithelium, is undoubtedly

very limited. Random distribution must be rejected, since it would seriously
reduce though not destroy the discriminating ability of the olfactory system.
However, GESTELAND'S argument against chaos, i.e., that people judge compounds
as similar or dissimilar in much the same way, is not convincing. Random distri-
bution of site types would mean that the response pattern resulting from stimu-
lation by a certain odorant would consist of a random assembly of millions of
points, each with its own shading between extreme inhibition and extreme ex-
citation. Such a pattern would have a uniform shade for a specific odorant, not
unlike the visual impression of a uniformly colored or dark or light field. It would
still be characterized by its intensity and its shading and so it would allow us to
distinguish many odors just as we are able to distinguish many colors. But it would
not have a topological structure.
As soon as, and to the extent in which randomness is replaced by organization
in the form of zones or patterns of receptor site types, the primary information
pattern acquires topological characteristics and its power of discrimination
increases enormously. This again can be compared to the visual system which can
process not only uniform colors and intensities but also differently colored and
shaded areas.
Organization of site types on the epithelium is highly probable, but we know
almost nothing of the nature of this organization. In a number of cases, we have
noticed some rough but sensible correlations in very limited material which cannot
possibly be representative of the whole population of receptor units. Also, we know
that the sense of smell may temporarily be impaired and even disappear, but it is
never distorted in case of abnormal secretion of mucus on the olfactory epithelium.
As long as we can smell at all, a musk is recognized as such and the impression
never changes into vanillin or amber. Both facts suggest that the olfactory system
and the brain are able to construct from the primary information pattern received
from any, even a small part of the stimulated epithelium, an odor impression hav-
ing all the characteristics of the one based on the information pattern of the whole
receptor area although it may be weaker and show fewer details.
This could mean either that the primary information pattern consists of a very
large number of areas with identically structured information or that it has
holographic characteristics.
3. Recordings from the Olfactory Bulb

ADRIAN (1950), recording from the olfactory bulb of rabbit and hedgehog,
concluded from his experiments that the olfactory signals originated in the receptor
membrane suppress the intrinsic activity in the bulb. During continued stimu-
lation, this activity gradually returns and swamps the transmission of the olfactory
signals. He (ADRIAN, 1949-1950) observed that stimulation of the receptors with
odorants possessing functional groups such as esters and ketones, gives pre-
dominantly responses from the oral part of the olfactory bulb whereas stimulation
with hydrocarbons is recorded mainly from the aboral part. Also the temporal
pattern of both types of responses is different; those caused by esters start and end
abruptly while hydrocarbon responses start slowly and outlast the inspiration.
Recordings from the Olfactory Bulb 281
These observations suggest that the olfactory information pattern in the bulb has
at least two dimensions, a spatial one and a temporal one.
Later work confirmed and refined ADRIAN'S conclusions. In the bulb of the
rabbit (ADRIAN, 1951), amyl acetate activity is mainly observed in the anterior
part, pentane activity in both regions but starting earlier in the posterior part and
coal gas activity mainly in the posterior region (see also MOZELL and PFAFFMANN,
1954). The bulb of the cat shows a similar topology. Here the posterior region also
responds to stimulation with decayed meat, fish and trimethyl amine.
The activity of the various mitral cells can be distinguished by the size of the
action potentials. Often, one series of large, uniform spikes and a number of
smaller ones of varying size are recorded. According to ADRIAN (1953) the large
spikes represent discharges in one mitral cell and the smaller ones those in the
neighbouring units, leading to other groups of receptors. It is nearly always possi-
ble to find an odorant which, in low concentration gives only the large spike
discharge. Units have been located having specific activity to a long list of sub-
stances such as trimethyl amine, esters, acetone, xylene, eugenol and cineole. With
increasing concentration smaller spike units start discharging although in a few
cases the smaller spikes appear first.
In later papers, ADRIAN (1956, 1959) attempts a further classification of the
units of the bulb into 5 main groups which are especially (but not exclusively)
sensitive to aromatic hydrocarbons (benzene, toluene), esters (ethyl- and amyl
acetate), alkanes (pentane), terpenes and terpenoids (limonene, cedarwood oil and
cineole) and sulfur compounds (mercaptans and hydrogen sulfide). He adds
(ADRIAN, 1959) that it is reasonable to draw the conclusion that the receptors in
the epithelium are arranged in small zones where the affinity to a group of struc-
tures predominates.
Using his technique with 6 electrodes permanently implanted in the olfactory
bulb of the rabbit, MOULTON (1968) found a progressive alteration in the relative
effectiveness of odorants such as pentadecanolide, butyl ether, a-ion one and a
homologous series of n-alkanols on proceeding from the front to the rear of the
bulb. Butyl ether was found to be more effective in the posterior part, a-ionone in the
anterior part of the bulb.
MOULTON (1965) applied a technique, developed by MOULTON (1963) and by
MOULTON and TUCKER (1964), to the study of the multi-spike discharges in the
secondary neurones of the olfactory bulb. According to this method a number of
electrodes are permanently implanted in the skull of a rabbit. The odorants used
can be distinguished by the sign as well as by the intensity of the response in each
site and by the time course of the response. Some structurally unrelated odorants
such as tert.butyl alcohol, heptanol and pentadecanolide; octane and propanol;
butyl ether and d-limonene show similar response patterns in the 6 locations from
which recordings were taken. Octane could be distinguished from propanol by
temporal differences in the evaluation of the response. In a later paper (MOULTON,
1967) the method was applied to a larger number of odorants. Fig. 8 shows the
integrated response patterns from 6 sites for 20 different odorants.
Odorant pairs such as benzothiazole-anisole and l-menthol-d-limonene have
similar patterns but they can often be distinguished by the different time course of
the responses. On the other hand, stimulants which are closely related in structure
as well as in odor such as cyclopentadecanone and pentadecanolide (exaltolide)

have totally different response patterns. However, as MOULTON (1965) observed
correctly, a number of 6 out of perhaps 103 -10 4 sites may reveal pattern similarities
which disappear when recordings from a larger number of sites are compared.
D6vING developed a method, based on statistical analysis of electrophysiological
data, which promises to become a fertile source of information on response-
structure relations. Also its results permit a comparative analysis with the results
of psychological methods and with profile parameters of odorant molecules.
~ 8
Amyl AcOlO" 10- 2 BU'yl E er 10- 2
~ d
Cyclopen anon. 10- 2 C,'ronollal 10.15
~
OClane 10. 1 5
~
Propanol 10- 1
0P.n,ono l 10. 2
~
Hep'anol 10. 2
~
,f" /.
'.rl BUlanol 10- 2
~
lonon. 10 - 2
~
120lchlofoechone
~ ~
I.rt- Bulyl M.,hyl Elher CIn.ol. 10- 2
~
1- Menlhol 10.1 5
~
2- Butanonr 10 - 2
&
10- 15 10' 2
~
AnISole 10 . 2
~
B.nZolh,azole 10. 15
~
d - L,mOMne 10- 2 Cyclopontadoconono 10. 1
~
E,aItQI,d 10- 2
Fig. 8. Integrated response patterns from 6 sites in the olfactory bulb of the rabbit for 20 dif-
ferent odorants. (From MOULTON, 1967)
In this work the olfactory epithelium of the frog is stimulated with odorants
and responses are recorded from a large number of units in the olfactory bulb. The
stimulus concentration is monitored in such a way that the amplitudes of the
simultaneously recorded KO.G., and, consequently the stimulating efficiencies,
are approximately the same for all odorants within a series of experiments.
The response patterns obtained, consisting of excitatory, inhibitory and zero
responses were statistically processed by means of a chi-square test for pairs of
odorants, starting from the assumption of odor independence within each pair.
The chi-square values computed are used as a criterion for the degree of similarity
between the information patterns of the two stimulants in each pair.
In his first paper on this technique, D6vING (1965) used a series of 5 stimulants,
consisting of I-menthol, coumarin, salicylic aldehyde, a mixture of the two
stereomers of citral and a somewhat impure mixture of geraniol and nerol. Cou-
marin, geraniol and salicylic aldehyde were selected as representatives of AMooRE's
(1962) floral primary odor. Menthol belongs to the minty group and citral is,
according to AMooRE, a complex type of odorant. Recordings were made from
85 bulbar units. The chi-square test was applied to all (10) pairs of odorants. The
initial assumption of pattern-independence was rejected statistically only for the
pairs citral-geraniol and citral-l-menthoL
Recordings from the Olfactory Bulb 283
The chi-square test indicates that the response patterns of coumarin, geraniol
and salicylic aldehyde are independent. This would be inconsistent with AMOORE'S
classification. However, we should keep in mind that the latter has a semantic
basis and for this reason can only be applied to man.
D6vING points out that citral, geraniol and menthol have similar molecular
profiles. This, however, only holds good for one of the stereomers of each geraniol
and citral and for this reason it is not unexpected that the highest value was found
for the pair geraniol-citral.
In a later paper, D6VING (1966a) applied the same technique to homologous
series of alcohols (3-8 carbon atoms; recordings from 93 units), acetates (of
alcohols with 1-4 carbons; 93 units) and 2-alkanones (4-7 carbons; 80 units).
In the alcohol series it was found that the degree of pattern similarity decreases
with increasing difference in chain length. The highest values were obtained for
the pairs hexanol-heptanol and heptanol-octanol. A significant rank correlation
coefficient was obtained from the chi-square values and the rank order of the
ratios of molecular weights for each pair.
In the series of acetates very low values were found except for the pairs propyl-
butyl, butyl-pentyl and, to a lesser extent, propyl-pentyl. Also in the alkanone
series the values decreased with increasing difference in chain length. Calculations
with pairs of which the members belong to different groups do not suggest a
significant dependence of the response patterns. Since the three series have
widely different molecular profiles this is not surprising.
The next step (D6vING, 1966b, 1968) was the application of the method to
33 odorants belonging to the seven primary groups proposed by AMOORE.
For the camphoraceous class, the highest chi-square value was found for the
pair tert.butyl alcohol-tert.amyl alcohol. Low values were obtained for structurally
heterogeneous pairs such as p-dichlorobenzene-camphor, camphor-tert.amyl
alcohol and dichloro benzene-tert.butyl alcohol.
In the ethereal class 7 of the 10 pairs gave chi-square values indicating that the
response patterns are significantly associated. The highest value was obtained for
the pair carbon tetrachloride-trichloro ethene, the lowest for pyrrole-trichloro
ethene.
In the floral group containing such divergent structures as geraniol, salicylic
aldehyde, benzophenone and anisole, it is not unexpected that generally low values
were obtained. The highest value was obtained for the pair acetophenone-salicylic
aldehyde. In the same way it is not surprising that in the so-called minty class
high values are obtained for pairs consisting of cyclic ketones with 5- 8 ring
members and for those consisting of the structurally closely related stereomers of
menthol and menthone. For pairs consisting of a member of each of these two
structurally unrelated groups low values were obtained.
In a series of musks, comprising musk ketone,5oc-androstan-3f'1-01, 1,1-dimethyl-
6-tert.butyl-4-acetyl-indane, 3 isochromane musks and pentadecanolide, the chi-
square values indicate a significant association between all response patterns.
Other characteristics of this series are an unusually large number of unaffected
units, which could suggest a fairly homogeneous population of interaction com-
plexes on the receptor level, and a relatively small E.O.G. amplitude.
Of the 5 classes studied by DaVING, only the musk group shows sufficient
homogeneity in its response patterns in the bulb to be considered as a type which
remotely resembles the ideal of the mono-osmatic odorants and which could be
grouped as a primary class. The other classes may contain one or two smaller
groups of odorants with related response patterns but as classes they are clearly
heterogeneous.
In the major part of the cases showing a significant pattern relatedness we also
find a corresponding similarity between the oriented molecular profiles.
The correlation between the chi-square values obtained with DaVING'S method
and the values resulting from two psychological methods (similarity judgments
and confusion measures) was studied by DaVING and LANGE (1967). Table 2 shows
the results.
Table 2
Odor pair Mean similarity Mean confusibility Chi-square

estimate (%) %
Menthol/salicylic aldehyde 30 6 0.22

Menthol/citral 40 19 4.93
Menthol/geraniol 59 24 3.76
Salicylic aldehyde/acetophenone 64 55 10.42
Salicylic aldehyde/anisole 56 48 0.24
Salicylic aldehyde/citral 37 14 2.12
Salicylic aldehyde/geraniol 31 10 1.41/0.34a
Acetophenone/anisole 51 72 6.29
Acetophenone/geraniol 42 24 0.23
Anisole/geraniol 38 12 4.67
Citral/geraniol 84 82 9.19
Camphor/tert. butanol 52 36 5.70
Camphor/tert. pentanol 67 56 0.54
Camphor/3-methyl-butanone-2 62 28 10.04
tert. butanol/tert. pentanol 80 70 15.86
tert. butanol/3-methyl-butanone-2 54 42 13.57
tert. pentanol/3-methyl-butanone-2 61 45 8.47
a Values obtained from two different experimental series.
Rank correlation coefficients were calculated which show that the agreement
between the two psychological methods is better than between each of these
methods and the chi-square method. With some notable exceptions the trends
between the pair-values in Table 2 agree with the trends of profile similarity
estimates. The high value found for the seemingly heterogeneous pair camphor/
methyl butanone is of particular interest since both have that part of their mole-
cular profile that surrounds the functional group in common.
The correlation coefficient between DaVING'S (1966a) electrophysiological data
on aliphatic alcohols and ENGEN'S (1962) psychophysical results with the same
series, was found to be 0.87, indicating a significance on the 0.1 % level.
LEVETEAU and MACLEOD (1966) studied the E.O.G. as well as the response
from the glomeruli in the bulb of the rabbit. Under the experimental conditions
The Verbal Expression of Odor Sensation 285
selected, the glomerular response was found to be a slow monophasic potential, in

shape resembling the E.O.G. The responses of 47 glomeruli in 23 rabbits to stimu-
lation with 9 arbitrarily selected stimulants were described. Of the 47 glomeruli,
12 responded to all odorants and were not taken into account. Only 3 pairs of
glomeruli exhibited the same response spectra.
There is no obvious relationship between the response patterns and the struc-
tures of the odorants. Two structurally unrelated chemicals as fJ-ionone and
propanol have a considerably higher pattern similarity than related ones such as
propanol-butanol and a-ionone-fJ-ionone.
HUGHES and HENDRIX (1967) recorded responses from 4 locations in the olfac-
tory bulb of the rabbit and plotted response amplitude against frequency. This
revealed significant differences in the responses to various stimulants. Especially
the frequency of the major peak seems to be distinctive for each odorant. The
logarithms of amplitude and frequency of the major peak for the odorants tested
were found to be inversely related. Also an inverse linear relationship was detected
between the frequency of the major peak and the molecular weight of the stimu-
lant, which means that low molecular weight compounds are generally associated
with high frequency and low amplitude of the major peak and vice versa.
The authors suggest that the higher frequency components which are maximal
in amplitude at the beginning of the response, signal only the presence of an odor
since they do not vary significantly between odorants and that the major peak and
the low frequency peaks, which vary between odorants, signal the identity of the
stimulant.
An attempt was made to correlate the major peak frequencies as well as the
total peak frequency pattern with AMOORE'S classification of odorants into primary
classes characterized by odor type and molecular dimensions. If all peaks would
help to define a given primary class, a significantly larger number of common peaks
should be found within such a class than across classes. It was found that, on the
average, for a certain site in a certain animal, 26% of the peaks are common to
the response patterns of two chemicals belonging to AMOORE'S floral type, whereas
21.5% of the peaks are common to representatives, one of which belongs to the
floral and the other to the camphoraceous type. Although this result does not seem
impressive, a chi-square test proved it to be significant. It seems unfortunate that
much of this work was done with complex and sometimes unidentifiable mixtures.
In a later study which confirms their previous results, HUGHES et al. (1968) applied
the same methods to 3 human patients.
This necessarily brief and incomplete survey of electrophysiological con-
tributions to our subject shows that in many cases the structural chemist must be
satisfied with the crumbs from the electrophysiologist's table but also that a few
methods have been developed which hold great promise for our insight in structure-
response relations. However, also in these cases the supply of information tends to
stop when it becomes most interesting.
E. The Verbal Expression of Odor Sensation

The primary information pattern resulting from interaction between odorant
molecules and receptor sites is subjected to processing steps in the receptor cells,
in the glomeruli and the mitral cells of the bulb and in the brain until the final
product of the olfactory process, the odor sensation, is obtained. This can be used
in several ways as a source of information on the discriminatory mechanism of
olfaction. In man as well as in animals the odor sensation is compared automatic-
ally with memory contents, i.e. with experience, and this results frequently in
recognition. In animals such recognition influences the behavior and this effect can
be studied by means of statistical or psychological methods. In man the recognition
of the sensation pattern or part of it can be translated into a verbal expression
which can be studied semantically or statistically under normal as well as under
abnormal conditions. The latter may be present in the subject (specific anosmia) or
induced by the experimenter (adaptation). These cases will be discussed in the
paragraphs G and H; here we shall consider the information which can be obtained
from the verbal expression under normal conditions.
Verbal expression of an odor sensation is the result of recognition and con-
sequently it can only be used for comparative analysis. It can tell us whether two
odorants elicit similar or different sensations. In the latter case a limited degree of
analysis is possible, always resulting in the identification of the non-identical parts
of the sensation patterns with sensations elicited by other odorants. This suggests
two different approaches. The first one involves a search for important similarities
between sensation patterns and tries to correlate these with structural similarities.
This method boils down to a classification of odorants. The second approach
compares the difference between the sensation patterns elicited by two closely
related odorants with their structural difference. It is applicable to homologs,
isomers and stereomers. Enantiomers are a special case which is discussed in
paragraph F.
The value of any conclusion based on these two methods depends on the
validity of the assumption that similarity between two sensation patterns repre-
sents similarity between the primary information patterns from which they have
been derived or, in other words, that transformation of the informational content
of the primary information pattern in the olfactory system is similar for different
odorants. This assumption seems safe enough for odorants with closely related
structures but it may not valid when we compare odorants with similar odors but
widely different structures such as nitro musks and macro cyclic musks.
1. Odor Classification
An imaginary attempt to describe the odors of all known organic chemicals
with molecular weights up to about 300 in terms of key-words and to process the
descriptions in the form of a huge table would result in a surprisingly small number
of key-words and a remarkably large number of odorants under each key-word.
Many odorants would be found under several key-words and although many others
would occur under a single key-word we would find that not two of these have
exactly the same odor quality. This shows that the semantic tools at our disposal
are severely limited by our vocabulary and by our odor experience but also that
even odorants occurring under a single key-word produce more or less complex
sensations in which one component predominates but which are never free from
secondary ones. In this respect the facts observed at the terminus of the olfactory
process agree with theoretical considerations on the composition of the primary
Odor Similarity 287
information pattern (par. B). Both indicate that the true mono-osmatic odor and
the true mono-osmatic odorant, eliciting a primary information pattern and a ~en
sation pattern consisting of a single type of information generated in a single type
of receptor site, do not exist. But although a frequency diagram representing the
distribution of the total population of odorants over an imaginary continuum of
odor types would not show any isolated peaks, it would certainly show a con-
tinuous curve with a number of peaks of varying height. It is not unreasonable to
assume that each of these peaks, some of which would correspond with a key-word,
represents a single type of response originated in. a single type of receptor site.
Consequently, a catalog of such peaks would, if it could be made complete, contain
a representation d all the fundamental components of any olfactory information
pattern.
AMOORE (1952, 1962) has applied this principle by recording the key-words
found in a large collection of published odor descriptions. This method evidently
limits the number of fundamental odors we can discover to those which can be
characterized by one or more words but at least it is a beginning and a serious test
of a theoretical principle. AMOORE has pursued this approach with great energy.
An important aspect of his work is discussed briefly in paragraph H and in detail
in his chapter on olfactory genetics and anosmia in this book. The theoretical con-
cept, called the stereochemical theory of odor, which AMOORE based on his research,
is described by DAVIES in this volume. Here we shall merely touch a single aspect
which is relevant to the problem of structure-odor relations.
As a result of his semantic literature analysis, AMOORE found 7 primary odor
classes, characterized by the key-words: camphoraceous, musky, floral, pepper-
minty, ethereal, pungent and putrid. The structural basis of this classification was
tested statistically (AMOORE, 1967b; AMOORE and VENSTROM, 1967) by correlating
the odor similarity (estimated by a panel in terms of numerical values between 8
for "extremely similar" and 0 for "not similar") and the profile similarity of each
of 107 odorants with each of 5 standard odorants (each representing a primary
class). The pungent and putrid types, which are believed by AMOORE to depend on
the nature of the functional group rather than on the profile, were omitted.
In spite of the inevitable errors involved in both similarity assessments, a
satisfactory degree of correlation was obtained for all 5 classes.
2. Odor Similarity
Accepting the subjectivity inevitably associated with selection, we shall discuss
in this paragraph only the most characteristic examples from the large volume of
published odor descriptions. One possible approach which we shall follow here is
to see what we can change in a specific structural detail without changing the odor
beyond recognition or, in other words, without changing the odor quality to such
an extent that the major component of the information pattern of the odor sen-
sation is pushed into a secondary position. This means that we can study and
draw conclusions from the variations of any structural detail within the bonds of
the same odor type. Our definition of the latter will often, though not always,
coincide with that of AMOORE'S primary classes; odor types such as amber and
bitter almond, which do not occur among AMOORE'S primaries, are assumed to cover
groups of odorants with a reasonable consistency in their information patterns.
In the following discussion of odor similarities we shall see that many examples
are taken from the musk class. We know far more representatives of this odor type
than of any other type, if we exclude heterogeneous classes with inordinately broad
and vague odor descriptions. Although no two musks have exactly the same odor,
the main character of all musks is easily identifiable. The large population of
known musks seems to suggest not only a continued commercial interest and con-
sequentlya concentrated research effort but also that it is much easier to find a
new musk than to find for instance a new amber. This may be due to the rather
broad and simple structural requirements for the musk odor, i.e. a rigid, closely
packed structure of a certain size and a well accessible, preferably weakly basic
functional group. THEIMER and DAVIES (1967) have refined the profile criterion for
musks further by defining the ratio between two dimensions of the oriented profile
as 2.8-3.3 and the cross-sectional area as 40-57 A2.
Extensive reviews on the musk odor have been published by BEETS (1957,
1967) and by WOOD (1968). All examples of musk odorants mentioned in this
paragraph are discussed in these reviews where also the original literature refer-
ences can be found.
The structures I-XI, representing some of the most important musks, give an
impression of the extremely wide structural scope of the musk odor.
0
(CH 2 ) 14
I
c=o (CH2 )14
CHa
I
CH---CH2
I I I I
c=o
I I
(CH2 ) 1 2 - - C=o
II III
IV VI VII
Ac
VIII
Ac
IX
~ Ac
X
0/ Ac
XI
Also the odor of some androstane derivatives is reminiscent of musk (BEETS,

1962).
The molecular weight of odorant molecules in general has no lower limit.
Chemicals with small molecules such as methanol and formaldehyde elicit odor
sensations consisting of olfactory as well as of strong trigeminal effects. The upper
limit seems to lie just beyond 300.
The molecular weight limits for specific classes of odorants, however, seem to
be characteristic for each class and even for each sub-class. Taking musks as an
Odor Similarity 289
example we see that the limits are somewhat different for the various structural
types (structures XII-XVII).
I
--0
'--1
H
0 = C t r N02
C=O (CH2 1ts
T'O-Me
1 L . . 1_ _ _ c=o
XII MW = 210 XIII MW = 296 XIV MW = 237 XV MW = 311
Ac
XVI MW = 216
560
XVII MW = 284
The most direct demonstration of the importance of the molecular profile for the
odor type can be given by replacing certain atoms or groups in an odorant struc-
ture in such a way that the molecular shape and size remain virtually unchanged.
The materials available for such a remoulding of the profile are rare since we can
only replace carbon and hydrogen by other atoms and this usually introduces a
factor influencing polarity and orientation pattern. Also the vapor pressure may
be reduced by the replacement, leading to disappearance or weakening of the
original odor. Obvious examples are the replacement of hydrogen by fluorine or
deuterium and the replacement of methyl by bromine.
THEIMER (1968) synthesized the hexafluoro derivative (XIX) of the well.
known odorant dimethyl phenylethyl carbinol (XVIII) and found the odors to be
very similar with a somewhat harsher note for XIX.
-9-
Me CF 3
o-CH2CH2-~-OH o-CH2CH 2 0H
Me CF 3
XVIII XIX
XX R = Me XXII R = Me
XXI R = Br XXIII R = Br
XXIV R = Me
XXV R = Br
WOOD (1968), reviewing the work of CARPENTER et al., mentions several

examples in which the musk odor is retained by replacement of methyl by bromine
(XX - XXIII). The effect is not specific since replacement of methyl by other
19 IId. Sensory Physiology, Vol. IV/1
groups such as methoxy or chlorine in XX and cyano or methoxy-carbonyl in

XXII also produces musks. However, this principle has a limited validity since
the musk character of musk ambrette (XXIV) is destroyed when methyl is
replaced by bromine (XXV).
KLOUWEN (1959) observed that the odor character is retained when a condens-
ed cis-cyclohexane ring is replaced by a tertiary butyl group; XXVI and XXVII
both possess a sandalwood odor. Model studies show that the profiles of cis-
decalin and tert.butyl cyclohexane are similar.
(XX) 0 )X>=o
XXVI XXVII
In the initial paragraphs of this chapter we have mentioned the probability

that a characteristic, easily recognizable odor type which is shared by a group of
odorants with different structures, is associated with the predominance of one, or,
in some cases, a few orientations in the orientation pattern presented to the receptor
membrane. If this is true, we may expect the elimination of the polar group from
an odorant structure with a single, well-accessible functional group, to result in
degeneration of the orientation pattern towards randomness and to complete loss
of the odor character. This is indeed observed when the carbonyl group in the
~ Ac
yo Et
XXVIII XXIX
strong musk XXVIII is eliminated. The hydrocarbon XXIX, which has largely
the same molecular profile, but which cannot possibly have a homogeneous orien-
tation pattern, is odorless. Similar changes in smaller molecules lead to the for-
mation of hydrocarbons which are not necessarily odorless but which have the
atypical "blurred" hydrocarbon odor.
According to the same hypothesis we may expect that functional groups are,
to a certain extent, interchangeable as long as the structure of the orientation
pattern is largely retained. A classical example of this phenomenon is the bitter
almond odor which is shared by benzaldehyde, nitrobenzene and benzonitrile. Also
replacement of the aldehyde group in vanillin by a cyano- or a nitro group results
in compounds with a weak vanilla odor.
xxx XXXI XXXII
Qroo
XXXIII XXXIV XXXV
Odor Similarity 291
The ring-oxygen in the musk XXX, which has a medium intensity, may be
replaced by an imino- (XXXI) or a methyl-imino group (XXXII). Both are weak
musks. The same substitution in the very strong musk XXXIII, gives a musk of
intermediate intensity XXXIV (BEETS, 1967).
The corresponding ketone XXXV, however, was found to be odorless. This
may be due to reduced volatility as well as to the changed direction of the dipole
vector.
Since the orientation of molecules at an interface between two phases of differ-
ent polarity is associated with the coordination- or solvation tendency of their
functional groups, the steric accessibility of the latter is a decisive factor for the
homogeneity of the orientation pattern. We have some reason to assume that this
also holds good for the situation at the receptor membrane. An obvious consequence
would be that decreasing accessibility of the functional group of an odorant mole-
cule results in degeneration of the orientation pattern towards randomness and in
loss of odor character.
y
Me"C~O
~
Me"C~O
A
Me
/c~
0
AO~C'H
XXXVI XXXVII XXXVIII XXXIX
~
Me"C~O
~ Me/C~O
~ ~ Me"C~O Me"C~O
XL XLI XLII XLIII
000 CW Q6l0 dO?

MeC~O Me"C~O
XLIV XLV XLVI XLVII
Qro
XLVIII
Qo6XLIX
Qov Qoy
L LI
XXXVI is a musk of medium intensity. Its isomer, XXXVII, is odorless. Its

carbonyl group, which does not oximate at room temperature, could, according to
the molecular model, just be coplanar with the aromatic nucleus but probably it
has rotated slightly out of the plane of the latter. Also XXXVIII, where co-
19
292 1\1. G. J. BEETS: Olfactory Response and Molecular Structure
planarity and oximation are impossible, is odorless. The aldehyde XXXIX, which
oximates slowly and in which coplanarity between carbonyl and nucleus is just
possible, is a fairly weak musk.
The ketones XL-XLIII, in which the functional groups are freely accessible,
are all strong to very strong musks. However, XLIV in which the quaternary
alkyl groups are in the position which seems to be optimal for this group of musks
but where the accessibility of the functional group is reduced by two vicinal
methylene groups which prevent its coplanarity with the aromatic nucleus, is a
fairly weak musk. Its isomer XLV, in which the accessibility of the earbonyl
function is still further diminished, has a very weak musk odor.
Two other examples of ketone musks have been described by CARPENTER
(1955). The ketone XLVI, in which the carbonyl group is well-accessible, is a musk,
but its isomer XLVII, in which the accessibility of the functional group is diminish-
ed by the bulky quaternary group in ortho position, has no musk character.
The isochromane derivative XLVIII is a musk of medium intensity. The
introduction of a methyl group in position 4 (XLIX) increases the intensity
dramatically but the two isomers Land LI, in which the methyl group is in a
position vicinal to the functional group, are both odorless.
It is interesting to observe that the same phenomenon can be reproduced after
replacement of the functional group. The introduction of a 4-methyl group in the
weak musk LII increases its intensity considerably (LIII) but the same methyl
group in the 3-position (LIV) results in complete loss of odor.
Qo:)H
LII LIII LIV
The presence of a second functional group in the molecule may influence the
odor in a variety of ways but since it alters necessarily direction and value of the
dipole vector and consequently also the orientation pattern at the membrane, the
introduction of a second functional group generally decreases the odor intensity
or changes the original character to an extent which depends strongly on nature
and position of the second group. A few examples may serve to illustrate the various
types of effect we may observe.
M~~
II
o
LV
M~~ o
II
LVI
)Or)
LVII
o
m LVIII
o
The ketone LV is a musk of medium intensity. Replacement of methylene by

oxygen in the non-aromatic ring (LVI) weakens the musk odor considerably and
introduces an anisic note (BEETS, 1967). Both LVII and LVIII have similar odors
reminiscent of burning sandalwood (Buu-HoI et al., 1966).
For theoretical reasons we may expect the influence of a second functional
group upon the odor to depend strongly upon the distance between the two groups
Odor Similarity 293
and upon their nature. We consider in the first place the situation in which the
functional groups Fl and F2 in a hypothetical structure LIX have widely different
polarities and solvation tendencies. As long as the two groups are fairly close, they
can cooperate more or less effectively as a single group which defines the pre-
dominating component of the orientation pattern.
LIX
The profile of this component is determined mainly by the bulk of the molecule
(B) since orientations in which the minor part (A), enclosed between the two func-
tional groups, determines the molecular profile, can be assumed to playa negligible
role. As the distance between Fl and F 2grows, they may continue cooperating but
since part A increases in size at the expense of the part B, available for the mole-
cular profile in the predominating orientation, the odor may be expected to weaken
or to deteriorate. Beyond a certain distance cooperation ceases and the functional
group with the lowest coordination- or hydration-tendency becomes part of the
profile (AF 2B) in the predominating orientation. The competing orientation with
the profile AFIB can be assumed to have a minor influence in this case. Since the
bulk of the molecule is again available for the profile, the odor intensity can be
expected to increase.
(CH)
I
--C=o {CH2)14_n-- C=O
I
2 13
o o
CH:
I
CH2 o
I
(CHi)n
LX LXI
THEIMER and DAVIES (1967) described the odor intensities of a series of macro-
cyclic oxa lactones with 17 ring members which seem to corroborate this theoretical
picture. The parent compound, hexadecanolide (LX) is a powerful musk. The
corresponding oxa-Iactones LXI with n = 2, 3 and 4, representing the cooperative
stage, have odors of intermediate intensity. Beyond n = 4 cooperation ceases and
the oxa-atom becomes part of the profile. The lactones with n = 5 and n = 6 have
strong to very strong musk odors.
When both functional groups are identical we may expect the situation in the
cooperative stage not to change. However, when both groups are located in
opposite parts of the structure, each may govern its own oriented profile (AFIB
and AF2B) or, in case the configuration is favorable, Fl and F2 being sterically
adjacent (LXII), cooperation may continue. Here, the profile of the predominat-
ing orientation consists of two competing parts A and B and we may expect
complete deterioration of the odor. Molecular models show that this situation may
easily occur in a group of macrocyclic diones (LXIII) described by OHLOFF et al.
294 M. G. J. BEETS: OUactory Response and Molecular Structure
(1967) of which the dione with n = 3 is a musk of undescribed intensity whereas

those with n = 5 and n = 6 are odorless.
II
/C'"
(H2 C)n (CHz)13-n
""-c/
II
o
LXII LXIII
In some odorants it is not possible to decide whether a group functions as part

of the molecular profile or as a functional group. This is particularly the case with
the nitro groups in the nitro musks. Some of these seem to have the ability to play
both parts with equal ease.
o,r;-gNo, ~No,
DOMe
O=C-gNo,
H
OMe OMe
LXIV LXV LXVI
yr:::
H
\Yc,o H HC=O
~OM'
LXVII LXVIII LXIX
NOz NOz Ac
o,r;*NO, O,N*AO o,r;,*No,

LXX LXXI LXXII
LXIV represents a musk in the structure of which one nitro group is sterically
unhindered and which consequently lies in the plane of the aromatic nucleus
whereas the other is forced out of this plane by the neighboring groups. The free
nitro group can be replaced by tertiary butyl (LXV; weak musk) or by formyl
(LXVI; musk) without destroying the musk character. Replacement of the remain-
ing, hindered nitro group in LXV by formyl (LXVII) gives an odorless compound,
although both LXVIII, in which the methoxy group is lacking, and LXIX, in
which the position of formyl has been changed, have odors in which the musk
character is blended with other notes (BEETS, 1957; WOOD, 1968).
Both types of nitro groups in musk xylene (LXX) can be replaced by acetyl.
LXXI and LXXII are both musks. The musk character of LXX is also preserved
when either of the two types of nitro groups is replaced by bromine, cyano or
methoxy-carbonyl (WOOD, 1968).
Odor Similarity 295
An example of an apparently minor structural change which destroys the

strong musk character of a nitro musk, has been described by BEETS (1961&).
LXXIII LXXIV LXXV
LXXIII (Musk Ambrette) is a strong musk. LXXIV, in which the methoxy-

and the tert.butyl group have been fused into a 5-membered ring, is odorless. The
molecular models, however, show that the methyl group of methoxy in LXXIII is
forced out of the plane by the neighboring groups whereas the 5-membered ring in
LXXIV is only slightly puckered.
We have already mentioned (XXV) that the methyl group in LXXIII cannot
be replaced by bromine without loss of the musk odor. It is remarkable that the
compound obtained by interchanging methoxy and bromine in XXV (LXXV) is
a musk (WOOD, 1968). This is surprising since XXV does and LXXV does not
obey CARPENTER'S (1955) "ortho-rule" according to which the ortho position of
tertiary alkyl- and alkoxy groups is a requirement for musk odor in alkoxy-nitro
musks.
These examples, arbitrarily selected from the abundant material described in
the literature, show that the class of nitro musks, with their large variety of
frequently interchangeable and replaceable substituents represents one of the most
complex problems for research on odor-structure relations.
In other series of musks profile characteristics are somewhat easier to define.
R
LXXVI Rl = R. = H; R. = Me : odorless
~~~ LXXVII Rl = R. = Me; R. = H : weak
LXXVIII R1 =R.=Ra =Me : very strong
Ac
LXXIX Rl = R. = H; R. = Me : extremely weak
LXXX Rl = R. = Me; R, = H : weak
~ Ac
LXXXI
LXXXII
LXXXIII
Rl = R. = Me; R. = Et : strong
Rl = R, = Me; R. = Et : strong
: weak
LXXXIll
~ Ac
~ Ac
LXXXIV LXXXV
LXXXVI Rl=R.=H : medium
Qd)' LXXXVII ~= H;R.= Me

LXXXVIII R1 = H;R.= Et
LXXXIX Rl=R.=Me
: very strong
: medium
: medium
296 M. G. J. BEETS: OHactory Response and Molecular Structure
XC : weak
XCI Rl = Me; R2 = Rs = H : medium
XCII Rl = Rs = Me; R2 = H : powerful
XCIII Rl = R2 = Me; Ra = H : odorless
BEETS (1967) reviewed the development of the musk odor in series of bicyclic
and monocyclic keto musks (LXXVI-LXXXV) and showed that in these series
no musk odor of usable intensity can be obtained without the presence of two
quaternary carbons in both meta-positions with respect to the carbonyl group. The
monocyclic musks LXXXIV (medium intensity) and LXXXV (weak) should be
compared with the strong musks XLII and LXXVIII.
In the tricyclic isochromanes (LXXXVI-XCIII) the presence of two quater-
nary carbons in ortho-position was found to be a requirement for musk odors of
reasonable strength. The dramatic increase of odor intensity obtained by intro-
duction of a methyl group in the 4-position has already been mentioned (BEETS,
1967). Also in acyl tetralines (XCIV) and in acyl indanes (XCV) summarized by
WOOD (1968) a musk odor of useful strength is only found when two quaternary
carbons in ortho position are present. In these classes also the presence of a lower
alkyl group (R2 = Me or Et), ortho to the acyl group, and of a methyl group in
the non-aromatic ring (Ra = Me) are important for the odor intensity.
XCIV XCV
Profile studies in other series of odorants are still rare. The profile XCVI
(F.G. = functional group; BEETS, 1961a) seems to be favorable for the amber
odor; XCVII-C are examples of odorants belonging to this class (R = H or,
preferably, Me).
0'
t1:)
.I
, ,
F.G.
XCVI XCVII XCVIII XCIX C
PRELOG and RUZICKA, in a series of papers, summarized by BEETS (1957)

described a series of steroid-ketones and alcohols of which a limited number pos-
sess odors with associations of musk as well as of urine. Of the latter type CI is a
representative example. The urine-like odor has since been found in at least one
material of different structure of which the profile is related to part of the steroid
structure. Characteristic examples of specific anosmia which have been observed for
the urine-like odor suggest that this type of odor may be one of the fundamental or
primary odors (BEETS and THEIMER, 1970).
Odor Transition 297
PRELOG and RUZICKA (1944) and PRELOG et al. (1944) mentioned the remarkable
formal resemblance between the structures of these steroids, some of which are
musks, and the macro cyclic musk civettone (ClI). The bicyclic ketone ClIr in
which one ring has been closed, is a weak musk (PRELOG et al., 1947). The odors
and structures of a number of steroids have been summarized by BEETS (196la).
CI ClI cm
The profiles of the mono-stubstituted phenyl group and the 4-hydroxy-3-

alkoxy-phenyl group have already been mentioned as representing the bitter
almond and the vanillin odor. In the same way the profile of the 3-4-methylene-
dioxy-phenyl group seems to represent the heliotropine odor which is shared by the
aldehyde CIV and the azide CV. This profile seems to be sufficiently preserved
when methylenedioxy is replaced by the isothiocyanate group, since also CVI has
a similar odor type.
0'0 0,
s
C C
o
II \
H oII \ H
CIV CV CVI
It should be kept in mind that the methylenedioxy-, the hydroxy- and the
alkoxy groups in these classes cannot be expected to function exclusively as profile
groups.
The importance of orientation of the molecular profile for the odor type is
demonstrated by several of the examples mentioned above. BEETS (1968) sug-
gested that the odors of the aromatic aldehydes CVlI (medium intensity) and
CVlII (weak musk) and those of the nitriles CIX (very weak musk) and CX (non-
musk) could point in the same direction.
HA HA A
0 0 C
N
~C
N
CVIl CVIII CD( CX
3. Odor Transition
Gradual changes in the structure of an odorant may produce either a gradual
transition from one odor type to another or the odor type may change more
abruptly. The first case is observed in homologous series of open chain compounds
298 M. G. J. BEETS: OHactory Response and Molecular Structure
where no configurational changes play a part. The second case occurs when the
addition of one or two chain-members elicits a transition from one configuration to
another or results in an abruptly increased conformational freedom of the molecule.
A classical example of the latter case is presented by the macrocyclic lactones. In
this series we observe a certain parallelism between odor- and stereochemical
characteristics, observed in molecular models. This is shown in Table 3.
Table 3. Macrocyclic lactone8
Carbon Odor type Stereochemistry

atoms
9, 10 Camphoraceous Considerable ring tension; circular structure

11 Harsh, terpeney, leBS camphor Less tension, circular structure
12 LeBS harsh and camphor, trace of Tension free, circular structure
cedar note
13 More cedar, faint musk Circular structure
14 LeBS cedar, more musk Circular or oval
15,16 Pure musk Circular or stretched
17 Musk, trace of civette Circular or stretched
18 Faint musk, trace of civette Circular or stretched
19 Nearly odorless Circular or stretched
20 Odorless Circular or stretched
We see that the musk note appears simultaneously with the increased con-
formational freedom. RUZICKA et al. (1930) have been the first to point out the
correlations between odor and configuration in the macrocyclic series. The same
authors also described several physical characteristics of these series, such as
molecular volume, density, melting points and refractive index.
Another interesting study on the transition of odors in a series of homologs has
been published by KLOUWEN and Ruys (1963). Benzaldehyde, nitrobenzene and
the three mono methyl homologs of both possess a bitter almond odor. In most of
the ethyl-homologs, this odor is still recognizable but it is blended with a cuminic
note. The latter is only absent in both ortho isomers. In the iso propyl series the
para isomers have strong pure cumin odors, in the meta-isomers these are mixed
with a weak note of sassafras and the odors of the ortho compounds are only weakly
reminiscent of cumin. Finally, in the tert.butyl series, the para- and meta isomers
all have cumin odors with carrot-like secondary notes but in the ortho isomers
only a non-characteristic odor reminiscent of tert.butyl-benzene remains. The
"hydrocarbon" odor in the latter case is, as we have seen, associated with a
strongly reduced steric accessibility of the functional group and consequently with
degeneration of the orientation pattern towards randomness.
RUBIN et al. (1962) studied a series of homologous lactones of structure eXI
and compared the odors with predictions made on the basis of AMOORE'S stereo-
chemical theory of odor. In two short series in which ~ is hydrogen and methyl
and in each of which RII increases in size from ethyl to octyl, the odor changes from
the minty-camphoraceous type to the floral type. According to AMooRE's theory,
Odor Transition 299
the camphor- and the minty note should decrease from very strong to very weak
whereas the floral note should increase in strength with increasing size of Rs.
err
RI
R2
CXI
~
CXII CXIII
~H CXIV
Although stereoisomers of odorant molecules are structurally closely related,

their oriented profiles and, consequently also their odors, may be totally different.
Numerous examples demonstrating this effect have been described in the literature.
Here we shall only mention a single example described recently by DEMOLE (1964)
who studied the stereochemistry of hydrogenation products of the isomers obtained
by condensation of camphene with phenol. He found that, out of all isomers and
stereomers, only one, CXII has a strong odor of sandalwood. All others, parti-
cularly the stereomer CXIV of CXII, have weak odors or are odorless. The example
is of particular interest since the molecular shapes of CXII and CXIV do not differ
much but the oriented profiles, viewed from the direction of the polar group, are
vastly different. The natural constituents of sandalwood oil, IX-and p-santalol have
partly flexible molecules, which may assume, in the interaction stage, a con-
formation which resembles that of CXII (CXIII; p-santalol).
The oriented profile of odorant molecules with a bivalent functional group is
determined by two parts of the molecule A and B (CXV). When A is small (hydro-
gen or methyl), the oriented profile is almost entirely defined by the dimensions of
B but as A grows in size it starts contributing to the profile of the predominating
component of the orientation pattern. Consequently we may expect gradual
changes in odor quality and intensity.
o
II
Me{CHa )nC{CH2 )a -nMe
CXV CXVI
R,C
II~
o
6
CXVIII
An example of this effect has been described by VON BRAUN and KROPER (1929)
who studied the odors of a series of undecanones (CXVI).
Undecanone-2 (n = 0) has the strong odor of rue in which this ketone occurs.
As n increases from 0 to 4, the rue odor becomes weaker and is replaced by a fruity,
amyl acetate-like odor of increasing intensity. A second example is taken from the
musks (BEETS, 1968). When R in the mono cyclic musks CXVII is hydrogen, we
observe a musk odor of medium intensity, blended with an anisic note. The latter
disappears for R = methyl. In the ketones with R = ethyl and iso propyl a sweet
note of increasing intensity appears while the strength of the musk odor decreases.
A similar effect is observed in the bicyclic ketones CXVIII where increasing size of
R results in the appearance of a floral note.
F. Optical Antipodes
The chirality of pharmacon- and substrate-molecules is known to have a strong
influence upon the effect of their interaction with enzymes and receptors, often to
the extent that one antipode is active and the other inactive. This is comprehensible
in view of the high stereospecificity required for the lock and key relation between
both partners, of which one has a rigidly defined chirality, in the interaction
complex.
Although the molecules of two optical antipodes have closely related structures,
their oriented profiles can be vastly different. Consequently, the effect of chirality
on pharmacological activity should be considered as an aspect of the general
importance of molecular shape in biological processes.
At first sight we would expect a difference between the odor characteristics of
two optical antipodes similar to the one found for other pharmacological properties.
Also in this case a stereospecific interaction between an intruder molecule and a
component of the organism is involved and, although we know very little about
nature and structure of the olfactory receptor sites, we can be fairly certain that
they are proteinaceous molecules or part or combinations of such molecules. The
protein part of such molecules is constructed of L-amino acids and has, consequent-
ly a well-defined chirality. Also, even in the absence of conclusive evidence we
have reasons to assume that the efficiency of the interaction depends on the
closeness of fit between the two partners in the interaction process. If the steric
requirements for participation in the latter were as critically important as they are
in enzymatic processes, olfactory interaction could be expected to be highly
specific with respect to the chirality of the odorant molecule.
However, the picture is much less clear where olfaction is concerned. Any type
of olfactory receptor site is probably able to accommodate a rather wide range of
odorant structures with varying efficiency and the closeness of fit required for
olfactory interaction seems to be a much cruder criterion than it is in the case of
enzymatic processes.
Finally, a fairly large number of examples, which are discussed in other para-
graphs of this chapter, indicate that position and sterical accessibility of the func-
tional group in odorant molecules seem to be more important than its nature,
whereas in various other pharmacological and in enzymatic processes the naturc of
the functional group plays a critical part in the lock and key relationship.
Optical Antipodes 301
All this suggests that the mechanistic analogy between olfactory interaction
and the interactions involved in other pharmacological and in enzymatic processes,
while it does exist, is probably a limited one.
The study of olfactory response of optical antipodes presents us with a unique
opportunity to isolate the aspect of molecular shape from all other structural
aspects. Enantiomers have the same scalar properties, physical as well as chemical.
Especially they have the same functional groups with the same steric accessibility
and the same chemical reactivity. They have the same spectra and the same dipole
moments. Only their molecular profiles are different. To find the answer to the
question of odor differences between optical antipodes is probably one of the
most important single problems to be solved by research on odor-structure re-
lations.
Any reliable study of the olfactory characteristics of enantiomers should satisfy
a number of requirements. In the first place asymmetry should preferably be
restricted to a single center or, if more asymmetric centers are present, the chirality
of all should be opposite for both compounds. Diastereomers are just cases of
molecules with entirely different configurations and consequently with different
chemical and physical properties. They can be expected to have different odors
(VON BRAU~ et al., 1925, 1926, 1927). Also, both antipodes must be rigorously pure.
Any trace of impurity with low olfactory threshold, originating from different
sources or different isolation techniques for both antipodes, may cause misleading
odor differences. Consequently, only optical antipodes obtained by resolution from
the same racemic material under mild conditions and purified with excessive care
can be expected to yield reliable results. Finally, trigeminal and taste effects must
be avoided as near as possible since they may easily cause perceptual differences
which havc no basis in olfaction. We know that many pairs of enantiomers (amino
acids, carbohydrates) have different tastes (BEIDLER, 1966). This can be realized
best by studying electrophysiological response to stimulation of the olfactory
membrane with enantiomers.
In the earlier work these requirements are not fulfilled.
TIEMANN and SCHMIDT (1897) reported l-citronellal to have a sweeter odor than
its antipode but both were obtained from different sources and crudely purified.
Similar results of doubtful value were obtained by VON BRAUN and KAISER (1923)
who also prepared the corresponding dimethyl octanols by hydrogenation. Also
here, impurities may be responsible for the odor differences found in both cases.
WERNER and CONRAD (1899) prepared the antipodes of dimethyl trans-hexa hydro
phthalates by resolution of the racemic acid via its quinine salt, followed by rather
careless esterification and purification. One of the antipodes was found to be
odorless whereas the other had a fairly strong odor. POSVIC (1953), repeating these
experiments under carefully controlled conditions, found both enantiomers to have
identical odor qualities and comparable intensities. Also this result is not con-
vincing since a difference which a single observer fails to detect may still turn out
to be significant after statistical analysis of data obtained from a large pancl. In
all other examples reported by VON BRAUN'S group (VON BRAUN et al., 1925, 1926,
1927) molecules with 2 or 3 asymmetric centers are involved. These stereomers
have not only different odors but also different configurations which are not symmet-
rically related and, consequently, are irrelevant to the present subject.
SINGH and LAL (1939, 1940) prepared the antipodes of 3-nitro- and 5-nitro-
ortho-toluidino methylene camphor and of 2,5-toluylene- and 2,3-toluylene bis
amino methylene camphor. The odor intensities were evaluated by panels consist-
ing of 10-13 observers. For all 4 compounds the odor intensity was found to
decrease almost invariably in the sequence l > dl > d. However, in spite of the
apparently sound basis of this work, the results are far from convincing since the
two latter compounds have a molecular weight of 446 and for this reason must be
odorless in pure form. Consequently, purification for all 4 compounds must have
been unsatisfactory.
Trigeminal- and taste-effects can not always be distinguished easily from pure
odor effects. The cooling effect of menthol is a characteristic example and for this
reason menthol is perhaps not the most favorable example for odor comparison of
enantiomers. No critical evaluation of this complication is possible for the data
published by READ and GRUBB (1931) who reported identical odor qualities for d-
and I-menthol but a higher intensity for the latter. Later, DOLL and BOURNOT
(1948) made a careful attempt to evaluate the various effects separately. The two
antipodes were prepared by resolution of the racemate as well as by isolation from
natural sources and the odor identity of both qualities was checked by expert
evaluation for the two antipodes. The threshold values were found to be 1.3 0.5 Y
per 1.51 of air. Up to a concentration of 25.2 y in 1.51 of air no difference in
quality or intensity of the odor was observed. At higher concentration the cooling
effect appears, the intensity of which increases more rapidly for I-menthol than for
d-menthol while the quality of the total sensation remains the same. The same
difference was found when solutions of equal concentration of both antipodes were
applied to the back of the hand.
A valuable contribution to the solution of the problem was described by GUIL-
LOT and co-workers in a series of papers (GUILLOT and BABIN, 1949; GUILLOT and
THrnAUT, 1951; GUILLOT, 1955). Octanol-2 was resolved via the brucine salt of its
mono phthalate. Antipodes were obtained with identical rotation and 1.R. spec-
trum. The odor intensity was measured by repeated evaluation of a series of
smelling strips with alcoholic solutions of increasing concentration and counting
of the positive, negative and uncertain answers. On the resulting S-shaped curves
the concentrations giving 50% positive answers were determined. The ratio be-
tween these concentrations for 1- and for d-carbinol was found to be 3.3 for one
subject and 3.2 for a second one. No difference in odor quality was found. Similar
work was carried out for isoborneol. Although l-isoborneol was obtained in pure
form, the d-antipode contained about 12 % of l-isoborneol. In this case a significant
difference in odor quality was observed by all members of the panel. Both forms
have a camphoraceous odor but in the case of d-isoborneol the odor is reminiscent
of celluloid and rosemary oil whereas the bynotes of l-isoborneol are sweet and
musty. The intensity ratio, in favor of l-isoborneol, was estimated to be 1.25 for
the celluloid-note and 2 for the musty note.
STUIVER (1958) repeated GUILLOT'S experiments with octanol-2 and measured
the threshold for both enantiomers by means of an olfactometer. The threshold
concentrations found, viz. 5.2 x 109 molecules per cm3 for the d-carbinol and
1.5 x 109 molecules per cm3 for the I-carbinol, show a ratio of 2.9 which agrees well
with the values reported by GUILLOT.
Optical Antipodes 303
RIENACKER and OHLOFF (1961) prepared the two antipodes of p-citronellol by

pyrolysis or (+)- and (-) pinane (obtained by hydrogenation from (+) p-pinene
and (-) IX-pinene or (-) p- pinene isolated from natural sources) followed by
hydroboration. No purification technique is mentioned but the rotation values for
both alcohols are comparable with the highest values found for the corresponding
natural alcohols. The odor qualities were found to be different; (- ) p-citronellol is
reminiscent of citronellol isolated from geranium oil, (+) p-citronellol of the alcohol
obtained from citronella oil. Intensities or threshold values were not given.
NAVES (1947) resolved dl-IX ionone via its I-menthyl amino carbamate. Both
antipodes and the racemic mixture have the same odor quality but the threshold
concentration of the antipodes is about 12 times higher than that of dl-IX ionone.
Similar observations were published on the neo-IX irones (NAVES, 1953). VELDSTRA'S
observation (1961) that the two liquid and practically odorless enantiomers of
IX-allyl phenyl acetic acid form a crystalline racemate with a clear honeytype odor
belongs to the same category. Assuming that these observations are correct, it is
interesting to consider that they demonstrate clearly that the interaction patterns
of the two antipodes with the olfactory membrane are different since a mixture of
equal parts of two identical compounds can only give the same information pattern
as the component parts, either directly or after any number of processing steps.
This means that, although the information produced by both enantiomers on the
membrane level is different for both, the odor sensations, i.e. the final products
obtained after processing of the primary information pattern in the olfactory
system and the brain, are similar or identical. In this connection it is interesting to
mention LETTVIN and GESTELAND'S (1965) observation that in their electro-
physiological work they found one axon in which little change was produced by
stimulation of the membrane with either ethanol or musk in any concentration.
Stimulation with a mixture of both in moderate concentration resulted in strong
exaltation.
Two recent reports seem to bring this problem closer to its solution. FRIEDMAN
(1969) converted pure d-carvone into l-carvone after which the latter was recon-
verted into d-carvone. During these steps the odor character changed from
caraway to spearmint and from spearmint to caraway.
THEIMER and McDANIEL (1970) used a different technique to eliminate the
possible influence of impurities as the source of odor differences. These authors
converted d-IX-pinene from Greek turpentine oil by borohydride isomerization in-
to d-p-pinene and lop-pinene from American turpentine oil by palladium catalyzed
isomerization into I-lX-pinene.
The two pairs of pinenes were converted into d-trans pinocarveol (d-CXIX1 ;
d-CXIX 2) and I-trans pinovarveol (1-CXIX1 ; I-CXIX 2) of each of which two
duplicate qualities were obtained in this way via different synthetic paths from
the same source. Each of the four pinocarveols was converted into trans-pino-
carvyl propionate (CXX), myrtenal (CXXI), myrtenal diethyl acetal (CXXII) and
pinoacetaldehyde (CXXIII). The materials were presented on a blotter to each
panel member in groups of 4 coded, randomly arranged antipodes. The results
were processed statistically and gave significant evidence that for the selected
odorants the human nose can discriminate between two enantiomers.
The available evidence, although limited, seems to suggest that, at least in

some cases optical antipodes have different odor qualities or odor intensities or
both and that even in cases where the odor characteristics are indistinguishable,
enantiomers may elicit different response patterns on the membrane.
6r
.OCEt
o
II
1 - CXIX1 1 - CXIX2 1-CXX1 I-CXX2

d-CXIX1 d-CXIX2 d-CXX1 d-CXX2
HC=O
@
1 - CXXI1 1 - CXXI2
d-CXXI1 d-CXXI2
.... OEt
HC
~OEt
1- CXXII1 1- CXXII2 1- CXXIII1 1- CXXIII2

d - CXXII1 d - CXXII 2 d-CXXIII1 d-CXXIII2
Electrophysiological data on enantiomers are still deplorably scarce. SCHNEI-

DER (1963) determined the electro-physiological thresholds for both enantiomers
and the racemate of the lure substance ((+) lO-acetoxy-hexadecen-7 cis-ol-l) of
the gypsy moth, and found the same value, 10-2 microgram for all three. Behavioral
thresholds were determined for the (+) enantiomer (10- 5 microgram) and for the
racemate (10- 3 microgram) but JACOBSON et al. (1960) found the same value
(10- 7 microgram) for both.
Perhaps the only way towards confirmation or rejection of our preliminary con-
clusion may be the accumulative evidence from a large collection of experimental
material since the influence of minor factors can be expected to be phased out as
the evidence grows. Also, and perhaps first of all, electrophysiological response
patterns obtained by recording from receptors or nerve fibers may help us to solve
this problem since they permit the exclusion of other than olfactory contributions.
For both techniques a careful selection of materials is needed. It is suggested to
continue experiments with rigorously pure enantiomers of molecular weight
between 120 and 280, and preferably with a single center of chirality. Such ma-
terials should comprise flexible as well as rigid structures and the asymmetric
center should be close to as well as far removed from the functional group.
G. Adaptation
Olfactory adaptation is the temporary loss of sensitivity as a result of con-
tinuous or repeated stimulation. When the conditioning stimulus is not identical
with the tested stimulus the phenomenon is called cross-adaptation.
Adaptation 305
Experimental work suggests that the process of adaptation consists of two

super-imposed components. The major component, characterized by fast recovery,
is located in the olfactory bulb and is comparable to our inability to perceive,
after a certain time, the ticking of a clock. According to ADRIAN (1950), it is
caused by the loss of capacity of the olactory signals to disorganize the resting
activity of the bulb. The existence of a minor component, characterized by slow
recovery, located in the receptor membrane is suggested by experiments reported
by STUIVER (1958) and by OTTOSON (1956).
In a few cases cross-adaptation has been used as a technique to obtain infor-
mation on structure-odor relations on the basis of the idea that cross-adaptation
between odors is related to their mechanistic or structural resemblance (ZWAAR-
DEMAKER, 1895).
LE MAGNEN (1942/1943) used cross-adaptation in an attempt to classify a
number of odorants on the basis of their cross-adaptation effects. He measured the
thresholds of a certain odorant before and after 10 minutes of adaptation to a
second odorant. The difference between both threshold levels was assumed to be
directly related to the degree of cross-adaptation and through this to the degree of
similarity between the patterns of interaction between both odorants and the
olfactory system. The materials used for this study comprise vastly different
structures such as terpineol, vanillin, safrole and tetralin and the results present a
complex picture which is difficult to analyze. Adaptation to vanillin reduces the
sensitivity to both terpineol and safrole whereas adaptation to safrole does not
influence the sensitivity to vanillin. The latter is destroyed by adaptation to
terpineol. The choice of materials in this work, although sufficient for a demon-
stration of the principle, is too unsystematic from the structural point of view to
permit analysis of the results.
In a later paper, LE MAGNEN (1948) describes the application of cross-adapta-
tion to some odorants belonging to the bitter almond-, the camphor- and the musk-
type.
Strong cross-adaptation effects were found when the benzaldehyde odor was
evaluated after adaptation to benzonitrile, leaving an odor sensation resembling
safrole; to a mixture of benzonitrile and safrole, leaving an odor sensation resembl-
ing indole, and to a mixture of safrole and indole, leaving merely a benzene-like
note. Similar interrelations were found in a group of odorants with camphor-odors.
However, it was found that musks belonging to two structurally unrelated
series, i.e. musk ketone and pentadecanolide showed no cross-adaptation. This
could mean that the two structural types of musks of which the olfactory similarity
is easily recognized are not only structurally but also mechanistically unrelated.
This confirms similar findings of GUILLOT (1948a).
CHEESMAN and co-workers (CHEESMAN and MAYNE, 1953; CHEESMAN and
TOWNSEND, 1956; CHEESMAN, 1960) studied the quantitative aspects of cross-
adaptation with a group of 4 odorants. The results suggest that the logarithm of
the threshold concentration of the tested odorant is a linear function of the
logarithm of the concentration of the conditioning odorant.
The slope of this linear function may be considered as a measure for the degree
of similarity between the interaction patterns of both odorants with the olfactory
system. The interesting aspect of the method is that, although the results are
20 Hdb. Sensory Physiology. Vol. IV!l
obtained from human observations, they express a similarity of information pat-

ters on some level, possibly that of the membrane, in the olfactory system, rather
than a similarity between the odor sensations.
The results are summarized in Table 4.
Table 4
Conditioning odorant Tested odorant

Isopropanol Dioxan Cyclopen- Cyclopentanol
tanone
Isopropanol 0.68 0.51 0.24 0.72

Dioxan 0.59 0.74 0.48 0.50
Cyclopentanone 0.18 0.54 0.67 0.04
Cyclopentanol 0.64 0.59 0.17 0.68
They show that some of the highest values are obtained when the tested and
the conditioning stimulus are identical and that these values are approximately
independent (0.7) of the nature of the odorant within a single experiment.
At first sight it seems surprising that two structurally dissimilar odorants such
as isopropanol and cyclopentanol (0.64 and 0.72) should give high figures whereas
structurally similar odorants like cyclopentanol and cyclopentanone (0.17 and
0.04) give low values. This becomes less surprising when we compare the oriented
profiles rather than the shapes of the molecules. In Fig. 9 the molecular models of
isopropanol, cyclopentanone and cyclopentanol are shown, viewed in the direction
of the carbon-oxygen bond (as a reasonable approximation of the direction of the
polar axis). This demonstrates that the oriented profiles (as they may be presented
to the receptor sites) of cyclopentanol and isopropanol have a higher similarity
(percentage superimposability) than those of cyclopentanol and cyclopentanone.
In these experiments a simple sniff-bottle technique was used and, owing to the
rather cumbersome procedure, the range of materials studied was necessarily
limited. In the meantime (CHEESMAN and KIRKBY, 1959) an olfactometer has been
developed which is expected to enable this group to test a larger series of odorants.
MONCRIEFF (1956), using undiluted conditioning odorants, determined thresh-
olds before and after adaptation in order to obtain indications on the relationship
between cross-adaptation and odor similarity.
ENGEN (1963) measured cross-adaptation in a series of straight-chain alkanols
of which each served both as adapting- and as test-stimulus. No systematic
relationship between magnitude of the effect and chain length was found. Possibly
experiments with an uninterrupted series of homologs would have resulted in
additional information on the latter.
KOSTER (1965) studied adaptation effects for a number of odorants with widely
varying structures by measuring the shift in the percentage of positive answers to
a stimulus rather than the difference in threshold. The results suggest that
olfactory adaptation may become a tool to discern different types of receptor site
which could be the basis for a comparison with electrophysiological data. Part of
the odorants used in this work were selected in order to test AMOORE'S stereo-
Adaptation 307
chemical theory of olfaction (see DAVIES in this volume). According to the latter,
the odor of benzaldehyde is based on an interaction pattern in which receptor
sites responding to odorants of the camphoraceous, the pepperminty and the floral
primary classes are involved. In KOSTER'S experiment, benzaldehyde was pre-
sented together with geraniol and dioxan. The interaction pattern for geraniol,
Isopropanol
Fig. 9. Molecular profiles of isopropanol, cyclopent:1none and cyclopentanol
which belongs to the floral class would, according to AMOORE, overlap with that of
benzaldehyde whereas the pattern of dioxan, which belongs to the ethereal class,
would not. The experiment showed that both benzaldehyde and geraniol have an
effect on the sensitivity for dioxan, the effect caused by benzaldehyde exceeding
all others found in KOSTER'S series. Also, dioxan showed a much greater influence
on the sensitivity for geraniol than could be expected on the basis of AMOORE'S
theory. The oriented profiles of the three structures have very little in common
although the superimposability of those of dioxan and benzaldehyde is higher than
of benzaldehyde and geraniol.
Electrophysiological work on adaptation effects is still rare (OTTOSON, 1956;
BOECKH et al., 1965) and has so far not yielded any material for the study of
structure-response relations.
20
Cross-adaptation could very well become one of the most useful tools for re-
search on the relationship between structure and olfactory response provided
rapid olfactometric techniques are developed permitting the testing of large
series of odorants. CHEESMAN'S approach, which results for each pair of odorants
in a numerical value expressing the extent of cross-adaptation, seems partic-
ularly suited for the detection of correlations with structural parameters.
H. Specific Anosmia
This subject is discussed in detail in AMOORE'S chapter "Olfactory genetics and
anosmia" in this volume. Here we shall only consider some aspects which are of
special importance for the subject and the general philosophy of the present
chapter.
Specific anosmia is the inability in persons with otherwise normal olfactory
sensitivity to detect the odor of one or more odorants. The obvious explanation is
that information which in normal observers is generated in a population of receptor
sites belonging to a single type is either not generated or not transmitted to the
higher centers, e.g., because the corresponding type of receptor site which is present
in the membrane of normal persons, is lacking or inactive.
This explanation immediately creates a dilemma. On the one hand, it is hard
to believe that there is such a thing as a true mono-osmatic odorant, the odor of
which is generated by the interaction of its molecules with a single type of receptor
site. This situation may be approached closely in some cases but even then we may
expect a primary information pattern in which one type of information pre-
dominates whereas others occur with far lower frequency. On the other hand it is
highly improbable that in a person with specific anosmia for a certain odorant, all
types of site able to accommodate its molecules in any orientation or conformation
with varying efficiency, are inactive. Not only would the chance of such a co-
incidence be almost negligible - it would make specific anosmia one of the rarest
phenomena in nature - but also it would seriously impair the whole olfactory
sense of the anosmic observer since the receptor sites involved to a minor extent in
the interaction pattern of the membrane with one odorant can be expected to play
a major part in its interaction pattern with other odorants. We know persons with
specific anosmia to have a normal sense of smell for other odorants.
If this reasoning is approximately correct, it leads to the tentative conclusion
that specific anosmia can be expected to occur with odorants of which the inter-
action with normal membranes involves mainly a single type of receptor site. Con-
sequently the resulting information pattern will show a high degree of homogeneity
and consist of one predominating type of information and other types in low
frequencies.
For reasons of probability we must assume that in the anosmic membrane the
site types responsible for the generation of the minor components in the information
pattern are functioning normally. The frequency of these components is either too
low to generate an odor sensation (total anosmia) or they cause a weak sensation
with a quality differing widely from the odor perceived by a normal person
(parosmia).
Specific Anosmia 309
This would mean in the first place that molecules for which a specific anosmia
has heen observed can be expected to belong primarily to the classes of fairly
rigid, polar structures with an easily accessible functional group and consequently
homogeneous orientation- and conformation-patterns. It would also mean that
specific anosmia will be hard to detect by electrophysiological recording from
groups of units in membrane, nerve fibers or olfactory bulb since in the anosmic
system most units will respond normally.
These considerations suggest that specific anosmia may be a valuable tool in
research on structure-odor relations. Its occurrence, even in a single subject,
indicates that the odorant is a primary (AMooRE, 1952) and that the predominat-
ing component lacking in its information pattern represents a mono-osmatic type
of information and a single type of receptor site.
Not only detection and labelling of mono-osmatic types of information and
receptor sites but also a meaningful classification of odorants is made possible by
application of anosmia. Groups of odorants with related odors for which anosmia
occurs can be assumed to belong to a single primary class since the same component
predominates in their information patterns (fundamental similarity). On the other
hand, two odorants with similar odors for only one of which a subject is anosmic,
do not belong to the same primary class (pseudo-similarity).
A few authors have seriously attempted to make practical use of these principles.
GUILLOT (1948 a, b) describes cases of specific anosmia for macrocyclic musks,
steroid musks, hydrocyanic acid, ambergris, benzyl salicylate, methyl ionone,
farnesol and sclareol. The latter case is of particular interest since here specific
anosmia is the rule rather than the exception.
Although macro cyclic musks, steroid musks and nitro musks have similar
odors, their anosmias seem to be independent. One of GUILLOT'S subjects was
anosmic to macrocyclic musks, a second one to steroid musks and a third one to
both. However, the latter had a normal sensitivity for nitro musks. A similar
independence was observed between the anosmias for hydrocyanic acid and
benzaldehyde, both of which have similar bitter almond odors.
Apart from specific anosmia, GUILLOT (1953) describes a few cases of parosmia,
in which the odor sensation obtained widely differs from the normal one. In such
cases the odors of the steroid musks are characterized as weak, fruity, floral, musky
or ionone-like notes. Another subject does not perceive the violet odor of the
ionones and describes the odor as being woody.
GUILLOT, on the basis of these observations, concludes that a list of odorants
for which specific anosmia has been observed may consist of representatives of the
"odeurs fondamentales" and also that the identity of odor sensations caused by
different odorants is not a criterion for identity of the sensory mechanisms
involved.
AMOORE'S (1967a, 1968; AMOORE et al., 1967, 1968) excellent research on the
detection of primary odors by means of specific anosmia, especially his work on the
fatty acids and the sweaty primary odor and his efforts (AMOORE, 1969) to increase
the number of known primaries, are described in great detail in his chapter in this
book.
Recently, BEETS and THEIMER (1970) reported an interesting application of
specific anosmia for the detection of fundamental odor similarity between two
structurally unrelated types, the first of which is represented by the steroid

ketones eXXIV and ex XV and the second type by the mono cyclic ketone
om:o
eXXVI.
CXXIV C,{XV
CXXVI
Out of a panel of 100 observers, 51 described the odor of all three as extremely
intensive, using the descriptors urine and perspiration most frequently. 35 observers
were unable to smell the major note. Of this group, 6 were total anosmics for the
three ketones while 29 were parosmics, describing the odor as weak and using
descriptors that were totally unrelated to those used for the major note. Although
the frequency of occurrence of descriptors used for the secondary notes varied
widely between the ketones, the same key-words were used for all three.
Obviously, the major note in all three ketones is derived from the same mono-
osmatic component in their information patterns and can be labelled with the
descriptors "urine-perspiration."
H H
o
('XXI\' ( 'XX\' I
Fig. 10. Molecular profiles and configurations of ,116-androstenone-3 (CXXIV) and of 2-methyl-
2-(4'-tert.butyl-cyclohexyl)-pentanone-4 (CXXVI). (From BEETS and THEIMER, 1970)
Indirect Relations 311
Fig. 10, showing the steric formulae and the molecular models of one of the
steroid ketones (CXXIV) and the mono cyclic ketone CXXVI, demonstrates the
rather unexpected profile similarity.
I. Indirect Relations
In the introduction to this chapter the statement was made that olfactory
response is necessarily related to other characteristics of the odorant since both are
derived from the molecular structure. In the course of this chapter several examples
of such indirect relations have been mentioned. Others are briefly discussed in this
paragraph.
Before World War II, DYSON (1928, 1937, 1938) described a few cases in which
the odor type seemed to be related to certain Raman frequencies in the range
between 1500 and 3000 cm-I . As WRIGHT (1954) observed, Raman frequencies in
this region are correlated with specific functional groups and consequently any
relations found would only indicate that the odor type is somehow related to the
nature of the functional group. For this reason WRIGHT (1954) suggested studying
the Raman lines below 1000 cm-I since these are associated with molecular con-
figuration and he actually found some frequencies which a number of odorants
with bitter almond odor seemed to have in common. This has been the starting
point for extensive research by WRIGHT and his group on the far infrared spectra
of odorants below 500 cm-I . The theory of olfaction which he proposed on the
basis of these findings is described in another chapter of this volume. Here we shall
mention merely a few of the most significant correlations.
WRIGHT (1967) studied the far infrared spectra between 80 and 340 cm-I of
45 musks and 15 chemicals which are structurally related to the musks although
they do not possess a musk odor, at any rate not at room temperature and not to
human subjects. He concluded that a necessary although not a sufficient condition
for the possession of a musk odor is a combination of three vibration frequencies
near 100, 160 and 260 cm-I . In addition to this, the molecule must have adequate
volatility, one or more functional groups to give it the correct orientation with
respect to the receptor surface, a minimum molecular size and certain other steric
characteristics. According to the reviewer this is rather a forced order of causality;
an odorant has certain frequencies in its spectrum as well as a musk odor and many
other physical, chemical and physiological properties because its molecules have
the steric and orientational characteristics mentioned by WRIGHT. This comment
is not given to criticize WRIGHT'S interesting work on odor-spectrum relations but
only to point out that the existence of such relations does not necessarily mean
that the frequencies shared by a group of odorants playa role in the interaction
mechanism of such odorants with the olfactory receptors.
The statistical significance of the correlations described by WRIGHT for the
class of musks is criticized in this volume by DAVIES who also pointed out that the
spectrum of only one of the eight macrocylic musks studied shows the character-
istic frequencies while one of these is lacking in the spectrum of the strong musk
ambrette. The latter argument does not entirely destroy the basis for WRIGHT'S
correlations since it could be interpreted to confirm that the olfactory response
patterns of nitro musks and macrocyclic musks are independent. As we have
discussed in the previous paragraphs this independence is also indicated by research

on specific anosmia and cross-adaptation.
WRIGHT (1966; WRIGHT and ROBSON, 1969) also studied the far infrared
spectra of nitrobenzene and benzaldehyde and their mono alkyl derivatives. The
odors of this series have been described by KLOUWEN and RuYS (1963) who found
an interesting transition from the bitter almond odor to the cuminic odor with
increasing size of the alkyl substituent. From the far infrared spectra of this col-
lection WRIGHT and ROBSON (1969) conclude that odorants possessing a bitter
almond odor show bands near 175, 225 and 345 cm-I while the cuminic odor is
associated with frequencies near 175, 265 and 310 cm-I . A peak near 395 cm-I is
shared by both classes.
The odor-structure relations in this group of compounds have been discussed
in paragraph E of this chapter.
Also the extensive research of DAVIES on the relations between the odor and
certain physical properties of the odorant has been closely associated from the
beginning (DAVIES, 1953) with a theory on the mechanism of odorant-receptor
interaction and stimulus formation. In this case theory and experimental work are
closely intertwined and both are discussed in extenso in DAVIES' chapter. Only a
few of the most characteristic results are mentioned here.
THEIMER and DAVIES (1967) studied a large number of musks belonging to
various structural classes, such as macrocyclics, isochromans and polycyclic
ketones and formulated two structural requirements for the musk odor.
The cross sectional area of the oriented molecule lies in the range 40-57 A2.
The ratio of the length of the oriented molecule to the breadth (LIB ratio) lies
in the range 2.8-3.3.
A molecular structure possessing these characteristics is, in principle, a musk
but its odor intensity may vary from zero upwards. Musk intensity is covered in
DAVIES' philosophy by a third, this time a physical, requirement.
The desorption rate from a water surface lies in the range 0.4-1.7.
Recently also the nitro musks were demonstrated by DAVIES (1968) to satisfy
the two structural criteria. Cross-sectional areas lie in the range 42-56 A2 and
LIB-ratios in the range 2.8-3.1. DAVIES observes that, since the LIB ratios are at
the lower end of the range found for macrocyclic, isochroman and polycyclic keto
musks, one might predict that the odor quality is not quite the same as for other
classes of musks. It should be noted that not less than five independent arguments
(anosmia, adaptation, 1.R.-spectra, intensity estimates and molecular dimensions)
have been found suggesting that the nitro musks and perhaps one or two other
groups occupy distinguishable sub-areas in the wide territory of the musk odor.
Also a steroid musk, 5 oc-androstene-16-01-3 oc, was studied by DAVIES (1968)
and found to have a cross-sectional area of 55 A2, an LIB ratio of 2.9 and a rate of
desorption of 0.43.
Similar studies have been carried out by DAVIES (1965) on the amber odor and
have led to tentative criteria for cross-sectional area and LIB-ratio. In this case the
picture is complicated by several factors. Although the number of amber odorants
available is fairly large, it is only a fraction of that in the musk field. The stereo-
chemistry of part of the ambers, all of which have a polycyclic structure, is still
Conclusions 313
unknown. Finally, the amber odor is less distinct than the musk odor and overlaps
strongly with other types such as the woody odor.
Finally an interesting approach should be mentioned reported by LAFFORT
(1963a) who calculated for the homologous series of alkanes, alkanols, ethyl esters
and fatty acids the molar concentration of the odorant in an aqueous phase in
equilibrium with an air phase carrying odorant molecules in threshold concen-
tration. This was done on the basis of critically evaluated literature data on thresh-
old values (LAFFORT, 1963b), water solubilities and vapor pressures at 37.
The negative logarithm of this concentration was found to be linearly related
to the logarithm of the molar volume at 20 which changes concurrently with the
chain length. The four regression lines found for the series gave correlation coef-
ficients in the range 0.95-0.98. The regression lines representing the three non-
ionising species seem to converge approximately in a single point.
LAFFORT developed an equation for the olfactory potential, i.e. the negative
log. of the molar threshold concentration, using the characteristics of the regres-
sion lines, the partition coefficient between water and air, the molar volume and
the dissociation constant. Between the (semi)-theoretical values obtained from
this equation and the experimental values a correlation coefficient of 0.97 was
found. The general validity of the equation was demonstrated by prediction of
threshold values for other homologous series on the basis of the known threshold
of a single representative. The excellent agreement reported for a few examples is
somewhat surprising in view of the rather heterogeneous literature data on which
this work rests and also because the threshold values of ethane thiol and diethyl-
sulfide were calculated from the known value for hydrogen sulfide which can hardly
be expected to behave as a lower homolog of the thiols and the thio ethers. Some
careful checking of the scope and reliability of this interesting method, starting
from reliable threshold data, would be desirable.
K. Conclusions
Most of the information on structure-response relations reviewed in this
chapter has been obtained as a by-product of research on olfaction. Consequently
it shows large gaps, often in areas where the most interesting data could be ex-
pected, and it presents the thinnest possible evidence for any concept we may
devise.
However, gaps as well as information have their own value. The former suggest
what should be done in the future. We shall revert to this later in this paragraph.
The information, thin though it may be, does not contradict and often supports
and adds to the tentative concepts from which we started. It seems to be consistent
with the following rough outline of olfactory interaction and discrimination.
The primary information pattern in olfaction is the result of physical inter-
action between a population of odorant molecules with a population of receptor
sites of molecular dimensions, followed by summation of the resulting energy
effects in the receptor cell.
The odorant molecules are presented to the receptor sites in a range of orien-
tations (and, for flexible molecules, conformations), which are distributed at
random over the olfactory epithelium.
The statistical composition of this range - the orientation pattern -, is mainly

determined by nature, position and environment of the functional group or groups.
The statistical composition of the orientation pattern and the steric character-
istics of the predominating oriented profile or profiles in this pattern are the major
criteria for the odor quality.
Characteristic, identifiable (primary, fundamental) odors are associated with
homogeneous orientation patterns in which one or a few orientations predominate,
with rigid molecular structures and with the presence of a single sterically accessible
functional group.
Non-characteristic, blurred odor types and lack of odor are associated with
random distribution of orientations. A random orientation pattern is closest
approximated by non-polar molecules and by molecules with sterically hindered
polar groups.
The influence of the functional group on the orientation pattern is determined
by direction and dimension of the molecular dipole and by its solvation tendency
(basicity). Further, its own steric requirements contribute to the molecular profile.
The parameters of the interaction between a receptor site and an odorant
molecule, defining the effectiveness of the interaction and its role in the primary
information pattern, are probably the closeness of fit, the life time of the inter-
action complex and its location on the epithelium.
Closeness of fit is determined by the steric characteristics of the accommodating
site and by the molecular profile (shape and size) of the molecule in the orientation
in which it is presented to the site.
The conformation of flexible molecules in the interaction complex is adapted to
the steric requirements of the accommodating site. Consequently such molecules
have a higher chance of interacting effectively with suitable sites than rigid mole-
cules but the composition of the information pattern tends to be more heterogeneous
for flexible than it is for rigid molecules.
For biochemical and other reasons we must assume the number of types of
receptor sites to be limited (rough estimate: 20-30).
The distribution of site types over the epithelium is not random, i.e. com-
binations of site types of different composition are arranged in zones or patterns
which are shading off into one another. This results in a primary information
pattern with topological characteristics. For the individual receptor cells, the
receptive membrane of each of which contains a number of sites, this means that
they differ in the nature and in the numerical ratio of their site types as well as in
their position on the membrane.
The structure of the information contained in the primary information pattern
seems to be identical with that in any fragment of this pattern. Only the resolution
of its details decreases with the dimensions of the fragment. Possible explanations
are that the pattern consists of a large number of areas with identical information
characteristics or that it has a holographic structure.
All primary information patterns are heterogeneous. Although one component
may strongly predominate, other components are invariably present in lower
frequencies. Consequently no odorants with different structures can exist which
elicit exactly identical response spectra in any part of the olfactory system.
Conclusions 315
Although the information bits generated in the molecular level probably do not
survive processing in the cell, the information elements originated in the receptor
cells retain their individuality up to the level of the olfactory bulb. This means that
also the topological information present in the primary information pattern is
delivered to the bulb. Although the details of the primary information pattern, its
topology and its shadings, are subjected to at least two strongly convergent pro-
cessing steps in the olfactory bulb, we may assume that all its essential information
is preserved in the olfactory code message which is presented to the higher centers
of the brain.
Information on response quality is mainly represented by the topological
characteristics of the primary information pattern, response intensity by the
shadings of its various zones.
Impulse frequency of receptor cells (shadings; intensity) and topological
structure (quality) suffice for the construction of a satisfactory, although specu-
lative model of olfactory discrimination. At this stage of our knowledge we are
unable to interpret other aspects such as amplitude and temporal differences
between responses satisfactorily in terms of odor quality and intensity. Also we do
not know whether the resting activity in the olfactory cells has a fundamental
significance for the olfactory information and consequently whether inhibition of
this activity forms an intrinsic component of the primary information pattern.
Some of the gaps in our knowledge of the olfactory process are the consequence
of the biological and technical complexity of the problem and of the low scientific
value of many of the early contributions. Others could have been avoided if the
various disciplines involved in olfactory research had started cooperating or, at
least, communicating at an earlier stage.
Especially the subject of this chapter could have benefited enormously by close
cooperation of the electrophysiologist, the psychologist and others with the chemist.
As it is, excellent chances to obtain a maximum of information from experimental
work have been lost by the inexpert selection and the poor quality of the stimulants.
Olfaction is the result of interaction between a chemical substrate and a
biological system. Like pharmacology and enzymology, olfactory research is
impeded by the complexity and by our imperfect understanding of the biological
system. However, it has some unique advantages over the other disciplines. The
choice of stimulants is almost unlimited. Their structures are perfectly known and
fairly simple since their molecular weights can hardly exceed 300. Also these
structures can be adapted to the requirements of the intended experiment. In
order to make full use of these advantages, stimulant structures should be planned
as carefully and as expertly as other aspects of the experiment. Any team involved
in olfactory research should have available the expert knowledge of the organic
chemist, facilities for organic synthesis and modern purification techniques.
In all experiments involving stimulant concentration, the physical chemist
should be consulted. Excellent experimental work has been wasted and question-
able conclusions have appeared in the literature because concentration aspects
were imperfectly understood.
Future research on olfactory response-structure relations requires painstaking
purification of stimulants, using modern techniques. Where possible the use of
mixtures of stereomers should be a voided; stimulants should be stereo chemically
pure and their stereochemistry should be known. In planning the structures of

stimulants, profile similarity as well as systematic profile differences should be
taken into account.
According to the author the following aspects require increased attention since
they offer the most favorable chances of success in the area of structure-response
relations as well as in olfactory research in general.
The development of objective methods for the expression of oriented profile
parameters in numerical values by means of computer techniques.
Resolution of large numbers of racemic odorants of varying structures into
chemically and optically pure enantiomers and the measurement of olfactory
responses by electrophysiological and psychological methods.
Measurement of electrophysiological response spectra of large numbers of units
in the epithelium, the nerve and the bulb of a single species by stimulation with
carefully selected series of odorants and research on correlations between profile
similarities and response spectrum similarities for pairs of odorants.
Research on threshold values and on correlations between these values and
structure parameters, physical properties and odor category (primary class).
Development of practical methods based on cross-adaptation, similarity
estimates, intensity estimates and confusibility and research on correlation of these
data with profile similarities.
Continued research on specific anosmia as the basis of a classification of types
of mono-osmatic information.
Acknowledgement
I thank Dr. K. B. DaVING, Oslo and Dr. J. T. DAVIES, Birmingham for reading part of
the manuscript and for valuable suggestions.
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21 Rh. Sensory Physiology, Vol. IV/1

Chapter 13
OHactory Theories
By
J. T. DAVIES*, Birmingham (Great Britain)
With 10 Figures
Contents
Introduction . . . . . . . . . . . 322
Stimulation According to the "Penetration and Puncturing" Theory . 323
Mathematical Model . . . . . . . . . . . 327
Predictions from the Theory. . . . . . . . . . . . . . . 328
Comparison with Transduces for Other Senses . . . . . . . 330
Stimulation Related to Vibrations and/or the Olfactory Pigment 331
Stimulation Theories Involving Enzymes and Specific Sites. . . 334
Odor Type According to the "Penetration and Puncturing" Theory 335
Odor and Desorption Rates . . . . . . . . . . . . . 337
Intensity in Relation to Concentration of a Given Odorant 341
Discrimination over Large Concentration Ranges. . . . . 342
Odor Type According to the Molecular Vibration Theory 342
Odor Type and "Primary Odors" According to the "Specific Site" Theory 344
References . . . . . . . . . . . 347
Introduction
Almost exactly 2000 years ago, LUCRETIUS (27. B.C.), tried to unify and explain
the concepts of heat, light and odor by an atomic theory. He wrote, "You cannot
suppose that atoms of the same shape are entering our nostrils when stinking
corpses are roasting as when the stage is freshly sprinkled with saffron of Cilicia
and a nearby altar exhales the perfumes of the Orient ... You may readily infer
that such substances as agreeably titillate the senses are composed of smooth
round atoms. Those that seem bitter and harsh are more tightly compacted of
hooked particles and accordingly tear their way into our senses and rend our
bodies by their inroads."
Thus over the centuries have investigators been puzzled by the extraordinary
sensitivity of the sensory nerves of living creatures. The human sensory systems,
for example, are sensitive to as little light as a few quanta (Le., a total energy of
about 10-11 erg), to sound waves of amplitude of the order of atomic dimensions,
* Formerly Beit Fellow for Medical Research.
Stimulation According to the "Penetration and Puncturing" Theory 323
and to quantities of odorous substances (called odorants) as low as 10-14 gram. The
latter quantity, although it still contains a very large number of molecules, is far
below the level of the best analytical balances. This astonishing sensitivity of the nose
cannot be understood until we know the detailed mechanisms by which the primary
impulses (the quanta of light, the wavelike vibrations of the sound and the adsorp-
tion of the molecules of the odorant, etc.) are converted into the electrical impulses
which constitute the excitation of the nerve. A device for the conversion of light,
etc., into an electrical (or other) impulse is known as a transducer, and these biolo-
gical sensory transducers are characterized by their extremely sensitive built-in am-
plifiers, which increase the energy of the input signal by a factor of several thousand
times. This is rather like the firing of a gun by a relatively small "triggering" action.
For many years it was believed that the chemical senses, smell and taste, were
among the more complicated. Certainly their behaviour had long defined numerical
analysis, while studies of light and sound have been more readily amenable to
quantitative investigations. One of the most striking facts about the stimulation
of the olfactory nerve fibres is the excessive sensitivity of the olfactory cells. Even
in man, whose sense organs are not particularly sensitive, quantities of certain
odorants (e.g., {3-ionone) as small as 10 8 molecules are normally detected, while for
moths the quantities are claimed to be much smaller. How is it, we may ask, that
such minute, unweighable quantities, containing a calculated 10-14 gram, can
affect the nerves ~ Direct experiments are urgently required, yet the receptor sites
on the ends of the olfactory nerves are too small to study directly, the result being
that several different theories of olfactory stimulation are current at the present
time (1968). Any theory of odor should explain the sensitivity of the nerves to
odorants, the enormous range of olfactory thresholds found (over a range of nearly
1010 times between large and small molecules!) and the very high discriminating
power of the nose to the odors of different quality.
The minute quantities of odorant necessary for stimulation have been mention-
ed above. These figures apply only to the most powerful odorific substances such
as musk, artificial musk (trinitro-tert-butyl-xylene), and {3-ionone. The answer to
the problems of the sensitivity of the detectors must lie partly in the structure of
the molecules causing the nervous excitation. What do these three types of mole-
cule have in common that makes them so peculiarly effective ~ They are, of course,
all sufficiently volatile for molecules to reach the receptors via the air. But, apart
from that, their common property is that the molecules are all bulky and fairly
strongly adsorbed at interfaces.
Olfactory theories are as numerous as pebbles on the beach. In the space
available here we have chosen to concentrate attention upon a few recent theories.
First, we shall discuss them as they apply to the stimulation of the sensory nerves,
and then in relation to the type or quality of the odor (e.g., fruity, floral or musky).
Stimulation According
to the "Penetration and Puncturing" Theory
(DAVIES, 1953a, b)
During the excitation of the giant axon of the squid, it was shown (HODGKIN
and KATZ, 1949) that the normal excess concentration of potassium ion inside the
21*
324 J. T. DAVIES: OHactory Theories
nerve cell is able to escape through the lipid (fatty) plasma membrane which
bounds the nerve cell. Simultaneously, the deficiency of sodium ion within the
nerve is made up locally by an intake of sodium from the higher concentration in
the aqueous phase surrounding the cell. During its resting period the nerve cell
somehow "pumps" sodium out of the cell and allows in excess potassium, both
processes occurring against the concentration gradients.
This excess concentration of potassium ion inside the cells and the consequent
electrical potential may be likened to the excess air inside a balloon: energy is
stored in the system by the inequality of concentrations, and this energy is released
upon "puncturing" the wall. Through the wall of the nerve cell, ions then exchange,
and the electrical potentials inside and out are momentarily equalized with a
release of energy. Just as when a balloon is pricked with a pin the energy released
breaks the wall farther along, so in some way the stimulation of one part of the
nerve wall is able to transfer the breakdown of the fatty lipid layer to a point
farther along the fibre. This progressive breakdown of the cell wall constitutes the
nerve impulse.
Recent work (TAKAGI et al., 1966) suggests that the process of depolarization
of the olfactory membrane depends primarily on an influx of chloride ion. Potas-
sium ions may also contribute to this process. Studies of a series of anions showed
that only F', Cl', Br' and HCO' 2 ions can penetrate olfactory membranes. Ions
such as I' or CIO'3' which are larger (in the unhydrated state) cannot pass across
the membrane (when the latter is stimulated by chloroform vapour). In their fully
hydrated states, the size order of these ions is HCO' 2 > F' > ClO'3 > Cl' = I' > Br',
so that on the assumption that punctures in the membrane act as sieves, the
anions which pass through cannot be fully hydrated. The relative sizes of the
naked ions are: CIO'3 = I' > HCO' 2 > Br' > Cl' > F' consistent with a hole or
sieve hypothesis, though it seems unlikely that such strongly hydrated ions are
completely dehydrated at the receptive membrane before they pass through it.
To the present writer it seems reasonable that the anions should lose some of
their water of hydration before passing through a hole in the membrane. Indeed,
to minimise the energy involved when the ions pass through a predominantly
hydrocarbon environment, they may well lose all their outer layers of water of
hydration, keeping only a strongly held single layer of water molecules arranged
around each ion. In this way the observed size sequence is explicable, since the
naked ion radii are all increased by the diameter of two water molecules.
The lipid layer in most cells is not thick. Even bimolecular "sandwiches" of
the type shown in Fig. 1 would be enough to confer, under normal circumstances,
the necessary impermeability to the cell wall and maintain the concentration
gradients. Such a layer preserves its continuity by being partly liquid. The hydro-
carbon chains of the fatty molecules can move about with a certain degree of
freedom, although this will be limited by the presence of rigid molecules such as
cholesterol.
Suppose that into this membrane there penetrates a bulky, awkward-shaped
and rather rigid odorant molecule (Fig. 2a). This may either desorb again out of
the membrane, or it may diffuse through (Fig. 2b). If the latter occurs, the hole
left behind the diffusing odorant molecule may heal only relatively showly, so that
ions can pass through the hole. Once this leakage occurs, it initiates (as is generally
Stimulation According to the "Penetration and Puncturing" Theory 325
accepted) the nervous impulse in the nerve. This puncturing theory (DAVIES,
1953a, b) is the modern counterpart of the atoms of LUCRETIUS, "tearing their
way into our senses and rending our bodies by their inroads."
Outer fluid, high Na+
nnll\ ~nl1
Protein
lUll) i11111Inner fluid, high K+
Fig. 1. Mosaic representation of cell membrane. The protein (enzymic) parts of the surface act
as sodium pumps, while the lipid parts are normally impermeable to ions. Hydrocarbon chains
represented by - ; polar groups by .; and the protein backbone by a spiral. The idea of the
biomolecular nature of the lipid part of the membrane was due originally to DAVSON and
DANIELLl (1943) and DANIELL! (1958)
Outer Channel left behind

aqueous phase diffusing odorant moleculel
Inner fluid, high K+ Inner aqueous phase

a b
Fig. 2. In a an organic odorant molecule is adsorbed, penetrating a lipid region of the cell wall.
In b, the odorant molecule is shown diffusing rapidly through the biomolecular lipid membrane,
with a sharp hole being left behind it. Through this channel ions may then exchange, initiating
the generator current which in turn releases the nervous impulse (DAVIES, 1968)
Recent work has shown that the olfactory nerve impulses as such do not begin
in the cilia or even at the tips of the olfactory receptors to which they are attached.
The neural impulses originate where the olfactory receptor narrows to the olfactory
fiber. Presumably, just as with the better studied mechano-receptors (LOEWEN-
STEIN, 1959, 1961), the tips and cilia of the receptor cell become more permeable
to ions. This would occur where the odorant molecules penetrate, and the resulting
increased permeability would partially short-circuit the membrane resting poten-
tial, setting up a generator current. Such currents do not themselves travel right
along the nerve fiber, but serve merely to trigger the nerve impulses (which start
at the first node of RA..~VIER in the mechanoreceptors, the frequency of these nerve
326 J. T. DAVIES: Olfactory Theories
impulses depending on the intensity of the generator current, i.e., on the number
of channels formed in any given membrane).
If the puncturing mechanism is realistic, we should expect some correlation
between the numbers of odorant molecules necessary for threshold stimulation and
the number of cells lining the olfactory epithelium in the nose. The latter number
about 2 x 107 in the human species, a figure in remarkable agreement with the
value of 10 8 molecules for threshold stimulation by large molecules such as {J-
ionone. This suggests that for these, the most effective of odorific substances, only
a few molecules (and perhaps only one) of the odorant are sufficient to cause
breakdown of certain cell walls.
For less powerful odorants such as ether or chlorophenol (whose molecules are
smaller), it may be necessary for several of the odorant molecules to penetrate the
cell wall side by side. This would explain the enormous differences in sensitivity to
molecules only slightly different in solubility and chemical properties; the shape,
size and rigidity are important. For example, if two molecules of odorant have to
penetrate the cell wall at the same point before stimulation occurs, the concen-
tration of odorant required must, by probability theory, be greater by several
powers of 10 than if only one molecule were required. The known million-fold
differences in sensitivity of the cell wall to (say) phenol and synthetic (xylene)
musk are thus explicable, as is the experimental finding that the olfactory thresh-
old (molecules of odorant per cubic centimetre of air, required for the odor to be
just detectable) of ethane is about 1010 times greater than the threshold for a
strong odorant such as {J-ionone.
It is not necessary, of course, that all the olfactory nerves should be stimulated
before an odor is perceived. It may happen (and this seems to be true for moths)
that only a few fibres need stimulation before the smell is detected (BOECKH et al.,
1965), whereas humans need a large fraction of ours to be affected. Perhaps this
greater amplification within the central nervous system explains also the high
olfactory sensitivity of dogs. The latter, however, have also a great total area of
the olfactory epithelium, up to 150 cm2, compared with only about 5 cm2 in man.
On this view that large organic odorant molecules puncture the lipid part of the
cell membrane, the mechanism of odor is different from that of taste. Recent work
suggests that adsorption on to specific protein groups in the taste buds is reponsible
for bitter and sweet tastes, whereas for odor the theory involves a rather non-
specific adsorption into the lipid part of the membrane. Perhaps this difference
accounts for the known differences between taste and smell: the more lipid-soluble
materials have an aroma (or a "flavor" which is lost if we have a heavy cold), while
many water-soluble materials (mineral acids, salts and sugars, for example) have
no appreciable odor. Others such as ammonia, H 2S, S02' etc. do have strong odors
presumably originating in the enzymic and proteinaceous parts of the receptor
membranes (Fig. 1). Such molecules, particularly sulfides, may well poison the
sodium pumps, resulting in back-flow of ions (just as there is a back-flow of water
when a centrifugal pump is stopped). One might also say that the odors of the
larger organic molecules could be correlated with the OVERTON-MEYER-COLLANDER
theory of penetration rates through the plasma membrane and of narcosis. The
effects of the more oil-soluble molecules depend on their oil solubility. Small polar
Mathematical Model 327
molecules, such as salts and urea, apparently permeate cell membranes VIa
channels in proteinaceous parts of the surface.
Mathematical Model
To place the "puncturing" theory of odor on a quantitative basis, DAVIES and
TAYLOR (1957) and DAVIES (1962) assume that two factors control the extent of
dislocation of the nerve membranes. The first is the adsorption energy of the odorant
molecules from the air to the lipid-water interface, constituted by the lipid-wall of
the hairs (which form the sensitive tips of the olfactory nerves) and the thin layer
of mucus in which they are bathed. This adsorption may be calculated from the
free energies of the odorant molecules in passing first from air to water and,
secondly, being absorbed from water at the lipid-water interface. For stimulation,
these adsorbed molecules must be effective in "puncturing" the olfactory nerve
membrane at some point, either singly or with several adsorbed close together.
From these considerations DAVIES and TAYLOR derived a mathematical equation
which predicts olfactory thresholds in terms of the molecular sizes and adsorption
energies of the odorant molecules. The appropriate adsorption energies can be
approximated from laboratory experiments on model systems, so that olfactory
thresholds can be predicted and compared with experiment.
The theory assumes (DAVIES, 1953a, b) that stimulus is initiated in an olfactory
nerve ending only when and where a critical number of odorant molecules is con-
centrated within a small region of the olfactory cell wall. Fewer odorant molecules
than this critical number will fail to puncture the wall. Whereas for very effective
molecules such as fJ-ionone (threshold only 1.6 x 10 8 molecules per cm3 ) very few
(perhaps only one) adsorbed molecules are necessary to cause the puncture, less
effective molecules such as phenol or ethanol stimulate only when several adsorbed
odorant molecules happen to be adsorbed adjacent to one another simultaneously.
In general this critical number of adsorbed molecules is designated p, and from
the theory below it appears that p lies in the range 1 to 13. The mean number of
molecules of odorant adsorbed per cm2 of surface is designated x; c represents the
concentration of the odorant in the air, and d represents the thickness of that part
of the membrane penetrated by the odorant molecules. In general c is rather small,
and the average adsorption x is also small; one can then write:
x = cdKLA (1)
where K LA is the adsorption coefficient for the odorant passing from air to the
mucus-lipid interface at the cell wall.
Now let n be the total number of "small regions" on each cell surface within
which the p molecules must be located to cause a puncture of the cell wall. Further,
let the area of each of these "small regions" (or non-specific sites) be IX cm 2 (see
Fig. 3), so that the average number of molecules adsorbed per site is x IX. How many
of the sites will contain the requisite p adsorbed molecules ~
To answer this question we have recourse to probability theory. The number N
of sites per cell, each containing p molecules, is given by the Poisson equation:
N = n(lXx)Pe-a."'jp! (2)
or, in logarithmic form,
log N = log n + p log IX + p log x - IXxj2.3 - log p ! (3)
Substituting for x from Eq. (1) and rearranging we obtain

log c + log KLA = (lip) 10gN - (lip) logn -log ~d + ~cd K LA12.3p + (lip) logp!
(4)
Fortunately, this equation simplifies considerably. At the olfactory threshold
concentration (0. T.) we assume there is just one puncture per cell, so putting
n sites,each of ex cm 2
Fig. 3. Diagrammatic representation of cell wall of olfactory cell, of thickness d em, and con-
taining n sensitive regions (or "sites"), each of area ex cm 2 On to the cell membrane, odorant
molecules (M) are adsorbed, to a mean coverage of x molecules per cm 2 According to the
"penetration and puncturing" theory, stimulation just occurs (i.e. at threshold) when
on one "site" (N = 1) there happen to be concentrated the requisite p molecules to cause
"puncturing." In this diagram, p is taken as 3
c = O. T. we have N = l. Further, the last-but-one term of Eq. (4) is found to be

always small (less than 0.001) and may be neglected.
Hence,
log O. T. + log K LA = - (lip) log n - log ~d + (lip) log p ! (5)
For p-ionone we take p = 1, and substituting log O. T. = 8.2 (the experimental
figure) and log KLA = 8.3 (from model systems in the laboratory), we find that
log nad = - 16.5. Further, for very weak odorants, the quantity (log O.T.
+ log K LA) is found in practice to approach a value of about 22. Under these con-
ditions p is relatively large, and (lip) log p! is about 1, so that log rxd = - 21.
Since log n~d = - 16.5, we see that log n = - 4.5 (a more elaborate treatment
gives - 4.64).
Predictions from the Theory

These theoretical results can now be compared with what is known of the
structure of the olfactory cells. Taking d = 10-7 cm, we have log n~ = - 16.5
+ 7 = - 9.5, i. e., ~n, the total active area of one olfactory cell, is calculated to be
about 3 x 10-10 cm 2 Though this is obviously only an order of magnitude, it is
clearly much smaller than the total area of a simple olfactory cell (perhaps 10-7 cm 2),
indicating that only certain patches on the cell surface have thin enough walls to
be easily punctured, i.e., to be regarded as "sites" in the present theory. The same
concept of "sites", i.e., of regions of the cell surface sensitive to activation, has
Predictions from the Theory 329
also been postulated (LOEWENSTEIN, 1959) to explain the generator potentials in

mechano-receptors; his use of the word "site" is identical with ours.
From log n = - 4.6 we deduce that there are 44,000 thin sites on each cell, and
from log IXd = - 21 (or more accurately - 21.2) we deduce that log IX = - 14.2,
i.e., the area IX of each "puncture area" is 64 X 10-16 cm2, i.e., 64 A2.
To predict olfactory thresholds, we use Eq. (5) with numerical substitution as
follows:
log O. T. = 21.2 -log KLA + {(lIp) log p! - 4.64Ip} . (6)
This treatment makes possible an understanding of the enormous range of
olfactory thresholds. This range is about 1010 times between a strong odorant such
as fJ-ionone and a very weak odorant such as methane. Of this factor about 105 can
be accounted for by difference in adsorption coefficients K LA of the molecules, and
about 105 times from the p factor [bracketed term in Eq. (6)]. The range of thresh-
olds is so great because both terms vary in the same way. Large molecules are both
more strongly adsorbed and also are more efficient at puncturing the membrane
than are small molecules.
From Eq. (6), making the further assumption that for any odorant molecule
IIp (i.e., its "puncturing power") is a linear function of its cross-sectional area A
when oriented in the membrane, one obtains
IIp = AI47 - 0.22 . (7)
The constants in this equation have been derived by taking p = 1 for fJ-ionone
(A = 58 A2) and p = 00 for water. The figure of 58 A2 is in fair agreement, inci-
dentally, with the 64 A2 quoted above (from an independent line of argument) for
the "site" area. Typical values of pare 1 for fJ-ionone, 1 or 2 for musks, 2 to 3 for
esters, 4 for butanol, 8 for ethanol and about 13 for methanol.
/..
8
9
.
10
"0
II> o .(
> 11
.~

II>
III
.0 12 o
0
13

III
c
I-'
14
d 15
Cl
.9

16
17
18
8
log O. T. (as calculated I
Fig. 4. Measured oHactory thresholds for humans are compared with those calculated from
Eq. (6) and (7). Open circles refer to normal alcohols, triangles to n.hydrocarbons, and closed
circles to other organic compounds of various types (DAVIES and TAYLOR, 1959)
We can now predict oHactory thresholds from Eq. (6) using laboratory in vitro
measurements of KLA and the value of p from Eq. (7). As shown in Fig. 4, there is
fair agreement with the measured thresholds, at least to an order of magnitude. In
particular the enormous range of the thresholds is explained by the theory. For a
group of rather similar molecules, p remains about the same (e.g., for musks p is
about 1.3), and so by Eq. (6) the oHactory thresholds will be lower the more
strongly adsorbed is the molecule. This is, however, not true of its strength as a
musk (see below).
Comparison with Transducers for Other Senses

Various extensions of the "puncturing" theory of oHactory stimulation have
recently been proposed for the stimulation of other sensory nerves, thus unifying the
primary mechanisms concerned. The "puncturing" theory explains both the
"triggering" mechanism and the release of more energy than is present in the input
signal.
In the light receptors of the eye, rhodopsin absorbs light with a change in
molecular shape of its chromophore (ll-cis retinene) to a more linear molecule (cis-
trans isomerization). Since the transverse nerve membranes are constituted in a
large part of visual pigment, WALD et al. (1962) suggested that " ... the attack of
the light on a molecule of visual pigment might in effect punch a unimolecular hole
in such a membrane. That might permit a large flow of ions, resulting in a local
depolarization or loss of resistance sufficient to excite." An interesting analogy
may also be drawn with the behaviour of monomolecular films. More linear mole-
cules (trans configuration) are well known to fit together much more snugly and
occupy less of the surface area. Consequently, holes could well be produced in the
membrane by such a change of packing under the influence of light.
The transducer action by which sound vibrations are converted into electrical
nerve impulses is also extremely efficient. Though amplitudes of as little as 10-11 cm
are reported (BEKESY, 1962) to be detectable by the ear, this figure seems to be
suspiciously low, as it is well below the size of a single atom. However, even if we
take a figure of 10-7 cm as reasonably well established, the problem still arises of
how a minute displacement can produce triggering of the nerve endings of the ear.
BEKESY'S suggestion of the mechanism again envisages a puncturing process;
" ... varying the free space between the molecules in a molecular layer may change
the equilibrium of flow of some ions - mainly the sodium and potassium ions -
across the molecular membrane, thus producing a change in the concentration
potential across both sides of the membrane ... The energy of the potentials would
not be obtained from the mechanical energy of the vibration but rather from an
electrolytic pool." Again, there is a close analogy with the behaviour of certain
monomolecular films. Orientated, close-packed molecules of hexadecanol can set
up a considerable barrier to the evaporation of water from a clean water surface
into air (reviewed in DAVIES and RIDEAL, 1963). The magnitude of the barrier is,
however, highly sensitive to the area occupied by each molecule in the film, and a
very slight expansion of the film allows water to pass through. The analogy with
the biological membranes is obvious.
Mechano-receptors, sensitive to mechanical pressure, may well function in the
same way; the increase in permeability leading to the generator current could
result a simple stretching of the receptor membrane (IsHIKo and LOEWENSTEIN,

1961; INMAN, 1962). Further, the temperature coefficient shows an energy barrier
of 16,000 calories per mole to ionic transfer. This is comparable with the 14,500
calories per mole for the diffusion of water through a fatty acid monolayer. The
so-called "sites" at which electrical generator currents originate in mechano-
receptors (LOEWENSTEIN, 1959, 1961) are analogous to those in the puncturing
theory of olfaction.
Temperature receptors in animals, it has been suggested (MURRAY, 1962), may
also operate through a change in the permeability of the nerve endings.
In olfaction, for which the "puncturing" mechanism of nervous stimulation
was first put forward (DAVIES, 1953a, b), monomolecular films again provide a
close analogy. In a hexadecanol film which would otherwise greatly inhibit the
evaporation of water, if there is a very small amount of benzene this drastically
reduces the barrier to the passage of water. Presumably, by rapid interchange
between dissolved benzene below the film and benzene molecules in the gas above,
holes are formed in the hexadecanol monolayer, and through these holes water can
easily escape. Here the parallel with the escape of ions in the "puncturing" theory
of nervous stimulation is again clear.
Stimulation Related to Vibrations and/or the Olfactory Pigment

The surface of the olfactory area is yellow, due to the presence of pigment. As
long ago as 1870, OGLE speculated that this pigment (situated in cells adjacent to
the nerve structures themselves) might be able to absorb the "vibrations of odor,"
affecting the contiguous cells in which the olfactory nerves end. "The pigment which
is so constant in the olfactory region is there to absorb the odorous emanations."
A different vibration theory, due to RANDEBROCK (1968), is that " ... most
probably the perceptors are composed of peptides, which have ample capacity for
forming hydrogen bonds" with the odorant molecules. It is assumed that an oc-
helix is the sole perceptor for odor; that the peptide chains in the oc-helix are
vibrating, and that a single odorant molecule attaching itself to one end of one of
the peptide chains modulates this vibration. The modified vibration is then trans-
ferred to a nerve cell by the other end of the helix.
In another form, as proposed by HAMILTON WRIGHT (1954, 1964, 1966)
and DEMERDACHE and WRIGHT (1967), the vibration theory supposes that
"triggering" of the nervous impulse arises from molecular vibrations: "We picture
the out-of-plane movements of the adsorbed odorous molecule as conforming with
corresponding out-of-plane movements of the sensitive surface. To conform in this
way, the two molecules must have nearly the same frequencies in nearly the same
place, so that the net effect depends both on shape and on frequency ... The
synchronous 'throbbing' of the odorous molecules and the receptor surface over
a considerable area would permit of a much closer approach than would otherwise
be possible, and hence, allow a closer than usual electronic (vander Waals) bonding
between them. This may be an important hint as to the primary event that
initiates an olfactory sensation." Thus, the odorant by its interaction with the
sensory surface (possibly the pigment) merely modifies the electroneural activity,
which receives its energy from some independent metabolic mechanism.
WRIGHT et al. (1967) have extended this idea, postulating that" ... a particular
vibration frequency in the molecule enables it to 'fire' one kind of receptor but
not another, and the receptors use a frequency coding of a very different sort to
distinguish their signals from those of receptors of other types." They relate
molecular vibration frequencies to the electrophysiological frequencies measured
in the olfactory bulb. The latter frequencies are, for example, mainly in the range
37-75 sec-1 for floral odors of high molecular weight, and mainly in the range
75-125 sec-1 for camphoraceous compounds of lower molecular weight (HUGHES
and HENDRIX, 1967). "Although each odor seems to be associated with a distinctive
major frequency component, various common components have been noted in the
responses to odors in the same stereochemical category. This finding would seem to
provide neurophysiological evidence for AMOORE'S stereochemical theory of
olfaction" (HUGHES et al., 1968).
However, WRIGHT et al. now claim that the relation of bulb frequencies to odor
type supports the molecular vibration theory of olfaction. They plot the electro-
physiological frequencies against the far infra-red frequency of odorant molecules
as in Fig. 5, admitting to " ... a small adjustment in the middle range ... ," but
100.----------------, C = citral
..............( B E = eugenol
A =anisole EU = eucalyptol
~ 90-/ ' \
B B = benzaldehyde F = fenchone
I = indole
80 - L = hmonene
M = methyl saticylate
:l: B ME = menthol
~ 70- ./\ B P = phenylacetic acid
o 60-
!
C
8
50 - A/~BB
A \.. . / B BB
iJ 40 - A -'4 _ . /B
f~
30 -
Ar)'?~
A A A /
Stimulus frf'quency (((j2 Hertz)
Fig. 5. "Best curve" of WRIGHT'S (1968a) claim of correlation of frequency components in

olfactory bulb in human subjects, plotted against the infrared frequencies of the odorant
molecules. The subjects were stimulated by (left) anisole and benzaldehyde; additional
compounds have been included (right). Reproduced by permission of Science Journal
claiming that for benzaldehyde and anisole on a human subject" ... most of the
molecular vibration frequencies can be correlated systematically with frequency
components in the discharge from the olfactory bulb [although] as pointed out
elsewhere, not all molecular vibrations are necessarily osmically active." They do
not claim, however, any direct mechanical coupling between the infra-red vibra-
tions of the odorous molecules and the frequencies of discharges which can be
measured in the olfactory bulb.
The present writer has checked the significance of the correlation of Fig. 5 by
taking 12 random numbers, setting these in ascending order and plotting them
against another 12 random numbers set in decreasing numerical values. This was
done twice, and the resulting curves (Fig. 6) clearly show a general resemblance to
WRIGHT'S plot (Fig. 5). Moreover, after a little selection and a small adjustment in
the middle range, quite a good "correlation" of the type obtained by WRIGHT could
100..-----------,
-\., .~
-\ .
'\
50 .\ ~\
.~. .\--..,
, ,
-.
, , : , ,
""
I -~.~
a 50 100 a 50 100
Fig. 6. Present author's plots of 12 random numbers (arranged in ascending order) against an
additional 12 random numbers (arranged in decreasing order). This has been carried out
twice, giving the two plots shown. They show a close resemblance to WRIGHT'S results in
Fig. 5. With some adjustment in the middle range, and a little selection, good "correlations"
of quite random numbers can clearly be achieved
obviously be obtained from these sets of random numbers. Consequently, the

reviewer is not convinced that the data of WRIGHT et al. support his conclusion that
there is a significant correlation of molecular vibration frequencies with those
measured in the olfactory bulb. In answer to this criticism, WRIGHT (1968 b) points
out that his attempted correlation " . .. was not intended to be a definitive state-
ment of fact but rather a challenging hypothesis worthy of (and capable of) further
investigation." He continues, "within the last few months we have made HUGHES-
type recordings from insect brains, constructed an infra-red correlation, and used it
to predict a combination of chemicals that should elicit a designated behavioral
response from intact insects - and it did."
That the olfactory pigment contains carotenes which playa dominant role in
stimulation was proposed by BRIGGS and DUNCAN (1961, 1962), who found ca-
rotenes in the olfactory region of freshly killed cows. Dogs also have carotenes in
the olfactory regions. Most of the pigment is present in the supporting cells and on
Bowman's glands. Whether physiologically significant amounts of pigment are
present in the olfactory receptors is still not known. BRIGGS and DUNCAN theorize
that while the mechanism of interaction with odorants is obscure" ... the known
ability of carotenoids to exist in numerous cis-trans forms (together with the
knowledge that the only action of light on rhodopsins is to induce a geometrical
isomerization) suggest that the exchange of energy from an olfactant molecule to

an olfactory protein-bound carotenoid involves a similar molecular change."
However, rabbits, sheep, rats and pigs apparently have no carotenoids in their
olfactory epithelia, and this (and other) reasons led MOULTON (1962) to challenge
the view of BRIGGS and DUNCAN. The latter replied that the analytical techniques
for detecting carotenoids may have been not sensitive enough to show carotenoids
from these animals.
Another carotenoid pigment theory has been proposed by ROSENBERG et al.
(1968), who envisage the formation of a weak bond or "complex" between the
odorant molecules and certain carotenoid pigments in the olfactory mucosa, giving
rise to an enhanced electrical conductivity of the pigments. The latter are sug-
gested to be incorporated to some degree into the resistance-controlling membrane
of the receptor cells. H such an increase of electrical conductivity occurred, it could
constitute an amplifying process leading to depolarization of the membrane and
signal generation. Experiments in vitro on solid crystallites of fJ-carotene showed
that the surface conductivity was increased by several powers of 10 in the presence
of oxygen and isopropanol, and particularly strongly (by factor X 105) for ethanol,
and (x 106 ) for methanol. Esters had but little effect. On the other hand, crystals
of vitamin A alcohol gave increases of surface conductance of 103 or 104 times in
the presence of ester vapors. A range of pigments, these authors suggest, could be
responsible for odor quality sensations.
Stimulation Theories Involving Enzymes and Specific Sites

It is quite widely believed that enzymes (constituting specific "sites" for
particular odors) playa direct part in olfactory perception, by a reversible adsorp-
tion upon them of odorant molecules. KISTIAKOWSKI (1950) assumed that various
different enzymes were involved in the sensory nerves, related to a number of
basic ("primary") odors. The action of the odorant is simply to inhibit one or
more of these enzymes, leading to a metabolic change which initiates the nervous
impulse. BARADI and BOURNE (1951) showed by staining methods that enzymes
were localized in the nasal mucosa of the rabbit, different enzymes being found in
different areas.
ASH (1968) has recently prepared a water-soluble fraction of proteins, taken
from scrapings from the nasal septum of the rabbit, and thus including water
soluble components of the mucus, the olfactory bipolar celis, the sustentacular cells
and the basal cells. This aqueous extract was mixed with aqueous solutions of the
odorants linalool or linalyl isobutyrate or both, this mixing resulting in a decrease
in the absorbance at 267 nanometers. This change ASH ascribes to a complex that
involves the odorant and the olfactory extract, with a conformational change that
might serve as the trigger to incite the change in membrane permeability needed
to institute the chemical sensing.
However, the change in absorbance at 267 mm is not unique to the olfactory
preparations: a brain tissue extract gave 41 %, and a liver extract 7 %, of the
change exhibited by the olfactory extract. Clearly further work with fractions taken
from only one source in the olfactory epithelium would be of the greatest interest.
Odor Type According to the "Penetration and Puncturing" Theory 335
MONCRIEFF (1967), however, wrote that the evidence in favour of enzyme

mechanism of olfactory stimulation is much less impressive than that afforded for
the taste sense, and DRAVNIEKS (1966) writes. "There are many valid arguments
against enzyme systems as the primary sensing elements. Molecules with similar
shape but totally different functionalities may have the same odor. The electro-
physiological response to an odorant is relatively independent of ionic strength and
pH in the olfactory region, whereas enzyme systems are usually sensitive to
changes in chemistry and pH. A large number of odor types would require many
enzyme systems, which seems unlikely."
Odor Type According

to the "Penetration and Puncturing" Theory
The "penetration and puncturing" theory postulates that the diffusing mole-
cules of odorant, after being adsorbed, cause a hole to be formed in the lipid
membranes of the olfactory nerve cells through which ionic flow may then occur,
this process initiating the nervous stimulation, as in Fig. 2. But whether the chan-
nels will persist long enough behind the diffusing odorant molecules for this ionic
exchange to occur must depend on the ratio of the diffusion rate of the odorant
molecule through the membrane to the rate of healing of the lipid cell membranes
in different parts of the olfactory epithelium.
In practice, not all types of molecules will be equally effective in leaving a
sharply defined hole in certain membranes, and so the quality of the odor must
depend on the rate of diffusion of the musk odorant through the cell membrane
relative to the subsequent rate of healing of the appropriate membrane (DAVIES,
1965); i.e. the intensity of odor must depend on a term such as {I - tdiff./th} where
t diff. is the time for diffusion of the odorant molecule through a certain membrane
and th is the time of healing of that membrane. Typical values for t diff. might be
10-6 sec for ether and 10-2 to 10-1 sec for a macro cyclic musk, but exact values are
not required at this point. If for a given odorant and membrane the time for dif-
fusion is small compared with the time for healing of the membrane, a sharp hole
will be left after the odorant molecule has diffused through the membrane. Through
this hole ionic flow may then occur, so initiating the nervous impulse. Under these
circumstances, the bracketed term above (called the "hole sharpness factor") tends
to unity.
But if the odorant molecule is larger or more strongly adsorbed, then its time
for diffusion through the cell membrane is relatively great, and the "hole sharpness
factor" is reduced. This factor will reach a limit of zero when the time for diffusion
through the membrane is equal to the time for healing of the lipid membrane. The
membrane would then heal completely behind the moving odorant molecule; i.e.,
the latter would not leave a sharp hole through which ionic exchange might then
occur.
On the other hand, tho the time of healing of the membrane, may have dif-
ferent values for different olfactory cells. Thus, some nerve membranes may be
relatively coherent and slow healing; also very resistant to penetration by the
odorant molecules (this is shown schematically to the left of Fig. 7). Other nerve
endings may have membranes which are very fluid and easily penetrated, so that
any "puncture" heals very rapidly. Such an assumption appears not unreasonable
in the light of the known tissue specificity of lipids in mammals; the molecules of
the membrane phosphatide have different distributions of their fatty acids
(palmitic and oleic) in different tissues of a given animal (VEERKAMP et al., 1962).
Very fluid membranes;

Semi - solid coherent fast healing.
membranes;
difficult to penetrate;
slow healing
1/k3 0.51 k3
th _10- 1 sec
. Ether molecules do not penetrate

. .. .. 1..
Ether stimulates;
sharp hole left.
Ether does not stimulate;
wall heals too quickly
..
tditt. _10-6 sec
ETHER
st~~:teJ Musk does not stimulate; wall heals too quickly
1
~ ~'I~"~--------------------------------------------------~"~
tdifC 1O-2sec
Musk-like MUSK
substances
cannot penetrate
these membranes
Fig. 7. Schematic representation of range of properties of sensory endings of different olfactory

nerve cells. The cell distribution may be such that musk odors (for example) form a fairly well-
defined group, with but little overlap with other groups (DAVIES, 1968)
Indeed, it has been suggested in a different context (DE GlER and VAN DEENEN,
1961) that there may be a relation between the lipid composition and the per-
meability properties of red cells, depending on the ratios of sphingomyelin, lecithin
and kephalin.
Even single olfactory nerves of the opossum and frog are known (BEIDLER and
TUCKER, 1955; GESTELAND et al., 1963, 1965) to exhibit some specificity; a given
fibre will not respond so strongly to some particular odor as will another fibre,
though the relative responses of the two fibres can be reversed if a different odor is
chosen. This is therefore some direct physiological support for a more or less con-
tinuous distribution of properties between different olfactory nerves; it appears
that no two cells have identical responses, but that every cell responds in one way
or another to a number of different odors. One observes no sharp specificity such as
would arise from a chemical or enzymic process, but only the rather blurred
specificity and overlap which are characteristic of physical adsorption.
Fig. 7 shows in detail why, on this basis of a distribution of physical properties,
an odorant such as a musk will stimulate only the cells with slow-healing mem-
branes. Smaller molecules (such as ether) are too weakly adsorbed to penetrate
these musk-sensitive strongly coherent membranes; just as laboratory experiments
Odor and Desorption Rates 337
(DEAN and FA-Sr Lr, 1950) have shown that benzene vapour cannot penetrate a
monolayer of stearic acid spread on a water surface at a surface pressure of 17 dynes
cm-I, although the more strongly adsorbed molecules of n-hexane can penetrate
into this stearic acid monolayer [see also Eq. (8) below]. On the other hand, musk,
for example, would not stimulate appreciably the cells particularly sensitive to
small molecules such as ether because the very fluid membranes of such cells will
heal much too rapidly behind the relatively slow-moving musk molecules, so
that no sharp hole is left behind them.
Odor and Desorption Rates

The treatment which follows was developed originally (DAVIES, 1968) to explain
musky odors, but the treatment is quite general. As before, N is the number of
stimulated sensitive regions (or "sites") on a nerve ending, these regions contain-
ing the necessary p adsorbed odorant molecules to induce ionic leakage across the
cell wall. We assume now that above threshold level the response R of any receptor
cell is proportional to N (of course, N = 1 at threshold). But by Eq. (2) for a given
constant response R (i.e., N constant) the mean adsorption x will also be constant
for any given value of p (for various musks p averages 1.3). From adsorption theory
(DAVIES and RIDEAL, 1963; DAVIES, 1968) for penetration into a layer of lipids
one writes that
x = ed K LA /(l + ke) (8)
where e is the concentration of odorant in the air, and KLA is the adsorption
coefficient for the odorant molecule between air and the lipid-water interface. As
before, d is the thickness of that part of the membrane penetrated by the odorant
molecules. The constant k depends on molecular cross-sectional area. Of course,
when e is relatively small (e.g., around threshold), Eq. (8) reduces to Eq. (1) above.
Eq. (8) approximates for any given odorant (i.e., for KLA fixed) to
(9)
where the exponent 'JI increases from 1 at very low adsorptions towards 10 near
saturation, but maintains a value of about 3 around the middle of the adsorption
range.
Given that x is constant for a constant response R and knowing that K LA is
reflected in z (the measured desorption rate [see THEIMER and DAVIES, 1967J of
odorant molecules from a monolayer-covered water surface into the air above) by
z ex: IjKLA' one finds that Eq. (8) reduces to
lje = k1jz - k2
where kl and k2 are constants relating respectively to the sensitivity of the cells
and the cross-sectional areas of the odorant molecules. Under these conditions of
constant response, e is the concentration of odorant in the air to give a constant
psychological response to a panel of trained judges. The study was undertaken
(THEIMER and DAVIES, 1967) with a range of substances having relatively pure
musk odors, and the corresponding reciprocal concentrations are conveniently
designated "Intensities of Muskiness," abbreviated to I.M. Hence,
I.M. = krjz - k2 (10)
22 Rb. SellOor~ l'h~siolog~. Vol. IV/l
So far this treatment has not allowed for the "hole sharpness factor," given
above as (1 - td1ff.jt,J This factor may also be expressed in terms of the measured
in vitro desorption rate z if we allow that the diffusion and desorption processes are
basically similar, involving similar activation energies. The "hole sharpness factor"
then becomes (1 - k3/Z) where k3 includes the term tit for the healing of the appro-
priate sensory membranes. It has been explained above that if this factor is zero;
no sharp passage is left behind the diffusing odorant molecule. Accordingly, Eq. (10)
should be corrected by multiplying by this factor:
I.M. = (1 - k3jZ) (~/z - k 2) (11)
or
(12)
Comparison with the intensity of muskiness data of THEIMER and DAVIES
(1967) shows (Fig. 8) that Eq. (12) fits the form of their results with the empirical
values of 8.1 for (~+ k 2 k 3 ), 3.26 for kl ka, and 3.0 for k 2 Hence, kl = 6.6 and
ka = 0.49, these relating to those cells particularIysensitive to musks. We should
expect the ka value in particular to be characteristic of such cells, and the musk
odor will be found only when the experimental desorption rates z are appreciably
greater than ka, of the order 1.5 ka to 2 ka. The LM. will be zero according to
Eq. (11), when z = k3; the corresponding times of diffusion being proportional to
Ijk3 (see Fig. 7). The I.M. will also be zero when z = k1/k 2; these limits of LM.
corresponding numerically to z = 0.5 and z = 2.2 for musks.
The "strongest" musks (i.e. those with the maximum intensity of muskiness)
should have desorption rates of 2~ k3/(~ + k2k3) if Eq. (12) is generally valid.
Numerically, this ratio is about 0.8, so that the strongest musks should have
desorption rates of about this figure. Very strongly adsorbed molecules (z low or
KLA high) will have lower olfactory thresholds [cf. Eq. (6) above], but under
perfumery conditions (far above the thresholds) the strongest musks will be those
with z around 0.8.
The data in Fig. 8 are from known musks of many different chemical types,
including macrocyclic musks, indane musks, tetralin musks, aromatic carbonyl
musks and isochroman musks. For nitromusks, the rather ready solubility in
water precludes direct measurements of desorption rates, but the energies of
desorption as found in other ways (DAVIES and TAYLOR, 1957) are indeed very
close to those for macrocyc1ic musks. Fig. 8, like the above equations, represents a
necessary but not a sufficient condition for muskiness. A known musk will give
data round the correlation, but some substances may give points on the correlation
yet be odorless or have a non-musk odor (e.g., a "burnt," "acetophenone" or light
floral odor).
For the existing distribution of olfactory cells, the limits to any given odor
quality will be imposed firstly by the ability of the odorant molecules to penetrate
certain nerve cells walls and, secondly, by the ratio of the rate of healing of the
membrane to that of diffusion of the odorant molecules. Desorption rates, giving a
measure of each factor, would therefore be expected to be a significant first factor
in correlating odor quality. But it is necessary to take another factor into con-
sideration; this second factor is the geometry of the odorant molecule - its size and
the balance between its non-polar and polar portions. The size can best be expressed
Odor and Desorption Rates 339
as A, the cross-sectional area of the oriented molecule (DAVIES, 1965), while the
ratio of hydrocarbon to polar material in the molecule is given (THEIMER and
DAVIES, 1967) by the ratio of the length (L) of the oriented molecule to its breadth
(B) measured at the polar end. For example, for a molecule to be a musk, besides
the desorption requirement, it is necessary that A lies in the range 40- 57 A2 and
that LIB lies in the range 2.8 to 3.3 inclusive. For the moth sex attractants whose
specificity is very high (BOECKH et al., 1965), the geometry factor is again clearly
5.0.,------------------,
Vl 2.0.
ell
c
5:
E 1.0
Vl
:?:: 0.5
iii
c
:s 0..2
ell
Desorption rate
Fig. 8. Points represent the experimental' 'intensities of muskiness" of known musk compounds
(of various chemical types) plotted against the measured desorption rates from the film-water
interface into air (THEIMER and DAVIES, 1967). The broken line represents Eq. (12), the
constants being chosen to give the best fit to the experimental points
most important. But for the smaller molecules, particularly those which form less
well-defined classes than musks, one would expect LIB to be much less significant.
This steric factor is perhaps rather reminiscent of the rigid sites into which a
molecule fits, according to Amoore's stereo-chemical theory (see below).
The geometrical factor for large odorant molecules may be interpreted in
physical terms in the following way. The odorant molecule (say a musk) penetrates
into the lipid layer of the cell wall. This is normally liquid, impermeable to ions,
and self-healing against disturbances because of the rapid free movements of the
hydrocarbon chains of the molecules of the fats, acids and esters in it. However,
when a rigid, bulky odorant molecule has penetrated into the film, several neigh-
bouring fatty chains are constrained to stand up straight, forming a locally
crystalline region. Thus, by its size and its inertia and partly by its shape, a large
odorant molecule reduces the fluidity of the surrounding membrane into which it
has penetrated. This explains how the relatively long times of membrane healing
('" 10-2 to 10-1 sec) can be achieved; the adsorbed musk molecule reduces the
fluidity of the surrounding membrane before diffusing through, thus enabling a
sharp hole to be left behind it.
Some support for this interpretation comes from the in vitro experiments of
ADAM and JESSOP (1928). They showed that large, rigid molecules such as choles-
22'
340 J. T. DAVIES: OUactory Theories
terol can, by their size and inertia, obstruct the movements of several neighbouring
"liquid" hydrocarbon chains in monomolecular layers of certain fatty acids,
nonylphenol and monomyristin spread on the surface of water. One molecule of
cholesterol can partly immobilize 8 neighbouring molecules of myristic acid; 4 of
them being completely immobilized in this way. As many as 16 molecules in a
spread film of nonylphenol are considerably immobilized by a single molecule of
cholesterol, and with a ratio of 1 molecule of cholesterol to 4 or fewer molecules of
nonylphenol the mixed film shows marked elastic after-working; i.e. the re-
establishment of equilibrium after a sudden compression or expansion takes up to
several minutes. Further studies showed that specific complexes are not responsible :
cholesterol formate and acetate have the 'Same effect as cholesterol, and in no case
is the two-dimensional "vapour pressure" of the original film altered by the
cholesterol, as would have happened if a complex were formed. This reinforces the
conclusion that it is the size, rigidity and inertia of the cholesterol molecules which
cause the effect: molecules with a single straight chain, even if this is as long as
22 carbon atoms, are ineffective (ADAM, 1944). In a similar way, large rigid odorant
molecules of the appropriate shape might reduce the fluidity of the cell membrane
before diffusing through, while other odorant molecules, if they are flexible, may
act as plasticizers and increase the fluidity of the membranes.
If now, however, as a first approximation we neglect LIB, the quality of any
odorant should depend on z and molecular cross-sectional area A; in Eq. (12) k2 is
a function of the cross-sectional area of the oriented odorant molecules as is also ka
(t" being affected by bulky molecules). Over the proposed range of olfactory cells,
one might therefore expect a correlation such as that of Fig. 9, the various
areas of which should denote uniquely the quality of the odor in physio-chemical
terms. The desorption rate z limits the regions in general as designated by Eq.
(12). This suggests (because of the proposed distribution of olfactory cells)
that the different odor qualities shade into one another rather than there being
sharply demarked "primaries." But, as shown in Fig. 7 the cell distribution may
be such that some odor qualities, such as musky, constitute relatively well-
defined groups compared with, say, floral. BEETS (1966) has pointed out how the
odors of most macro cyclic molecules shade gradually into one another in general
as follows:
0 8 , Og, 0 10 camphoraceous,
Oll terpenic,
012> 01a terpenic-cedary,
014 cedary-musky,
015> 016> 0 17 musky,
0 18,019 civetty-musky,
019' 0 20 odorless.
Another piece of evidence in support of the general merging of odors into one
another, as shown in Fig. 9, comes from the similarity studies of AMOORE and
VENSTROM (1966). They reported (from panel tests) psychological similarities
("" 2 units) between their minty and camphoraceous odorants; between floral and
musky; and also between floral and minty. On their scale, completely similar odors
score about 6.5 units of similarity.
Intensity in Relation to Concentration of a Given Odorant 341
Yet further evidence comes from electrophysiological studies of similarity

(DOVING, 1966a). There is a significant rank correlation coefficient of 0.684 between
his results (DOVING, 1966b) and those of a plot (DAVIES, 1965) which is an earlier
version of Fig. 9.
60~------------------------------------,
40
<r
20
1 I
4 0.3 rates
0 5000 10000
..1G
Fig. 9. Odor quality "map" i.e. a correlation of odor quality with measured cross-sectional
areas (in AI) of odorant molecules and with free energies (..1 G, expressed in calories per mole)
of desorption of the odorant from the lipid-water interface into air. These energies are related
to KLA by the relation L1G = RTInKLA ; and to desorption rates (z) by In z = constant
- ..1 GIR T. The various areas indicated should denote uniquely the quality of the odor in
physical terms (DAVIES, 1965, 1968)
However, among the various musks, DOVING (1966a) found a relatively close
similarity in electrophysiological response; materials studied included an indane
musk, several oxa-octahydro-anthracene musks, a tetralin musk and a nitro-
acetophenone musk. The latter showed rather less similarity to a macrocyclic lactone
musk, however, and androstan-3-ol was less similar than most others within the
group. But compared with other types of odor, DOVING found a "rather strong
relationship between musky odors" ; they constituted a "rather homologous group
with significantly high chi-square values between all pairs." GESTELAND et al.
(1965) likewise report in the epithelium of the frog those receptors particularly
sensitive to musk do not respond much to other odorants. On the other hand, the
other receptors show extensive overlap in their response to different odorants,
rather like poorly constructed optical filters.
Intensity in Relation to Concentration of a Given Odorant

To interpret odor intensities when the odorant used is well above its threshold
concentration (DAVIES, 1968), we employ Eq. (3) above, relating N the number of
"punctures" per olfactory cell by any given odorant (i.e., constant KL.~) to the
mean adsorption x of odorant:

log N = p log x + constant.
Substituting now from (9) above, we have for the given odorant,
log N = (plY) log c + constant
or
log R = (plY) log c + constant (13)
assuming as before that the response R, whether measured electrically or psycholo-
gically, is proportional to N (since the "hole sharpness factor" is constant for any
given odorant).
For many compounds p is about 2 and Y is about 3 in the middle of the concen-
tration range, as shown by the experiments on the adsorption of vapours on to
spread films. DEAN et al. (1953), for example, quote results from which one may
deduce that diethyl-ether vapour adsorbs on to a close-packed film of stearic acid
on a water surface with Y = 3 when the ether vapour pressure is varied from about
11 % of saturation of 34 % of saturation. Hence, with these values of p and Y, we
have
log R = 0.67 log c + constant.
Experimental values of the numerical coefficient are 0.49 (ENGEN, 1964), 0.6
reducing to about 0.2 very close to saturation (MOULTON and TUCKER, 1964), and
0.55 and 0.60 (STEVENS, 1961). Agreement with the theoretical value of 0.67 is
satisfactory, but it must be emphasized that our use of a constant value of 3 for Y is
only approximate; as the adsorption proceeds from c = 0 to high values, Y must
increase from 1 to perhaps 10. In this way, theory predicts that the exponent
(plY) must fall from a maximum of perhaps 1 (for strongly adsorbed large mole-
cules with p = 1, such as p-ionone, very close to threshold), to perhaps 0.2 for,
say, musks close to saturation. This is consistent with the findings of MOULTON and
TUCKER.
Discrimination over Large Concentration Ranges

The figure for ply of about 0.7, decreasing to about 0.2 close to saturation,
explains the known discrimination of the nose to any given odorant (such as
vanillin) over an enormous range of concentration, e.g., up to about 105 times the
threshold concentration. According to Eq. (13) the response R varies by a much
smaller factor - perhaps by only 100 times if ply is given a mean value of 0.4 -
for a range of odorant concentrations of 105 times. The ability of the nose to
measure and discriminate responses over these large ranges of concentration is thus
explicable in terms of the above process of physical adsorption.
Odor Type According to the Molecular Vibration Theory

The larger odorant molecules, vibrating as a whole, will have a rather low
frequency, which will be detected in the laboratory by measurements in the far
infra-red region. In various recent publications, R. HAMILTON WRIGHT (1964,
1967) has presented his case that odor type is related to the far infra-red spectrum
Odor Type According to the Molecular Vibration Theory 343
of the odorant molecules, and in particular that a necessary condition for musky
odor is a combination of three molecular vibration frequencies near 100, 160 and
260 cm-1 .
WRIGHT'S (1967) analyses of the far infra-red spectrum (85 cm-1 to 340 cm-I,
i.e. 2.55 x 1012 Hz to 10.2 X 1012 Hz) of 45 known musky odorants, of various
chemical types, and also of 15 compounds with structures generally similar to
certain musk perfumes, gives the results shown in Table 1, as collated by the
reviewer. The numbers of compounds with I.R. peaks within each band shown is
listed. The average number of I.R. peaks per compound is 4.3 in the region
85 cm-1 to 340 cm-1, so that (assuming a uniform distribution of peaks), there
would be, in any band of width 31 cm-1, 4.3 x 31/255 peaks, i.e., 0.52 peak per
compound. Thus for the 45 musks there would be a mean of 23.4 peaks per band
on this assumption of uniformity and for the 15 non-musks these would be a mean
of 7.8 peaks per band.
Table 1. Occurrence of far infra-red peaks in various bands for various musks and other generally
similar structures. The figures quoted here were obtained by the reviewer from data of WRIGHT (1967)
Band No. of musks %ofmusks No. of non- % of non-

(ou t of total having peak musks (out of musks having
of 45) in band total of 15) peak in band
I. 85 to 115 cm-1 inclusive 22 49% 6 40%

II. 145 to 175 cm-1 inclusive 38 85% 8 53%
III. 245 to 275 cm-1 inclusive 32 71% 14 93%
I, II, and III simultaneously 11 25% 4 27%
How do the figures in Table 1 compare with these averages 1 Before answering
this question, we must note that for 45 x 4.3 (= 194) peaks, we should not be
surprised to find by random sampling any number between 15 and 31 peaks
within anyone of the bands. For the 15 non-musks, with 15 x 4.3 (= 65) peaks,
we should not be surprised to find any number between 3 and 13 in anyone band.
Thus for Band I separately there is no significant deviation from random
sampling for either the musks or the non-musks.
For Band II separately, the figure for the musks is surprisingly high, while the
figure for the non-musks is close to the average expected from a random distribu-
tion of peaks.
For Band III separately, the figures are rather high for both the musks and the
non-musks.
Taking Bands I, II, and III simultaneously for the musks and the non-musks,
the reviewer found from the spectral curves that 11 of the 45 musks (i.e. 25 %)
have peaks in all three bands. By multiplying the separate observed band per-
centages together, one finds that 30 % would have been expected (on a random
distribution of peaks between the musky molecules). For the non-musks, 4 out of
the 15 (i.e., 27 %) have the three peaks simultaneously.
WRIGHT (1967) also includes in his correlations a set of 109 spectra of a wide
variety of substances with pronounced odors or distinctive insect-attracting
properties; none of them having a musk-like character. Taking the three bands
simultaneously he then claims a strong, statistically significant correlation of
peaks in all three bands with musk odor: of the total of 109 + 15(= 124) non-
musks, only 15 have peaks in Bands II and III simultaneously. But it appears
that, of these latter 15 compounds, seven are from the group of fifteen compounds
"with structures generally similar to certain musk perfumes."
So from the 109 various additional compounds, only eight have peaks in Bands II
and III simultaneously. The reviewer finds this highly significant, and concludes
that though the far infra-red spectra (reflecting molecular rather than group
vibrations) are not specific to muskiness among molecules of known musks and
compounds with generally similar structures; such compounds do have more infra-
red peaks in the regions concerned, as compared with widely different substances.
This conclusion is apparently rather similar to the profile-functional group concept
of BEETS (1957, 1964) (M. Wt. in range 220-280, closely packed profile and
sterically accessible functional group) or the cross-sectional area, orientation, and
LIB requirements of TImmER and DAVIES (1967).
WRIGHT (1968b) has answered these criticisms by saying that of his group of
15 "non-musks" (WRIGHT, 1967; and also Table 1) with chemical structures similar
to musks, no fewer than seven were described as "odorless." This, he says, may
simply imply "inadequate volatility, and a corresponding inability of the mole-
cules to reach the receptor organ which makes their spectra irrelevant. Another
seven are described as 'non-musks' without any indication of whether this means
'no odor' or 'an odor but not a musk odor' ... Under these circumstances to have
got a chi-square significance even at the ten per cent level is a considerable achieve-
ment."
Clearly further testing is essential here, possibly using dogs or other animals to
identify whether the "odorless" compounds that WRIGHT studied do or do not
have a feeble musky odor, perhaps below human threshold.
Other tests of the theory involve deuterated compounds (which have somewhat
different spectral properties): DOOLITTLE et al. (1968) measured the effect of
deuterating parts of the molecule of the male melon fly attractant substance 4-p-
hydroxyphenyl-2-butanone acetate. Though deuteration led to changes in the far
infra-red peaks of about 10 cm-1 (and for two peaks changes of about 15 cm-1), it is
not clear whether a significant change in odor is to be expected from (say) a shift from
328 cm-1 to 316 cm-1 on deuteration. WRIGHT claims that these shifts are small
enough to lie completely within the relevant bands of frequencies, so that no change
in odor should be observed. Experimentally, deuteration of the molecule made no
perceptible difference to the attractiveness of the substance to the male melon fly.
Clearly further work on deuterated (or better, tritiated) compounds would be
of great value, particularly if some of the spectral peaks could thus be moved out
of the "permitted" bands postulated by WRIGHT.
Odor Type and "Primary Odors" According to the

"Specific Site" Theory
It has long been realized that molecular size and shape are important in deter-
mining odor type: LUCRETIUS (47 B.C.) thought of sweet-smelling odorants as
Odor Type and "Primary Odors" According to the "Specific Site" Theory 345
having smooth molecules, and of pungent ones as having hooked or barbed mole-
cules. In modern times, TrMMERMANS (1954) has postulated that most substances
smelling like camphor have spherical molecules (though methylcyclopentanol does
not), but the converse statement that all spherical molecules smell like camphor is
not true. Evidently other factor must be taken into account. For the group of
odorants having musky odors, BEETS (1957, 1961, 1964) concluded that "the
combination of a sterically accessible functional group and a closely packed profile
in a structure with a molecular weight of roughly 220 to 280 are criteria which
probably suffice to obtain a musk odor." This "profile-functional group" concept
relates the orientation of the odorant molecule on the receptor site to the affinity
of the functional (polar) group. The relevant molecular profile is then that of the
odorant molecule as it is attached to and oriented at the receptor surface.
It was AMOORE, however, who explicitly related profiles to highly specific sites
postulated to exist on the receptor. In a series of papers beginning in 1952, he
postulated seven "primary odors" (rather as one has primary colors or perhaps
primary tastes) and went on to relate these primary odors to the external shape of
the odorant molecule (AMOORE, 1952, 1962, 1964). In this series of publications,
he proposed precise site profiles and dimensions corresponding to his postulated
seven primary odors. For example, musky odorants were able to fit into an oval
pan 11.5 A long, 9 A wide and at least 4 A deep. The polar group of the odorant is
relatively unimportant here. All that AMOORE suggested was that in this special
site there is some electron-seeking group which must be satisfied if a response is to
occur.
This theory had one difficulty at the outset - how is one to identify the seven
"primary" odors ~ AMOORE proposed a method based on frequency of occurrence.
Thus, since there are many camphoraceous and musky compounds known, these
rank as "primaries." But the objection could well be raised that these frequencies
of occurrence reflect more the activities of organic chemists (who have spent much
labor on making artificial musks, for example) than any basic properties of the
olfactory nerve endings.
Further, some of the molecular profiles of odorants give rather poor fits with
the postUlated sites, and DRAVNIEKS (1966) cited three further objections "Some
classes of compounds, e.g., glycols, fit sites but have no odor. The number of
primary odors (7) is too small to explain the total number of odors that can be
discriminated by humans. Observations on partial anosmia and olfactory fatigue
revealed that some people can differentiate or detect only certain classes from
among several musks (a primary odor by AMOORE'S theory) belonging to different
chemical classes ... " WRIGHT (1968b and elsewhere) has emphasized the enormous
discriminatory capacity of the nose: "I do not see how any of the "shape" or "site-
fitting" theories can ever begin to supply this degree of specificity ... "
In his latest papers AMOORE (1965) (see also AMOORE et al., 1967) adopts a
less rigorous approach. He now correlates statistically odor with silhouette photo-
graphs of molecular models of the odorants: comparison of profiles and odor
qualities for ethereal, musky, camphoraceous, floral and minty odors gives
correlation coefficients lying between 0.45 and 0.66, showing that here molecular
profile and odor quality are related, though not as explicitly as was formerly
believed (see Fig. 10).
Recent electrophysiological studies of similarity between different odorants

have cast further light this vexed question of "primary" odors. These studies have
been based on measured nerve responses at level of the olfactory bulb units
(DoVING, 1966a). Stimulation of the receptor epithelium with odorants generally
causes inhibitions at bulb level, and from the responses of about 80 bulb units, a
correlation between various odorants was obtained. "Floral" odors showed little
correlation in their electrophysiological responses, though "camphoraceous" and
6
SM
M M
Sf- M M
.... 4~
M
0
"0
M M
0 M
....0 3~
C M
~ 2-xx ~
x
'E x x x x
x
V5 x
1~ x x
Xx
XX
x
a I
0.5
I
0.6
I
0.7
I
0.8
I
0.9 1.0
Similarity of shape to that of standard
Fig. 10. Data taken from AMOORE and VENSTROM (1967) are plotted to show whether odor
quality correlates with a geometrical factor alone, i.e. the overall molecular shape relative to
the shape of a standard substance. Here this standard is a musk, 15-hydroxypentadecanoic
acid lactone (point S M). Other substances generally regarded as having musky odors are
indicated by M; and non-musks (with little similarity to musks) are indicated X. It is clear
that there is an overall correlation of odor quality with molecular shape, though within the
group of musky compounds the correlation is much weaker. Further, there is considerable
overlap of the shape similarity factor between musks and non-musks in the region aroundO.57
"ethereal" odorants formed reasonably well defined groups. However, substances

from different groups did not always show independence; for example, the cam-
phoraceous and ethereal groups showed some overlap. Musky odorants formed a
rather homologous group with high chi-square correlations between all pairs and
insignificant overlap with other groups, as has been mentioned above.
Comparison of DOVING's correlations with AMOORE'S theory gives a significant
rank correlation coefficient (r8 ) of 0.498; but comparison with the "penetration and
puncturing" theory (Fig. 2 of DAVIES, 1965) gives, according to DOVING (1966b) a
better correlation coefficient of 0.684.
From DOVING's work, and also from that of BEIDLER and TUCKER (1955) and of
GESTELAND et al. (1963, 1965) on single olfactory cells, it appears that the receptors
which are specially sensitive to musk odors constitute a particularly well-defined
group: the nerve endings sensitive to musks are relatively insensitive to other
odorants.
Partial anosmia has recently been used by AMOORE (1967, 1968a, b) to try to
identify "primary" odors. Certain persons, having an otherwise apparently
References 347
normal sense of smell, nevertheless lack sensitivity in preceiving some particular

odor. This condition, which is usually inborn but may be acquired, is known as
partial (or specific) anosmia. Earlier, GUILLOT (1948a, b) made the suggestion that
each type of partial anosmia might correspond to a "primary" odor, by analogy
with color blindness. Thus persons exhibiting partial anosmia are presumed to be
defective in one of the primary odor-detecting mechanisms of the olfactory
system. One of GUILLOT'S female subjects, though highly sensitive to other odors,
was unable to detect the violet characteristics of ionones and methyl-ionones.
Another subject, able to perceive perfectly three different nitro-musks, was insensi-
tive to various macrocylcic musks and a steroid musk. Other subjects perceived
the macrocyclic musks but not the steroid musks. Yet another subject, anosmic to
natural ambergris, perceived well an artificial ambergris. However, partial anosmia
may be associated with defects in several olfactory regions, and if so, the partial
anosmia would not necessarily define the corresponding odor as fundamental or
primary. GUILLOT ended with the statement (here translated) that "the identity of
the olfactory sensation is not therefore an indication of identity of the sensory and
psychophysiological mechanisms set in action."
Recently, AMOORE (1967, 1968a, 1968b) has carried out a systematic study of
human partial anosmia to isobutyric and isovaleric acid. About 2 % of the popula-
tion had this particular partial anosmia, and AMOORE concluded from his studies
that there is in the normal nose a detector" ... specialized to react to the carboxylic
acid functional group ... It has a distinct preference for certain branched-chain
acids and responds most exclusively to isovaleric acid, among the compounds so
far studied ... The smell of isovaleric acid ... is the nearest approach to the
primary odor sensation. Thus the first primary of odor ... has been experimentally
categorized. "
In general, writes AMOORE (1967) " ... the assignment of a primary odor by the
specific anosmia method has a much stronger experimental foundation than any
earlier proposal, and may be considered to supersede the older classifications where
discrepancies occur." AMOORE (1968b) suggests that a specific loss of odor sen-
sitivity may be due to " ... an absent receptor protein, missing olfactory cells,
broken nervous connections, delayed recovery from adaptation or a psychological
block, to name a few." "Proteins alone, among the constituents of protoplasm,
would appear able to confer enough selectivity on the detector system to respond
specifically to carboxylic acids and then to but a limited range of molecular shapes."
The reviewer believes that in view of the tremendous discriminatory power of
the human nose, the number of "primaries" would have to be in the tens or even
hundreds. And, since various olfactory cells have been found to be each more or
less sensitive to different odors (BEIDLER and TUCKER, 1955; GESTELAND et al.,
1963, 1965), the work ahead may be formidable before the "olfactory code" can
be "cracked" by this approach.
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Chapter 14
Insect Olfaction
By
KARL-ERNST KAISSLING, Seewjesen, Germany
With 62 Figures
Contents
Introduction . . . . . . . . . . . . . . . . . . . 351
A. The Biological Function of the Olfactory Sense and the Nature of Odors 352
B. Morphological and Functional Properties of the Antenna 353
C. The Morphology of Olfactory Sensilla. . . . . . . 358
D. Methods for the Study of Olfactory Organ Function 365
1. Behavioral Responses . . . . . . . . . . . 365
2. Electrophysiology . . . . . . . . . . . . . 369
E. Dynamic and Static Properties of Olfactory Cells 373
F. The Sensitivity of the Receptors. 378
1. The Relative Threshold 378
2. The Absolute Threshold . . . 381
G. Specificity of Receptors. . . . . 387
1. Odor Specialists of High Specificity. 388
2. Masking and Enhancing . . . . . . 397
3. Odor Specialists of Lower Specificity . . 400
4. Hyperpolarisation and Inhibition . 405
5. Odor Generalists. . . . . . . . . 405
6. COz - and Humidity Receptors. . 409
H. The Sensory Transduction .... 410
J. The Central Pathways of Olfactory Fibers. 421
References . . . . . . . . . . . . . . . . 423
Introduction
The behavior of insects is controlled to a great extent by olfactory stimuli.
Whereas a given insect species appears to be selectively specialized to a number of
odors, the list of substances odoriferous to insects in general is extensive although
352 K.-E. KAISSLING: Insect Olfaction
not completely known_ Many gaseous substances perceived by insects are odorless
to man. Into this category fall not only simple compounds like water vapor or
carbon dioxide but also complicated compounds such as bombykol or queen
substance and many others, some of wruch can be detected at very low concentra-
tions. The olfactory sense of many insects is highly developed. Moths, for instance,
have a more sensitive sense of smell than dogs, and the honey bee can distinguish
many flower odors at least as well as man.
Numerous papers have been published on the olfactory behavior of insects and the identi-
fication of the odor substances involved. Several excellent reviews cover this field from different
aspects (DETHIER, 1947, 1963; KARLSON and LUSCHER, 1959; HODGSON, 1965; JACOBSON,
1965,1966; Wn.SON, 1965; BUTLER, 1967; MOORE, 1967; SHOREY and GASTON, 1967; HOFF-
MANN, 1961).
A. The Biological Function of the OHactory Sense and the Nature

of Odors
1) For most insects, odors playa role as guides to food sources and to oviposition
sites. Many insects prefer very special types of food, which commonly produce
characteristic scents from numerous chemical compounds (FRAENKEL, 1969). These
scents attract the insects from a distance, contribute to the final recognition and
elicit feeding, oviposition and other specific behavioral responses. In many cases
these scents seem to be the byproducts of metabolism or decomposition of food
objects, as for instance grass odor, resins, the scent of blood or carrion odor.
Fungi are known to produce very specific scents which simulate carrion to several carrion
insects (SCHREMMER, 1963). The higher plants developed together with the insects, in evo-
lutionary interrelation, and the plants evolved special glands which produce the chemilJally
well known flower odors. An extreme adaptation are flowers which mislead male insects by
producing scents similar to the female sex attractant. Some orchids attract males of certain
species of Hymenoptera by this method, using them as a means of transporting their pollen
(KULLENBERG, 1961). Carrion odors are also simulated by flowering plants.
Apart from the abundance of scented chemical substances involved in food
behavior, CO 2 and H 20 vapor also attract insects (e.g. mosquitoes, WRIGHT, 1968;
GILLIES and WILKES, 1968). To date, the sense organs for CO 2 and H 20 cannot be
distinguished morphologically from the other olfactory organs, even with the
electronmicroscope (Fig. 12). Electrophysiologically they also have a similar
behavior (LACHER, 1964; WALDOW, 1970).
2) Besides the extensive group of odor substances emanating from the insect's
environment, a large number of odoriferous chemicals is produced by the insects
themselves. These compounds are called pheromones if they are involved in intra-
specific communication (KARLSON and LUSCHER, 1959; KARLSON, 1960; BUTLER,
1967; SHOREY and GASTON, 1967). To the olfactorily effective pheromones belong
the sexual attractants which are usually secreted by the female, but in some cases
also by the male or by both sexes (PYATNOVA et al., 1969). Sexual attractants
sometimes act over great distances, and can be species-specific (BORNEMISSZA,
1966), but they are mostly found to be identical in effect and probably also in
chemical structure for several closely related species (PRIESNER, 1968, 1969). Only
a few sexual attractants are chemically known.
Morphological and Functional Properties of the Antenna 353
Arresting pheromones called aphrodisiacs playa role in mating behavior and

are usually produced by the male (ROTH, 1967; APLIN and BIRCH, 1968; PILSKE and
EISNER, 1969). Apart from mating, many other types of behavior are controlled by
olfactorily effective pheromones, especially in the social insects (BUTLER, 1967,
1969; BLUM, 1969). Pheromones serve as congregating signals, trail markers, for
marking of flowers and other food sources, as alarm and alerting signals, as a colony
odor, for recognition of castes, as a releaser of food exchange and have many other
functions. Ontogenetic development can also be determined by olfactorially
perceived pheromones (LOHER, 1961 a, b).
Many pheromone scents including sex attractants consist of a number of compounds which
usually are effective singly but have possibly the highest effect in certain mixtures. On the
other hand single pheromone compounds may have more than one function, depending on the
particular situation in which they are delivered. For instance, 9-oxo-2 trans-decenoic acid
attracts swarming worker bees, attracts drones from a distance to the queen during the mating
flight and results in them mounting the queen, and also partially inhibits the development of
ovaries of workers and prevents them from building queen cells (BUTLER, 1969).
Secretions used as defence agents against other species (EISNER and MEINWALD,
1966) are not considered as pheromones. Some pheromones, however, such as
formic acid, function simultaneously as alarm substances for conspecifics and as
repellents against enemies of other species (MASCHWITZ, 1964, 1966).
Men have since developed powerful chemicals for attracting or repelling insects
for the purpose of pest control. Some of them are natural substances but others
have been discovered by testing large numbers of synthetic chemicals (BEROZA and
GREEN, 1963; VALEGA and BEROZA, 1967).
B. Morphological and Functional Properties of the Antenna

In general, insects perceive scent with a pair of segmented head appendages,
the "feelers" or antennae. The two proximal segments, scapus and pedicellus, are
supplied with muscles and move the antenna as a whole (Fig. 1).
Besides the olfactory organs, including those for CO 2 and humidity reception, the antennae
also bear sensory organs for other stimulus modalities such as taste, touch, air movement,
sound and heat (SCHNEIDER, 1964). In addition, antennae sometimes serve functions other
than sensory perception. They may be optical signals by means of their shape and colour,
processes for hovering, organs by which the animal may be carried, adhesive organs, senders
of mechanical or acoustical signals and in one case even serve as a light organ (WHEELER, 1960;
GRASSE, 1949).
All these functions have to be considered when we try to understand the shape
of the antennae as a product of evolution. Characteristic for antennae with most
sensitive olfactory function are an extended "outline area" (Foutd and a good
subdivision of form. The ideal is an "odor filter" which most effectively sieves out
the scent molecules from a large cross section of the odor stream.
The antennae of some night-flying moths (Saturniidae) come nearest to this
ideal (Figs. 1 e, 2, 3). The extended outline area, the "nostril" of the insect nose,
is in some of the saturniid moths larger than 1 cm 2 A marked subdivision is
produced by many side branches of the antennal segments. Many thousands of
olfactory hairs are arranged on these sidebranches in a characteristic manner
(Figs. 2-4, SCHNEIDER and KAISSLING, 1956, 1957, 1959; BOECKH et al., 1960;
23 lIb. Sensory Physiology, Vol. IV!l
STEINBRECHT, 1970a). Calculations made by ADAM and DELBRUCK (1968) show

that such antennae sieve almost all the scent molecules from the airstream. The
"diffusion space" is large in comparison to the diameter of the olfactory hairs. The
diffusion spaces of neighbouring hairs have a multiple and marked overlap and
because of this the molecules have an excellent chance of hitting the hair surface_
Fig. la-g. Antennae of male and female ('i') insects with well developed olfactory sense.
a Honey bee (ApismelliferaL.); b flesh fly (genus Sarcophaga); c carrion beetle (g. Necrophorus);
d scarabid beetle (g. Rhopaea); e saturniid moth (g. Antheraea); f hawk-moth (sphingidae,
g. Pergesa); g butterfly (g. Vanessa). Common scale (1 mm) for a-d and e-g
Fig. 2. Section of the male antenna of Telea polyphemus (saturniids). Shape of antenna
similar to the one of Fig. 1 e. Several types of sensilla on antennal stem and branches. The long
hairs (sensilla trichodea) contain the dendrites of the sex attractant receptors (from
BOECKH et al., 1960)
With radioactively labelled Bombykol, this prediction was found to hold true for the
antennae of the silkworm moth Bombyx mario The Bambyx antenna has an outline area of
6 mm 2 and adsorbs approximately 27% of all molecules transported through an airstream with
a cross section of 6 mm 2 Here one must take into account that, owing to the air resistance of
the antennae as a whole, a portion of the odorous air current is deflected around it (KAISSLING,
unpublished).
Fig. 3 Fig. 4
Fig. 3. Section of the male antenna of the silkworm moth Bambyx mari L. Branches of
the antenna with the sex attractant sensilla (approximately 100 fL in length, KAlSSLING
and PRIESNER, 1970)
Fig. 4. Single segments of the male antennae of Bambyx (above) and of a hawk-moth (below,
compare Fig. If), covered with sensilla trichodea. During flight the airstream comes from the
left side (original photograph)
The percentage of molecules the antenna adsorbs from an airstream cross

section equal in size to its outline area can be termed the "adsorption quotient"
(Qads). Only a portion of these molecules, namely the "effective fraction of adsorp-
tion" Qetf, impinge on the "effective surface area". These molecules have a
probability y (quantum efficiency) to affect the sense cells. In Bombyx, for example,
the total surface area of the hairs (sensilla trichodea) sensitive to bombykol
can be considered as the effective surface. All bombykol molecules impinging on
other types of olfactory hairs, mechanoreceptive bristles, or on the insensitive
surfaces of the stem and branches of the antenna do not register. According to the
calculations of ADAM and DELBRUCK (personal communication) the effective frac-
tion of the bombykol (Qeff) reaches 75 %, even though the surface of the sensilla
trichodea comprises not more than 25 % of the total antennal surface. This
prediction has been experimentally confirmed by STEINBRECHT and KASANG
(unpublished, see p. 384).
23*
356 K. -E. KAISSLING: Insect Olfaction
In summary, we can state that the efficiency of the antenna as an odor filter can
be defined by three terms which are mainly dependent upon the external geometry
of the antenna itself. The product of these terms: the outline area (Fautl ), the
adsorption quotient (Qads) and the effective fraction of adsorption (Qeff) can be
termed the "filter coefficient" Ga of the antenna.
(1)
The filter coefficient is defined on the basis of an odor stimulus within an air stream. The
product of the odor molecule concentration c, the air stream velocity v relative to the antennae,
and the stimulus duration t can be called the "particle transfer across unit area" N
mOleCUleS)
N=cvt ( (2)
cm 2
The number of adsorbed molecules N effa on the effective antennal surface is:
Neffa = N Ga (molecules) (3)
or
Neffa = c v' t Foutl Qads' Qeff.
This relationship shows that the particle transfer N is the adequate measure for
the strength of an olfactory stimulus as long as the filter coefficient Ga is a constant.
With the help of this filter coefficient the antennae of different insects can be
compared; in fact, any olfactory organs can be compared with one another in the
same way with respect to their filtering ability.
Here one has to consider, however, that the filter coefficient is not necessarily a
constant since Qads may depend on i) the direction of the air flow with respect to
the (maximum) outline area of the antenna Fauth il) the stimulus parameters:
concentration, air stream velocity and stimulus duration, iii) the odor saturation of
the antenna and iiii) the odor compound.
With Qads = 27%, Qeff = 75% and Foutl = 6 mm 2 one obtains a filter coefficient of
1.26 10- 2 cm 2 for the Bombyx antenna with respect to bombykol (and, of course, the bombykol
receptors). Here Qads was proved to be independent of the airstream velocity between 0.1
and 5 m/sec, of the odor concentration and the stimulus duration (KAISSLING, unpublished).
The adsorption quotient Qads of the honey bee's antenna was determined using C14-labelled
caproic acid and varied, in contrast to the Qads in Bombyx, with the stimulus duration
(1-8 sec) and with the air stream velocity; it decreased at 2 sec duration from 4.7% to about
1 % with increasing air stream velocity (from 1.6 to 6m/sec;KAISSLING, unpublished). The values
of Qads in the honey bee are smaller than in Bombyx, however, which can be explained by a
smaller ratio between the volume of the diffusion space of the antenna and its surface area,
which is due to lack of hair-like sensilla. With a Qads of about 5 % and a roughly estimated Qeff
of 5% and with Foutl = 1,12 mm 2 one obtains a filter coefficient of about 3.10- 5 cm 2 for the
drone bee antenna with respect to the perception of queen substance. The calculation of Qeff
is based on the assumption of 4 of 18 receptor cells per poreplate being sensitive to queen
substance and of 20,000 pore plates each with 40 fJ.2 effective surface on one antenna. Obviously
the bee's antenna is a much poorer scent filter than that of Bombyx with a filter coefficient of
1.26.10- 2 em 2 (see above). One can derive a Qads of 0.27% from the data of KAFKA (1970, see
p. 383) on the locust antenna which is similarly shaped to the one of the honey bee and also lacks
long sensilla trichodea. The air stream velocity was 5 m/sec see Table 5).
The antennal outline area varies in insects by about four logro units (Table 1).
Even with similar adsorption properties (Qads . Qeff) the Drosophila antenna would
still be 104 times Ie ss effective in molecule catching than the antenna of the male
saturniid moth Telea polyphemus.
The adsorption quotient Qads should, however, increase with very small
antennae because of the better proportion between the volume of the diffusion space
and the surface area of the antenna. The diffusion space around the antenna and
therefore the Qads should increase with lower air stream velocity since the mole-
cules then have a greater chance to hit the antenna by diffusion. This effect may be
detectable only in antennae with a small Qads (like the honey bee antenna, see
above) or in very small antennae.
Table 1. Outline area of insect antennae
Species area Type as

mm 2 in Fig. 1
Telea polyphemus ~ 85 e
18 e
Antheraea pernyi ~ 55 e
10 e
Bombyxmori ~ 6 e
5.5 e
Pergesa elpenor 9 f
Vanessa urticae 3.5 g
Polyphylla fullo ~ 36 d
4 d
Necrophorus vespilloides 0.7 c
Apis mellifera ~ 1.12 a
0.56 a
Sarcophaga sp. 0.5 b
Drosophila melanogaster 0.01 b
Hippelates 0.01 b
When the filter coefficient is independent from the airstream velocity as in

Bombyx, fast-flying insects may "get by" with smaller antennae than slow-flying
ones. For instance, the sphingids, which are fast flyers in relation to the saturniids,
have antennae with relatively smaller outline areas (Figs. If, 4) and therefore a
smaller filter coefficient.
It is obvious fOm insects with sexual dimorphism that the size of the antenna is
governed by the olfactory function. The outline area of the male antenna is several
times larger than in the females of some saturniids and beetles, the "macrosmates"
among the insects (Fig. Id, e).
The specialisation for absolute sensitivity reaches its extreme in some male saturniid
moths which can detect the sexual attractant of the female over a distance of several kilo-
meters (for references see PRIESNER, 1968; SCHNEIDER, 1965). Not only is the outline area Foutl
of the male antenna enormous (about 1 cm2) but, as in Bombyx, Qads is probably extreme
due to the subdivision of the antenna and the arrangement of the long olfactory hairs (Fig. 2).
Also Qetf is supposed to be near the optimum due to geometrical reasons (see above) and
due to the fact that most of the hairs are specialized to the reception of a single key sub-
stance. One of the two cells of each hair (sensilla trichodea) responds to the female pheromone
(SCHNEIDER et al., 1964).
In Bombyx, however, both cells of a hair respond to Bombykol. The Bombyx males are able
to respond to less than 1000 molecules of Bombykol per em" with an airstream velocity relative
to the antennae of 57 em/sec (KAISSLING and PRIESNER, 1970; see Table 5).
The filter coefficient of an antenna with respect to a definite odor substance is

less when several types of olfactory cells are specialized to different odor sub-
stances and, therefore, compete with each other for the effective surface area
(KAISSLING and RENNER, 1968). This results in a smaller Qelf' The honey bee, for
example, is able to distinguish between many odor substances but has com-
paratively high behavior thresholds of 10 8 to 1012 molecules per cm3 air (SCHWARZ,
1955). An additional reason for this high threshold is of course the rod-like form
of the honey bee antenna (Fig. 1a), the lack of hair-like sensilla and the small
outline area (see above).
Many insects have filiform antennae with a club-like thickening at the tip
covered with sensory cells. Probably these insects (e.g. butterflies) are specialised
for spatial odor perception (Fig. 1 g).
c. The Morphology of Olfactory Sensillae

The olfactory cells on the antennae of insects are primary sense cells. The cell
soma is part of the antennal epithelium and is directly connected to the deuto-
cerebrum by its axon (STEINBRECHT, 1969a). The distal dendrite of the olfactory
sense cell lies mostly within a thin-walled protrusion, the olfactory hair, which is
exposed to the surrounding air (Fig. 5). The cuticular hair together with one or
several sense cells and usually two formative cells and the accessory cells is called
the sensillum. Ontogenetically, the whole sensillum is derived from a single mother
cell (HENKE and RONSCH, 1951). Reviews on the fine structure of insect sensilla
have been written by SLIFER (1967, 1970), SCHNEIDER and STEINBRECHT (1968),
SINOIR (1969) and STEINBRECHT (1969b). For general organization of sensory
cells see THURM (1970a).
The olfactory cells of all insects studied to date are similar in construction
(Fig. 5). As in other sensilla of insects, the distal dendrite is separated into an
outer and inner segment (SLIFER, 1967; ERNST, 1969; THURM, 1965, 1970). Both
segments are connected by a thinner neck-like portion containing a ciliary struc-
ture. This part is always located proximal to the hair's base. Only the outer segment
of the dendrite lies within the hair lumen and may be several 100 [L in length
depending on the length of the hair (Fig. 6). Similar to other insect sensilla, the
outer segment contains no mitochondria. The only cell organelles found here are
neurotubuli and sometimes vesicular structures.
In the carrion beetle (N ecrophorus) these neurotubuli seem to have a morphogenetic func-
tion (ERNST, 1969). The outer segment contains about 30 neurotubuli. In the final stages of
ontogeny, the segment ramifies longitudinally into approximately 30 branches each containing
a neurotubulus. Presumably the tubuli limit the branching of the segment and prevent cross
division of the branches. These dendritic branches of the outer segment have a diameter of
500-1000 A and belong to the thinnest known processes of nerve cells.
The outer geometry of the cuticular parts of olfactory sensilla show.;; extremely
different types (Fig. 6). Obviously, one selective parameter in evolution is the
extent of exposure which the olfactory sense cell has to the stimulus-containing
medium (see below). The most sensitive of the antennae have hairs up to several
hundred microns long (Fig. 6a, sensilla trichodea). These hairs suggest a large filter
coefficient of the sensillum (G s ) in comparison to much smaller hairs which can be
interspersed with the long ones on the same antennae (Fig. 6b, sensilla basiconica).
The Morphology of Olfactory Sensillae 359
o e
air
--~
d
haemolymph
Fig. 5 Fig. 6
Fig. 5. Scheme of an olfactory sensillum with one sensory cell (sense c) and two formative cells
(tri c = trichogene cell, tor c = tormogene cell); epi c = epithelium cell; sept junct = septate
junctions; is = inner segment of the dendrite of the sensory cell; mit = mitochondria
(adapted from ERNST, 1969)
Fig. 6a-e. Types of olfactory sensilla: a sensillum trichodeum, long hair with the dendrites
of 2 sensory cells; b sensillum basiconicum, short hair with 3 sensory cells (Telea
polyphemus, saturniids); c sensillum coeloconicum, pit organ with the dendrites of 3 sensory
cells (grashopper Locusta migratoria); d sensillum ampullaceum, sunken peg with probably
only one sensory cell; e sensillum placodeum, poreplate with 18 sensory cells (d and e from
Apis melli/era)
~ .. wind direction
~-----
~~h"
.~..J
'~~
epithelium 50 Jl
Fig. 7. Olfactory pit of the fly's antenna, several sensilla basiconica with 2 sensory cells each.
Sarcophaga, from a photograph of SL1FER and SEKHON (1964)
360 K.-E. KAISSLING: Insect OHaction
Very often, small olfactory hairs (pegs) are set in pits with a small opening to
the outside (Fig. 6c, sensilla coeloconica and Fig. 6d, sensilla ampullacea). Even
several sensilla can be assembled within a common pit (Fig. 7). The location of an
olfactory hair within a pit suggests a low filter coefficient of the sensillum. Humidity
receptors and CO 2 receptors also belong to this type of sensillum (LACHER, 1964;
WALDOW, 1970), which function only at comparatively high stimulus concentra-
tions. A peculiar type of hair modification is the pore plate (sensilla placodea) of
bees (Figs. 6e, 11) and beetles (KRAUSE, 1960). This type of sensiIlum is obviously
very well protected against mechanical distortion, but by compromise, possesses a
lower effectiveness of adsorption (Qads) than a hair sensiIlum of equal surface area
standing free.
Analogous to the antenna as a whole the sensillum also has a filter coefficient Gs
(cf. formula I) which describes its efficiency as a target for molecules:
(4)
The adsorption quotient Qads (see above) of the hair-like sensilla is much greater
than 1 since the diameter of the hair is much smaller than the diameter of the
diffusion space around it (ADAM and DELBRUCK, 1968). The effective fraction of
adsorption Qeff is unity if one sets the total hair surface equal to the effective
surface from which all molecules have the chance to affect the sensory cells (see
p.355).
Calculating the filter coefficient of a sensiIlum, Gs ' it has to be considered
whether it stands alone or within a group of sensilla. A good approximation for Gs
is the filter coefficient of the antenna Ga divided by the number n of the respective
sensiIla.
G=~
s n (5)
For the long olfactory hair of the Bombyx antenna one obtains a filter coefficent Gs of 80 fLo,
for a whole poreplate of the drone bee a value of 0.63 fL' and for the coeloconic pit sensillum of
the locust only 0.015 fLo. These considerations show that due to the geometry of a sensillum
its filter efficiency may vary by at least 4 orders of magnitude.
In summary, two extremes of differentiation of olfactory sensilla have been
developed which are obviously adapted to the quantitative range of olfactory sense
cells either to very low or to very high concentrations: long hair-like sensilla with
well exposed surface suggest a high filter coefficient of adsorption, pit organs with a
small pit-opening a small one. Surprisingly, the wall structure seems to be correlated
with these properties: the free standing sensiIla are provided with a smooth
surface and a system of pores, cavities and tubuli as described below. The pit
organs have a ribbed wall with simple pores (without cavities and tubuli) filled
with electron-dense material. This correlation is not so obvious for very small
sensiIla and for pits with several sensiIla (Fig. 7) because here both types of wall
structure are present (MYERS, 1968; SLIFER and SEKHON, 1964; Du BOSE and
AXTELL, 1968; see also DETHIER et al., 1963).
The most striking speciality of olfactory sensilla is the fine structure of the hair
wall (Figs. 8-12). The hair wall of sensilla trichodea and basiconiJa is penetrated
by several thousand pores with an opening of 100-150 A in the minimum (SLIFER
et al., 1959; SLIFER, 1967; SCHNEIDER and STEINBRECHT, 1968; ERNST, 1969; Figs.
8a, 9, lOa). A fine channel leads from the pore opening to a widening, the pore
cavity or kettle. From here, 4- 8 tubuli go through the hairwall into the lumen of
the hair. Recently ERNST (1969) has proved that the pore system is open to the
Fig. Sa-c. Cuticular wall and dendritic branches of a sensillum basiconicum of the carrion
beetle Necrophorus. Longitudinal section, thickness of the section 500 A. Fixation with
osmium tetroxid, embedded in methacrylate. For explanation see Fig. 9. (Electronmicrographs
by ERNST, 1969). a section contrasted with phospho-tungstic acid, band c uncontrasted
section. The antenna was bathed in a protargol solution. Colloidal silver grains moved from
the outside up to the end of the tubuli (compare Fig. 9)
surrounding air
pore
cuticular
wall
QO[] pore keltle
pore tubule
receptor cell
dendrlle
L[=====J] IIOOOA
Internal flUid
Fig. 9. Schematic diagram of Fig. Sa. A contact between tubules and dendrites seems to be a
question of adequate preparation (see STEINBRECHT and MULLER, 1971)
362 K. - E. KAISSLING: Insect Olfaction
air. Colloidal silver grains of a diameter up to 100 Awere shown to have moved
from the exterior up to the end of the tubuli, but not into the sensillum fluid
(Figs. 8b, c). ERNST stated further, that the pore system as a whole is secreted by
the trichogen cell together with the cuticular hair wall. It is now understood that
Fig. lOa and b. Cross section of a sensillum basiconicum from the antennal surface (a) and
from olfactory pits (b) of the Florida Queen butterfly (Danaus gilippus berenice). The pore
tubuli in (a) running through the sensillum liquor, seem to contact the membrane of the
dendrite. 3 dendrites in a, 2 in b. Electronmicrograph (MYERS, 1968)
the well known pore tubuli (earlier called "pore filaments" , SLIFER et al., 1959;
SLIFER and SEKHON, 1964:; SILFER, 1967; RICHTER, 1962) are not processes of the
sensory cells' dendrites. It is however in question, whether the pore tubuli second-
arily come in contact with the dendrites. Electronmicrographs by SLIFER et al.
(1964:), MYERS (1968) and STEINBRECHT (1970, 1971) strongly suggest such a con-
nection (Fig. lOa).
Apart from these findings, ERNST (1969) discovered that the trichogene cell,
after forming the hairwall, withdraws from the hair lumen down to the hair base.
The free space which is left behind is then filled by an extracellular fluid, the sen-
sillum liquor. Finally, the outer segment of the dendrites of the sense cells
invades the hair (Fig. 5). Presumably this is the case in all the olfactory sensilla,
because no other cellular elements other than the dendrites are found within the
hair lumen. The liquor space continues below the hair base into a widening which
was earlier called a "vacuole" but is, in fact, an extracellular space surrounded by
the walls of the trichogen and tormogen cells. Both cells send microvilli into the
liquor space which suggests an exchange of material between cells and the
sensillum liquor (see below).
The pore plates (sensilla placodea) of the honey bee possess a pore system
similar to the one of the sensilla basiconica and trichodea (Fig. 11, cf. RICHARDS,
1952; KRAUSE, 1960; SLIFER and SEKHON, 1961; SCHNEIDER and STEINBRECHT,
Apis mellifera
1}J
Fig. II. Pore plate (sensillum placodeum) of the honey bee (Api8 melli/era). The oval plate
organ has a ringshaped pore field. The central plate is supported by radial ridges. Rows of
cuticular pores are arranged between the ridges. 4-5 tubuli belong to each pore. Only two
receptor cell dendrites running below the pore field are shown (from RICHARDS, 1952;
KRAUSE, 1960; SLIFER and SEKHON, 1961; SCHNEIDER and STEINBRECHT, 1968) (see Fig. 20)
1968). The pores are arranged in rows within a ringshaped pore field. Between the
rows of pores there are radial ridges connecting the surrounding cuticula with the
central plate. Pore plates of beetles show a divergent structure which so far is
unknown in detail (KRAUSE, 1960; IVANOFF, 1966). Another very exciting type
of plate organs was analyzed in aphids (Homoptera, SLIFER et al., 1964).
The pit organs (sensilla coeloconica and ampullacea) usually have a wall struc-
ture different from the free standing sensillae (Figs_ 10 b, 12). The wall of the pit
organs consists of complicated longitudinal ribs which are usually hollow in elec-
tron micrographs. Pores are located between the ribs. The pores are connected with
the inside of the hair by channels which are often filled with a very conspicuous
electron-dense material (Fig. 12). A similar material sometimes surrounds the
dendrites within the hair (STEINBRECHT, 1969b).
Fig. 12. Cross section of the cone of a sensillum coeloconicum of the locust (see Fig. 6c). The
three receptor cell dendrites lying in the center are surrounded by electrondense material,
which also fills the pores. The wall of the cone has a system of cavities and outer
longitudinal ribbons with some dense material on their inside (not in the scheme).
Electronmicrograph and scheme of STEINBRECHT (1969). The receptor cells of this sensillum
type respond either to water vapor or to odors (see p. 409)
The pore systems in the wall of olfactory sensilla probably are the routes by
which the odor molecules enter the olfactory hair. One of the problems arising here
is the avoidance of water loss. Experiments with Bombyx mori show that not only
the pores but also the total hair surface serves as a target for the odor molecules
cf. p. 384). Therefore the molecules adsorbed between the pores are able to reach
the pores by diffusion, possibly within the outer lipid layer of the cuticle (LOCKE,
1964, 1965; ADAM and DELBRUCK, 1968).
The minimum pore density in free standing sensillae, is about 5 pores per fJ-2 (Table 2). This
lower limit could be dependent on the effectiveness and velocity of diffusion of the odor mole-
cules within the surface layer. The lower limit for the total number of pores per sensillum seems
to be 3000- 5000. This figure is suggested by small basieonic sensillae with a pore density up
to 100 per fJ-2. A minimum number of pores per sensillum and, more important, a minimum
number of tubuli per sensory cell may be necessary to guarantee a good differential sensitivity
for odor concentrations (see below). The latter figure is at least 1000 and reaches 100,000, not
being correlated to the hair length (Table 2).
The functional significance of the pore cavities and tubuli is an open question.
These structures have amazingly similar dimensions in lepidoptera, beetles and
bees (summarized in SCHNEIDER and STEINBRECHT, 1968). In the sensilla trichodea
of these insect groups, the tubuli have a very constant diameter of about lOO A;
Methods for the Study of Olfactory Organ Function 365
the length depends on the wall thickness (1000-10000 A). Each pore has
4-8 tubuli. Some of the basiconic sensilla, however, have pore diameters up to
about 1000 A and up to 30 tubuli per pore (Table 2); these sensilla have many
sensory cells. No comparable data exist on the pore numbers of sensilla with a
ribbed wall structure.
Table 2. Morphological data of olfactory sensilla
Species sensillum length surface pores no. of tubuli no. of tubuli

([1) area ([12). ([12). pores pore cells cella
Antheraea trichodeum 300 3000 6 18000 4 (1-) 3 24000

pernyieS
Bombyx trichodeum 100 600 3-8b 2500b 5 2 6000
morieS
Necrophorus basiconic. I 40 150 100 15000 7 1 105000
vespilloides basiconic. II 15 50 100 5000 8 2 20000
Apis mellifera placodeum 40 125 5000 4 18 1100
Locusta basiconicum 10 150 20b 3000 20b 35 1700
migratoria
All values are rounded off; for references see SCHNEIDER and STEINBRECHT (1968, table 1) .
Newly calculated figures, b from STEINBRECHT 1970 and unpublished.
Olfactory hairs with a large surface area usually possess 1-3 sense cells, the
small sensilla and pore plates may have up to 30 cells. It has been shown electro-
physiologically, that different cells of the same sensillum often have a different
odor spectrum (SCHNEIDER et al., 1964; LACHER, 1964; KAISSLING and RENNER,
1968) or a different quantitative working range with similar specificity (PRIESNER,
unpublished results from Bombyx).
D. Methods for the Study of Olfactory Organ Function

1. Behavioral Responses
The function of olfactory organs can be studied indirectly through the obser-
vation of behavioral responses. In the simplest case, an application of a scent
stimulus releases a reflex-like response. For example Bombyx males respond to the
sexual attractant of the female by wing fluttering and - since they cannot fly -,
by walking upwind (positive anemotaxis). In cases of strong stimulation they show
a spiral searching pattern and movements of the abdomen (SCHWINCK, 1954, 1955).
The fluttering of the wings can be measured mechanically (Fig. 13) and can be
used for the determination of the male's olfactory threshold when stimulated with
the female sex attractant and other odoriferous substances (Fig. 14; SCHNEIDER et
al., 1967; KAJSSLING and PRIESNER, 1970). Other and similar reflex-like responses
to odors are known in many insects, for instance the alarm response or an oriented
approach flight.
The approach flight as elicited by an odor and directed to the odor source is a complex
behavioural response, in which olfactory as well as optical and other stimuli playa role. This
approach to odor sources (e.g. combined with traps) is often used in field experiments to test
the attractivness or the repellent properties of odor substances on insects. BOSSERT and W1LSON
(1963) developed mathematical models on the different types of chemical transmission in air
from an odor source. WRIGHT (1958, 1968), KELLOG and WRIGHT (1962) and TRAYNIER (1968)
studied the distribution of odor streams by smoke experiments and analyzed the complex
behavior reactions leading the insect to the odor source. GILLIES and WILKES (1968) suggest
that some mosquitoes are attracted from long distances by host odors, from a middle range by
CO 2 and finally by warmth, humidity and optical stimuli.
Bombyx mori
Bombyx mori 100 responses
0/0 I;/ i= -
1:/
50 / 1/
210'i1/ -
.
17 C
1.,. -
Fig. 13
0 / 1
C -6 -5 G -3 2
Fig. 14 I0910V9 Bombykol
Fig. 13. Registration of the fluttering response of the male silkworm moth. A thermistor
mounted above the head of the animal measures the onset of the odorous air stream (upper
trace). The response is registered by the pick-up system of a plate recorder (lower trace)
(KAISSLING and PRIESNER, 1970)
Fig. 14. Olfactory threshold of the male silkworm moth Bombyx mori. Percentage of animal
responses within 2sec, as a function of the load of an odor source (filter paper) with bombykol.
C = control responses with pure air. (KAISSLING and PRIESNER, 1970). The higher threshold
at 17 C is accompanied by a general sluggishness of the animal at low temperatures
Laboratory experiments usually allow a better quantification of the odor

stimulus. Insects are often made to select between the odor free and odor contain-
ing arms of a Y-maze_ In this way, MULLER (1968) for example could measure the
attractiveness and repellant properties of a number of substances and body fluids
in Aedes aegypti (Fig. 15).
During an insect's orientation to an odor source the olfactory organs not only
detect the presence of the odor but also its intensity and quality, as well as changes
in both parameters. The far-distance approach is usually an upwind orientation
(positive anemotaxis) which is elicited (in locusts, KENNEDY and MOORHOUSE,
1969) or maintained (in Drosophila, FLUGGE, 1934; in Bombyx, SCHWINCK,
1954) by the presence of an odor.
Drosophila melanogaster responds positive-anemotactically already with an air stream
velocity of 1.7 em/sec when the air contains odor of fermenting bananae (STEINER, 1954).
Other species of Drosophila respond to wind also without odor (KALMUS, 1942). Drosophila
melanogaster also responds osmotropotactically since flies with only one antenna turn to the
intact side (FLUGGE, 1934).
In the proximity of an odor source, bees can distinguish the spatial concentra-
tion gradient (MARTIN, 1964) but also the spatial pattern of odor quality (MARTIN,
1965) by comparison of simultaneous reception with both antennae. This ability
may be useful for orientation by means of spatial odor patterns of flowers (v. AUF-
SESS, 1960). KRAMER (unpublished) could show that honey bees are able to measure
the absolute odor concentration.
In his experiments honey bees walk on a motor driven ball which due to a feedback system
keeps the animal always on its top. During walking the odor concentration is controlled by the
position of the insect in the programmed field of odor concentrations. When a point is reached
with 90% of the odor concentration at which food was offered before, already 50% of the bees
stop walking and expose their abdominal glands "calling" other workers. Furthermore the
mechanism of the bees' olfactory orientation to food sources was thoroughly analyzed.
Aedes aegypti
100 responses
% formic attract ing
propionic
50 / neutral
repellent
O+-~--~~~~~~~~--~~~~~~
-5 -4 -3 -2 -1 o
10910 dilution steps
Fig. 15. Attraction and repulsion of mosquitoes by some fatty acids and lactic acid.
Percentage of animals walking into the odorant containing arm of a Ymaze. Abcissa:
degree of dilution of the odor substances on the source. 0 = undiluted substance. The
neutral behavior of the animals (dashed lines) at high concentrations could be an artefact
(redrawn from MULLER, 1968)
Many of the functional aspects of olfactory organs involved in orientation can

be studied behaviourally in conditioning experiments. VON FRISCH (1919)
demonstrated olfactory conditioning in insects with the honey bee.
He simultaneously offered to the bees several odorants each in a different box with an
entry hole. Only one of these boxes contained a dish of sugarwater as the reward. The bees
soon learned to fly to this box. When the sugarwater was withheld, the bees still flew to the
box with the odor substance to which they were conditioned, even though the positions of the
boxes were changed. Thus, the bees were shown to be capable of distinguishing between dif-
ferent odorants and odor concentrations (Figs. 16, 51; Table 9).
A combination of conditioning with an olfactometric control of stimulus
intensity shows that bees have acute behavioural thresholds for odors (SCHWARZ,
1955; Fig. 17) and CO 2 (LACHER, 1967a; Fig. 18). MARTIN (1964) conditioned
single bees to an odorant and thcn stimulated both antennae of the mechanically
fixed animal with different concentrations. By measuring the turning movements
of the animal he determined absolute and differential odor thresholds (Fig. 16,
for detection of absolute concentrations see above. See also p. 378).
Apart from the orientation behaviour of bees, the proboscis reflex can also be
used as a means of conditioning to odor and humidity (FRINGS, 1941; KUWABARA,
1957; 1961). In this way even drones could be trained to odor substances
TAKEDA,
(VARESCHI and KAISSLING, 1970 see p. 408). Proboscis reflexes are known in
many other insects but have not been used before as a means of training to odors.
Besides honey bees only a few insects have been conditioned to odors (a butterfly
by SCHREMMER, 1941; a beetle by SCHALLER, 1926 and ants by VOWLES, 1964).
Apis mellifera
100 + responses
%
o
0 Bromstyrol
.. A Methylheptenone
} distinguished concentrations
O+----.----,,---,,----,----,---~
-6 -5 -4 -3 -2 -1 a
10910 dilution steps
Fig. 16. Olfactory threshold of the honey bee stimulated with bromstyrol and methyl
heptenone. Percentage of correct choices, from values of v. FRISCH (1919, open symbols)
and MARTIN (1964, filled symbols). Explanation of the methods sce text. Abscissa: dilution
steps in paraffin oil (0 = undiluted)
Apis mellifera Apis mellifera

100 responses 100
_.-------------.-+
+
+ responses
% ,. n = 232 %

f 9
f~
. " ..
0
,
0 0 n=724
0
50 - - - - - - .---." -009' - -- - - - - neutral

__L___
nerol propionic acid 50
x=10 9 x=10 11
neutral
a lx 2x 3x 4x 5x 6x 7x
molecules / cm 3 air dist. concentr.
Fig. 17 U U U
a
0,1 10 100
Fig. 18 % CO 2
Fig. 17. Olfactory threshold of the honey bee for nerol and propionic acid. Percentage of
correct choices, from values of SCHWARZ (1955) using the olfactometer of NEUHAUS (1953)
Fig. 18. Sensory threshold of the honey bee for CO 2 , Percentage of correct choices between
three olfactometer outlets one of which was delivering CO 2 , Dist. concentr.: Concentrations
which have been distinguished by the animals preferring the higher one (redrawn from
LACHER, 1967)
2. Electrophysiology
A direct investigation of the function of olfactory organs, especially of single
sense cells, is possible by electrophysiological methods. Extracellular recording
shows graded receptor potentials (or "slow" potentials) as well as nerve impulses.
The recording of odor-induced summated receptor potentials (electroantennogram)
was first performed by SCIINEIDER (1957) in the antennae of the male silkworm moth
Bombyx mori. Nerve impulses from single olfactory cells were systematically
stimulus
recorded
receptor potentiat
+ j ~iff. el'
indiff.
0 ------ el.
----,,- _. - -_. - ... _- .. ---
- introce llu lar

el.
nerve impulses
o 0 o
Fig. 19. Schematic diagram of an olfactory sensillum according to THURM (1970a). Compare
Fig. 5. The sensillum liquor is positive (+), the cell's interior negative (-) with respect
to the haemolymph (0) (see text). The dashed line shows the potential between receptor
cell and haemolymph. This potential is thought to generate the impulses
recorded and quantitatively studied for the first time in the basiconic sensilla of
the Necrophorus beetle antenna (BOECKH, 1962).
For the recording from single olfactory cells, an electrode with a tip diameter
of about 1-10 [J. (different electrode) is inserted in the cuticle in the vicinity of one
of the olfactory sensilla (Figs. 19, 20). A second, broader, electrode (indifferent
electrode) is inserted into the antennal haemolymph. With excitatory stimuli
the different electrode usually becomes negatively charged by the receptor
potential. The nerve impulses, however, usually appear as positive deflections
(Fig. 21; BOECKH, 1962; LACHER, 1964). The same polarities have been observed in
insect mechanoreceptors and suggest a zone of high extracellular tissue resistance
between those parts of the receptor cell which cause the receptor potential and
those that generate the nerve impulses (THURM, 1963). This high resistance may
also explain the relatively small amplitude of the recorded nerve impulses when
compared with the amplitude of the receptor potential.
The extracellularly recorded slow potential reflects the receptor potential, which
is the first electrical response of a receptor cell to the stimulus. The receptor poten-
::!4 Rh. Sensory Physiology, Vol. IV/1
3iO K. E. KAISSLING: Insect Olfaction
tial is presumed to be caused by a change of the membrane conductance in the

more distal parts of the receptor cell under the influence of the stimulus (FUORTES,
1959). This potential change spreads out electrotonlcally to more proximal parts
ofthe receptor cell where it elicits a change of impulse activity. A depolarisation of
Fig. 20. Tungsten electrode penetrating one of the pore plates of a drone antenna
(photograph by courtesy of V. LACHER)
the receptor membrane causes an increase in impulse frequency, a hyperpolarisation

results in a decrease of the spontaneous activity often present in the unstimulated
cell. One has to remember that the extracellularly recorded receptor potential
differs in amplitude and polarity from the intracellular receptor potential which
triggers the nerve impulses (cf. Fig. 19).
ecrophor us \Ie splilio
--~----------~--- --- --- 100 msec
-2mVI
Fig. 21. Receptor potential and nerve impulses of an olfactory cell stimulated with carrion
odor. Extracellular DC-recording with microcapillaries from a sensillum basiconicum of
the carrion beetle N ecrophorus vespillio (BOECKH, 1962)
Recently THURM (1970b) found that the sensillum surface is positively charged
with respect to the haemolymph in the unstimulated state. This mostly neglected
permanent positive potential reaches up to + 80 m V in mechanoreceptive sensilla
and was also found in chemoreceptive organs (cf. WOLBARSHT, 1958). For instance,
the drone antenna, tightly paved with olfactory sensilla (pore plates), shows about
+ 50 m V between the outside of the antenna and its haemolymph space. This
permanent potential increases much the potential difference across the receptor
cell membrane in the resting state when compared with resting potentials of nerve
cells.
According to a highly attractive hypothesis of THURM (1970b), the permanent potential is
produced by an electrogenic ion pump which is located in the distal membrane of the tormogene
and trichogene cell adjacent to the sensillum liquor (Fig. 19). This location is suggested by the
special structure of the microvilli of this membrane and by the abundance of mitochondria
below it (Fig. 5). The permanent sensillum potential is quickly and reversibly abolished by
lack of oxygen, which indicates its origin from metabolic processes. The ion pump is thought to
produce the permanent potential by secreting potassium ions into the sensillum liquor.
Electrogenic potassium pumps are known from midgut epithelia of insects and show structural
and functional similarities (see THURM, 1970b). The stimulus controls the potassium perme-
ability of the dendrite membrane of the receptor cell. A short circuit between the positively
charged sensillum liquor and the haemolymph via the intercellular spaces is probably prevented
by the septate junctions always found between the cells of the sensillum and the epithelial cells
(Fig. 5). The opposite polarity of the recorded slow potentials and the nerve impulses (see
above) indicates that the impulse generation occurs proximal to the junction zone with its
high extracellular resistance.
By recording from the olfactory sensillum base, several receptor cells can be
registered simultaneously and distinguished as a rule by the amplitude of their
nerve impulses (Fig. 22). When recording receptor potentials from the sensillum
base it is usually impossible to distinguish whether they originate from one or
several simultaneously active cells. Several cells may be involved when the
relationship between the recorded slow potential and the impulse frequency varies
and shows a dependence on the odor substance (PRIESNER, unpublished).
Antheraea pernyi
a
i ,J i
c
Fig. 22a-c. Nerve impulses of the olfactory cells (large and small impulses) of a sensillum
basiconicum of Antheraea pernyi. Stimulation with terpineol (a), geraniol (b), isosafrol (c).
In a) one cell is excited, the other one inhibited. In b) and c) both cells are inhibited.
Extracellular AC-recording with tungsten wires. Impulse amplitude ca. 1 mV (SCHNEIDER
et al., 1964)
According to the hypothesis of THURM (1970b) sensilla with several receptor

cells would have a common battery (ion pump) which contributes to the membrane
potential of all cells. As a consequence, the stimulation of one special cell could
change the membrane potential of its neighbours and may influence this way
their impulse activity.
In many insects one can record an extracellular slow potential, the so-called
electroantennogram (EAG, SCHNEIDER, 1957) using broad electrodes, which are
inserted into the tip and base ofthe antenna (Fig. 23). The EAG is probably summat-
372 K. E. KAISSLING: Insect Olfaction
ed from the receptor potentials of many sense cells lying in series. This is indicated
by the fact that a depolarising stimulus negatively charges the distal electrode on
the antenna. This polarisation of the antenna is morphologically correlated with
the sense cell arrangement. The dendrites producing the receptor potential lie
partially within the lumen of the stem and branches before running into the olfac-
tory hair and are oriented in parallel to the antennal axis (Fig. 24).
Fig. 23. Arrangement for the recording of the electroantennogram (EAG). The glass capillary
electrodes, filled with ringer solution, are mounted on AgjAgCl-wires. The isolated antenna is
supported by the two glass electrodes which are inserted into its ends. On the upper right side
is the outlet of the olfactometer. At left two thermistors are mounted for measuring the
temperature and the air stream velocity (original photograph)
Fig. 24. Schematic longitudinal section through one branch of a saturniid antenna (from a
total antenna stained with methylene blue). The serial arrangement of the sensory cells
causes a summation of the receptor potentials of many cells between tip and base of the branch
or of the antennal stem (EAG). Each cell can be considered to become a small dipole under sti-
mulation, because the receptor potential depolarises only the dendrite of the cell. The
internal resistance of the antenna is in the range of 100 kQ between tip and base (KAISSLING,
unpublished).
SCHNEIDER (1957) has shown that the sexual attractant EAG of the Bombyx antenna
disappears when the animal is narcotized with ether, chloroform or killed with HCN vapour.
Female Bombyx antennae show a very weak EAG when stimulated with extremely high
bombykol concentrations (KAISSLTNG, unpublished).
No EAG-response of the female to its own pheromone was found in Periplaneta
americana (BOECKH et al., 1970) but in the honey bee all three castes resp:md to queen substance
(KAISSLING and REN~ER, 1968; RUTT~ER and KA1SSLING, 1968). Also the aphrodisiac
Dynamic and Static Properties of Olfactory Cells 373
compound of the male queen butterfly elicits EAGs in both sexes (Fig. 43, SCHNEIDER and
SEIBT, 1969).
Apart from the EAG, temperature-dependent potential changes which look like
an EAG occur that are not the result of a depolarisation of the cell membrane
(KAISSLING, unpublished). With extracellular recordings, one must always con-
sider that physical effects produced by temperature or even by odor substances
(KAFKA, 1970) can simulate slow cell potentials. EAGs of the noctuid moth
Trichoplusia ni were not influenced by light, day time and age of the animals
(PAYNE et al., 1970).
E. Dynamic and Static Properties of Olfactory Cells

With a rectangular stimulus, the receptor potential rises to a stationary plateau
level, often going through an initial phasic frequency peak (BOECKH, 1962; BOECKH
et al., 1965; KAISSLlNG, 1969; Figs. 25, 26, 27). The halftime of this rise can be
reduced to lO msec by strong stimulation (Fig. 56). The potential decays mono-
tonically after the stimulus end and never behaves phasically here. The lower
limit for the halftime of the decay seems to lie approximately at half a second, even
if a fresh air st,ream is directed over the antenna immediately after stimulation
Necrophorus humator
200 impulses/sec
100
o i I
o 500 msec 0 5 10 15sec
Fig. 25. Time course of the extracellularly recorded receptor potential and the impulse
frequency of an olfactory cell stimulated with carrion. Recording as in Fig. 21. Note the
different time scales at right and left (BOECKH, 1962)
Apis melli/era
odor stream no streom odor stream fresh Olr stream
odor 1 JJ9 queen substance -1 mV
0.6 sec
Fig. 26. Receptor potential recorded from a pore plate of the drone honey bee (thick line) and
the registration of the air stream velocity with a thermistor (thin line) (KAISSLING, 1969)
374 K-E. KArsSLING: Insect Olfaction
Thanatophllus rugosus Necrophorus

mY 200
-20 receptor potential carrion
i m~
sec mY
propion . ac . -15
-15
receptor
100 potentia l -10
-10
-5
-5 carrion
o 0
0 -.---,
sec 5
.5
.5
.10
.10
Fig. 28
.15
Locusta migrotoria
Fig. 27 impulses / sec .
100
k -
Apis mellifera
50
impulses/sec
100
~
50
50
1
0
O~Pj""
0 10 20 30 0 10 20 30 sec
1~ 1 ~ 95%
rei. humidity
L
J I , CI
I
5%r.h.
60 180 60 180
humld~
I I I
J LO% rei 0/. r.h
0 2 4 sec 6 0 2 4 sec 6
Fig. 29 Fig. 30
Fig. 27. Time course of the receptor potential of a depolarizing (carrion) and a hyperpolarizing
stimulus (propionic acid). (Recording as in Fig. 21, BOECKH, 1967b)
Fig. 28. Inhibition of a carrion-induced excitation by adding propionic acid. While the
receptor potential is hyperpolarized the impulse frequency becomes nearly zero. Recording
as in Fig. 21 (by courtesy of BOECKTJ, unpublished)
Fig. 29. Impulse response of a humidity receptor of the honey bee (above) stimulated by 95%
and 5% relative humidity of the air (below). Recorded with tungsten wires from a sensillum
ampullaceum (LACHER, 1964)
Fig. 30. Antagonistic impulse responses of a "humidity" receptor (above) and an "aridity"
receptor (middle) from the same coeloconic sensillum of the locust. Stimulus below. Recorded
with tungsten wires (WALDOW, 1970)
(Fig. 26). The time constants are often longer with hyperpolarising stimuli (Figs. 27,
28; BOECKH, 1967b, 1969).
The impulse frequency usually shows a strongly phasic initial peak up to
200 impulses per sec with a square wave pulse stimulus. The CO 2 -receptor of the
honey bee even reaches up to 500 impulses /sec (Figs. 25,31). When stationary, the
frequency does not exceed 50 impulses per sec and can be constant for ten or more
minutes. Contrary to thc slow potential, the impulse frequency often drops im-
mediately to zero when stimulation ends and returns after a short time interval to
l0.2.!!lsec -003
I -
I, I \ 2
, I I ~ I 5
, ~ I l 10
I 1
II I ~I. i 25
II III U 50 %
j- ,-j i I t I I ! 1100 C02
Fig. 31. Impulse response of a CO.-receptor of the honey bee. AC-recording with tungsten
wires from a sensillum ampullaceum (LACHER, 1964)
the spontaneous level. Such "off reactions" with phasic inhibition of impulses are
found in the queen substance receptors of the honey bee as well as in its CO 2 = and
in humidity receptors (Fig. 29,31; LACHER, 1964) and in the humidity receptors
of the grasshopper (Fig. 30; WALDOW, 1970). Contrary to the findings i.n these
cells, the impulse response of the highly sensitive Bombykol receptors has a more
tonic character without a marked initial peak and with a slow decrease of frequency
after the end of stimulation (Fig. 37; KAISSLING and PRIESNER, 1970).
Surprisingly, olfactory receptor cells of the generalist type in lepidopterous
larvae respond with complicated temporal patterns of spike-frequency which differ
depending on the odor quality (DETHIER and SCHOONHOVEN, 1969).
The reaction time from the beginning of the stimulus to the onset of the slow
potential or to the first nerve impulse, respectively, can with strong stimulation be
shorter than 10 msec and more than 100 msec with weak stimulation (Figs. 21, 31,
32,34,37). The reaction times between the end of stimulation and the reduction of
responses are usually not much longer (exception Fig. 37).
The stationary properties of the olfactory receptor cell can be described by the
stimulus response curve. Here the stationary amplitude of the response is usually
plotted against a logarithmic stimulus scale. The amplitude of the slow potential
can be considered as stationary 1 sec after the beginning of the stimulus (Figs.
25,32).
The stationary amplitude rises with the odor concentration at least over 2-3 log,o steps
(Fig. 33). The EAG of Bombyx rises over 5log,o steps (Figs. 42, 58), the EAG of the gypsy moth
was followed over 4log,o steps (STURCKOW, 1965). Also the mean of the impulse frequency
376 K..E. KAISSLING: Insect Olfaction
Apis melllfera Apis mellifera
mV receptor potential 0
c - 1mV -10
/0/
queen 0 caproic 1 0
acid
,
..i~ o
SUbstance! , 0 " ,0
3 "----------------------------
.---------.-----------
-5
,, .' / ~
'
'
1 I ,..-
."
0 capr. ac.
,,'
100 behavior . / i. '
jJ9 queen substance
1 sec . /
0/ 0 thresh .
.,- ,. '
0'
Fig. 32 o C 8 9 10 11 12 13 11.
Fig. 33 10910 molecules/em 3 air
Fig. 32. Receptor potentials (smooth line) recorded with glass capillaries from pore plates of
the drone stimulated with different loads of queen substance ([Lg) on the odor source. Rough
lines: Airstream velocity recorded by a thermistor (Anemometer from E. KRAMER,
Seewiesen; KAISSLING, 1969)
Fig. 33. Stationary amplitude of the receptor potential (after 1 sec stimulus duration) of
the queen substance receptor stimulated with queen substance and caproic acid. Dashed
lines : Response adapted by previous stimuli with high concentration of caproic acid
(KAISSLING, 1969). Behavior threshold for caproic acid according to SCHWARZ (1955)
Apis mellifera
Apis mellifera impulses
60 impulses SO

.-" .or
I. sec
50 ~ :
40 :;1'.--- 40
30 ~~. tests.
-- , ~.':.: : ::- ....1:::' ...
20
10 / spontaneous activity 0
0 0 0
0
0 0 8 0
0
0
0
0
msec reaction time
100 .....--0\. ' ___ " . tests , .
.

............ _\ ... . ---",. ...... .
SO ~., =- . --.---1-'
o ' .- .-:.
-1 0 .1 +2 m'V 10
log lO JJ9 Queen substance receptor potential ampl,tude
Fig. 34 Fig. 35
Fig. 34. Impulse response of a queen substance receptor averaged over 1 sec measured with
decreasing and increasing stimulus strength. In between two stimuli, test stimuli with
3 10- 1 [Lg queen substance were given. Below: Reaction time of the first nerve impulse.
Recorded with tungsten wires. (KAISSLING, unpublished)
Fig. 35. Relationship between the number of impulses within the 1St sec and the
stationary amplitude of the slow potential after one sec of stimulation with queen
substance (10' - 1011 molee/em 3 ) Circles: Values without counting the impulses of the first
100 msec (KAISSLING, unpublished)
(Fig. 31, 34) can have a broad quantitative working range (Fig. 36, 37). The impulse frequency
is not necessarily proportional to the slow potential amplitude, even if one excludes the initial
peak of frequency within the first 100 msec (Fig. 35). The relationship between odor concen-
tration and stationary reaction is markedly non-linear, at least for higher stimulus intensities,
and usually reaches a saturation level (for mathematical description see p. 415).
If different substances affect the same receptors unequally, then the stimulus
response curve can be shifted parallel to the abscissa on a logarithmic stimulus scale
(Figs. 33, 36) but can also change its shape (Figs. 36, 58). Apart from this the
stimulus response curve found with less effective compounds can also have a
lower saturation level (Figs. 42, 58).
Locusta migratoria 40 Locusta migratoria

impulses impulses

. l
500msec 200msec
50 ./ ..........-. 30 f-2-hexenoic a .
.........
I
./
t-2-hexenal
I
butyric acid
caproic / ./

./ ../ I ./
20
acid /
butyric
aCid /.
./. .1
10 / ........... .
/ / / ............ acrylic
............... ......-. ____:..........-. acid
O~--~--~~--~~~~ 0- - .-
10 11 12 13 15 14 16 2 3 4 5 6 7 8 9 10
10910 molecules/ cm 3 air 10910 mOlecules/sensiUum
Fig. 36a and b. Stimulus response curves of two single olfactory receptors (a and b) of the locust
(Sensillum coeloconicum). a Calibration of the stimulus scale with C14-caprioc acid and
gravimetrically for caproic acid and butyric acid (BOECKH, 1967 a). b Absolute calibration of
the stimulus scale using a gas chromatograph. Relative determination of the molecule
number adsorbed on the antenna using C14-caproic acid. The number per sensillum is the
average amount of molecules adsorbed per 15 [1.2 of the antennal surface, which corresponds
to the size of the sensillum opening (KAFKA, 1970). - The marked differences with
respect to shape and position of the curves of both cells (a and b) demonstrates the
natural variance between these cells. In a the impulses are counted for 500 msec from the
first impulse and in b within 200 msec during the maximum of frequency
The kinetics of the stimulus-response relationship have also been studied in the
dynamic phases of the receptor potential (EAG) in honey-bees and Bombyx
(KAISSLING, 1969). Here, the half-time of the potential increase was shortened with
increasing stimulus strength, but half-time of the decline in potential remains con-
stant over a wide range of stimulus intensities (Fig. 56). Decline time is found to
increase under strong stimulation (Figs. 56, 57).
Adaptation, defined as the reversible change of sensitivity produced by pre-
vious stimuli, ean take place independently at different levels. The first step lies
even before the receptor membrane level, at the stimulus conducting structures.
Under strong stimulation surface structures of the sensory hairs concentrate more
and more odor substance, so that after the end of stimulation, the stimulating
substance is still present, which can continue to affect the receptors and increases
the threshold for weaker stimuli up to several hours (Fig. 57; see p. 417). Further
adaptation certainly occurs by receptor potential build up in the membrane itself.

The impulse generation obviously adapts independently of the slow potential
adaptation, as PRIESNER (unpublished) has found in single Bombykol receptors.
Mter stimulation with 1 flog Bombykol, the slow potential can be elicited a second
time with the same amplitude. Impulses, however, completely fail unless there is
arestperiod of several minutes after the first stimulus. With the behavioral test
one has also to take into account central adaptation and fatigue of the motor
response as well as other factors influencing behavior (light, humidity, temperature,
time of day).
Extensive behavior studies on moths with respect to all of these factors are carried
out by SHOREY and his coworkers since 1962 (see SHOREY and GASTON, 1967; SHOREY et al.,
1968; BARTELL and SHOREY, 1969a, b). Adaptation of the EAG was observed by PAYNE
et al. (1970).
MARTIN (1969) found phenomena of adaptation and recovery in behavioral experiments
when honey bees were conditioned to an odor substance and afterwards a small amount of
the same compound was injected into the animal's blood system. It would be very interesting
to know which levels of adaptation were here involved.
F. The Sensitivity of the Receptors

The study of stimulus-response relationship in olfactory cells has two main
aspects, the aspect of sensory information and the aspect of sensory transduction.
The first concerns the data transfer from the "olfactory" environment to the CNS,
the latter deals with the biophysical processes which transform the chemical
stimulus into a nervous excitation. It is essential for the analysis of both aspects
to know the sensitivity of the receptor cell to a given odor substance.
1. The Relative Threshold

The determination of the behavioral threshold can give a relative measure of
the sensitivity of olfactory cells, as long one can be sure that the observed behavioral
response is elicited by the excitation of a single type of olfactory receptor cells all
of which have identical specificity. This type of behavioral response can be called
"one push-button" response in contrast to two or multiple push-button responses.
For instance, the fluttering response of the Bombyx male can be regarded as a one
push-button response (see below) with respect to olfactory stimuli.
Behavior thresholds can be expressed in numbers of molecules per cm3 air. For
the comparison of the effectiveness of different odor substances it is appropriate to
determine the odor concentration at which 50 % of the animals respond. This
threshold value is independent of the number of measurements in contrast to the
concentration value with the lowest significant percentage of responses.
Very precise measurements of insect behavior threshold have been performed
in the honey bee by SCHWARZ (1955).
He used the conditioning method of v. FRISCH (1919) combined with that of very accurate
odor measurement with an olfactometer developed by NEUHAUS (1953). This olfactometer
measured directly the evaporation rate of the odor substance from a capillary into a defined air
stream. The meniscus of the fluid in the capillary is controlled microscopically. This method
obviously reduces the error in determination of the odor concentration to less than 5%.
A graph of the data given by SCHWARZ shows that an increase of concentration
by 10 % over the just-subthreshold concentration produces maximal response
The Sensitivity of the Receptors 379
(Fig. 17). Far flatter threshold curves have been found in the honey bee by MARTIN
(1964), who placed glass tubes over the antennae. These tubes contained a different
concentration of odor solution on each side (Figs. 16). Then he measured the
animal's tendency to turn towards the side with the stronger odor. The conditi-
oning experiments of v. FRISCH (1919) as described above revealed even flatter
curves (Fig. 16). This comparison of results from different experiments on the same
species indicates the strong influence of experimental methods on the results
obtained.
The conditioned responses in the experiments of SCHWARZ (1955) probably
belong to the multiple button type of behavioral responses, since LACHER'S
(1964) electrophysiological studies suggest that several types of receptor cells are
involved in the perception of the compounds used by SCHWARZ. (see p. 409)
The lowest thresholds determined by SCHWARZ (1955) for several flower odors lie near
2 109 molecules/cm 3 Fatty acids have threshold concentrations nearly 100 times higher
(Table 3). Similar threshold concentrations were found in man for these substances. As is
the case in man and dogs, the honey bee also shows a greater sensitivity to butyric acid than to
any other homologous fatty acid. In contrast to this, the green.odor receptor of the locust is
more sensitive to caproic acid than to butyric acid (BOECKH, 1967 a).
Table 3. Olfactory threslwlds of man and worker honey bees (SCHWARZ, 1955) (Threshold
concentrations in molecules/cm 3 air)
Odour Man Author Bee
Propionic acid 4.2 . 1011 v. SKRAMLIK (1948) 4.3 . 1011

Butyric acid 7.0 10' v. SKRAMLIK (1948) 1.1.1011
Valerie acid 6.0 10'0 v. SKRAMLIK (1948)
iso-Valerie acid 4.5 10'0 SCHWARZ (1955) 1.6 1011
Caproic acid 2.0.1011 v. SKRAMLIK (1948) 2.2 . 1011
Ethyl caproate 1.3 . 1011 SCHWARZ (1955) 3.8 1011
Ethyl caprylate 3.7.10'0 SCHWARZ (1955) 5.4 10'0
Ethyl pelargonate 3.1 . 10'0 SCHWARZ (1955) 3.7 . 10'0
Ethyl caprate 4.2 . 10' SCHWARZ (1955) 5.6 10'
Ethyl undecylate 1.4 10'0 SCHWARZ (1955) 1.8 . 10'0
Methyl anthranilate 2.6 . 10'0 v. SKRAMLIK (1948) 1.9 . 10'
Phenyl propyl alcohol 6.5 10' SCHWARZ (1955) 2.210'
Nerol 5.710' SCHWARZ (1955) 3.2 10
Ionona 3.1 . 10 8 ZWAARDEMAKER 1.5 . 10'0
Eugenol 8.5 1011 Ohma 2.0 10'0
Citral 4.0 1011 v. SKRAMLIK (1948) 6.0 1011
When training honey bees with CO 2 , using a reliable stimulation method

similar to the one of SCHWARZ (1954) one requires a 100 %increase in concentra-
tion to obtain the maximum percentage of responses (Fig. 18; LACHER, 1967 a). In
this case we are dealing with a one push-button response since only one cell
type responds to CO 2 (LACHER, 1964). The threshold curve is flatter compared with
curves of SCHWARZ (Fig. 17), which may be due to the relatively small number of
CO 2 = receptors.
Recently odor threshold determinations have been reported from ants stimu-
lated with alarm pheromones. According to MOSER et al. (1968) the behavioral
380 K.E. KAISSLING: Insect Olfaction
threshold for 4-methyl-3-heptanone lies at 2.7.10 7 molecules per cm 3 air in the

ant Atta texana.
This value is based on a dilution procedure by means of a syringe. In this, a saturated
atmosphere was diluted with pure air by pushing and withdrawing the syringe plunger several
times, diluting the vapour concentration by a factor of 10 on each occasion. Then 1 ml of the
diluted vapour was released into a 6000 ml container with the animals. The variance of his
method is not yet known. Also it has not been proven whether the volumetric procedure
guarantees the correct dilution factor. VICK et al. (1969) used the same method and found a
threshold value of 9 . 1013 molecules of 4-methyl-3-heptanone per cm3 in Pogonomyrmex. This
value has to be corrected by the factor 1000 into 9 lO lD molecules (VIeK, personal com-
munication).
The threshold for the alarm pheromone undecane was on the average near 1010
molecules per cm 3 in Lasius alienus (REGNIER and WILSON, 1969) and near 1012
molecules per cm 3 in Acanthomyops claviger (REGNIER and WILSON, 1968, see
Table 4). The latter species responded to 1010 molecules/cm 3 of citronellol.
Table 4. Alarm response of the ant Acanthomyops claviger to compounds found in the
mandibular gland (M) and the Dufour's gland (D). Average threshold concentrations of n
behavioral tests and [Lg content of a single gland (REGNIER and WILSON, 1968)
molecules
Compound cm 3 air n [Lg
Citronellol 1.7.1010 5
2-Tridecanone 2.8' lO lD :3 1.88 D
Citral 7.0lO lD 3 0.3 M
Citronellal 1.6 . 1011 5 4.3 M
Undecane 1.1 . 1012 20 2.48 D
2,6-Dimethyl-5-hepten-l-01 1.2 . 1012 2 0.054 M
ClO. 12, 13-alkane ~2 . 1012 >20 (C 13 ) 0.02 D
Methyl decanoate 4.2' 1012 4
Nonane 1.5. 1013 ()
Butylether 1.4 . 1014 4
Heptane 4 .101 3
Pentane 9 . 101 n
As NEUHAUS did, these authors measured the loss of the odor substance evaporat-
ing from a capillary into a closed container holding several animals. They measured in addition
the distance of an animal from the opening of the capillary and the reaction time of the animal
after introduction of the capillary. They then calculated the dispersion of the odor by diffusion
neglecting convection. From this they obtained the concentration near the animal at the time
of its reaction (WILSON et al., 1969). The values for each compound varied by the factor 1000.
It is not known to what extent this variance depends on their cxperimental procedure.
In some cases the stereotyped alarm response of ants may be of the one
push-button type as for instance in Atta texana since 4-methyl-3-heptanone acts
very specifically (see below). Several receptor types may trigger the alarm response
of Acanthomyops claviger because of the rather unrelated compounds involved
(Table 4).
Also in the case of the electroantennogram one has to prove whether it is generat-
ed by one or several types of receptor cells. The latter may be true in the fly
Lucilia sericata. KAY et al. (1967) found thresholds between 1012 and 1014 molecules
per cm 3 by recording slow potentials from the antennae.
They injected saturated odor vapor from a motor-driven syringe into a defined air
stream. The odor concentration was checked by means of a gas chromatograph monitor. In
addition, they measured spectrophotometrically the time course of the stimulus concentration
within the experimental chamber. The stationary amplitude of slow summated potentials
(EAG) recorded from olfactory pits was found to be proportional to the logarithm of stimulus
concentration. The intersection of the stimulus response curves with the abscissa was taken as
the threshold concentration. The threshold of a particular substance varied within the limits
of one loglo step. The 18 tested substances were from different classes of odors.
The relatively high threshold concentration they found may suggest that no
substance of "interest" for the fly was included. On the other hand, the behavior
threshold of the fly is probably lower than the EAG threshold for the same sub-
stances. The relative effectiveness of these compounds differs from that in man.
2. The Absolute Threshold

The definition of the threshold stimulus applied in streaming air (only this is
considered here) contains the odor molecule concentration, c, the air stream velocity,
v, and the stimulus duration, t.
mOleCUleS)
1= c v ( cm 2 sec '
(6)
is the rate of particle transfer across unit areas and can be termed the particle
stream intensity I.
mOleCUleS)
N=cvt=It ( (2)
2 cm '
is the particle transfer across unit area during the stimulus. All three stimulus
parameters (c, v, t) may influence the number of adsorbed molecules Nads on the
antenna (see p. 356). Nads divided by N results in the product of the antennal
outline area Foutl> and the adsorption quotient Qads:
Nads_. 2
(7)
N - Foutl Qnds (cm ) .
As discussed above Qads is not necessarily a constant and may depend on c, v, t and on
the stimulus compound (see p. 356). Moreover the geometry of the antenna strongly influences
Qads (see p. 357).
From Nads one can calculate the number of molecules N elfs which impinge on
the effective surface F elfs of a single sensory cell. The surface area of sensory hairs
or plates or the area of a pit opening can be regarded as the effective surface of the
sensillum and has to be divided by the number of receptor cells per sensillum in
order to find F elfs (see p. 360). The fraction of Nads impinging on the total effective
surface of any particular sensillum type is Qelf (= effective fraction of adsorption)
which is to be divided by the number n of sensory cells in this type of sensillum
N elfs = N nds ' nQelf (molecules). (8)
Qelf is the effective surface of all concerned receptor cells as a fraction of the
total antennal surface Fa, when the adsorption is homogen20us all over the antenna:
Q _ Felfs 'n
(9)
elf - F. .
This may be true in the drone bee antenna, which has nearly exclusively pore
plate sensilla. Inhomogeneous adsorption has to be expected in the case of antennae
with long hair-like sensilla (see p. 355), which adsorb more molecules per surface
area than other parts of the antenna. With inhomogeneous adsorption Qclf has to
be determined directly
Q = h . F elfs . n (10)
elf Fa'
The factor h equals unity with homogeneous adsorption and is greater than
unity when the probability of adsorption is increased in the sensilla compared
with the rest of the antennal surface.
In summary, the number of molecules adsorbed per sensory cell can be
calculated for homogeneous adsorption
Feffs
Neffs = c V t . Fout! . Qads . ~ (11)
a
and for inhomogeneous adsorption
v . t F out!' Qads' - - .
N elfs Qelf
= C (12)
n
Threshold calculations were made for three types of receptor cells and are
summarized in Table 5. BOECKH (1967 a) determined the threshold concentration
of C14 -labelled caproic acid which elicits a few nerve impulses in single grass odor
receptors of the locust.
The odour source was a piece of filter paper soaked with the odorant + paraffin oil and
placed into a glass tube at the end of an air flow system. BOECKH blew the air stream carrying
the labelled compound into a plastic bag and washed out the radioactivity.
Table 5. Calculation of
c v I N Fout!
Definition c'v c.v.t
Dimension molec. cm molec. molec.

sec mm'
cm a sec cm" sec cm'
Bombyxm.,
bombykola 3.1 . 104 57 1.77 . 106 1 1.77 . lOs 6
behavior thresholdb 650 57 3.7 .104 0.86 3.2 .10 4 6
Locusta m.,
caproic acid (BOECKH) 1.1 . 1010 250 2.75 . 101 0.5 1.4 . 1012
(KAFKA)C 5.109 500 2.5 .101 0.6 1.5 101 2e
trans-2-hexenal (KAFKA)C 5 10 7 500 2.5 .1010 0.6 1.5 . 1010 2e
Apis m., Drone
caproic acidd 3 . 1012 160 4.8 . 1014 1 4.8 . 1014 1.12
9-oxo-trans-2-decenoic acidd 3 lOS 160 4.8 . 1010 1 4.8 . 1010 1.12
Stimulus: 10- 4 [lg bombykol on the odor source. Response: 0.35 impulses per stimulus.
a
Stimulus: 3 10-6 [lg bombykol. The initial odor cloud (30%) was prevented. Response:
b
significant percentage of fluttering males (see Fig. 38).
Response: about 0.2 impulses per stimulus.
C
The threshold concentration from C14-experiments was about 3 times larger than the one
found by measuring the weight loss of the odour source which has been exposed to a constant
air stream for several minutes (c = 3.3 . 109 molecules per cm3 air, extrapolated from 104 times
higher values). This discrepancy may be due to a decrease of odor evaporation during the first
seconds of the 5 min exposure to the air stream. The threshold concentration of hexenal deter-
mined by weight loss only was 3.6' 10 8 molecules per cm 3 air.
Recently KAFKA (1970) continued these experiments using a different method of stimu-
lation. He placed a small glass beaker containing C14-caproic acid diluted with paraffin oil
into a syringe. After several seconds the vapor within the syringe reached an equilibrium con-
centration. Then the plunger was pushed by a motor and the air stream was directed towards
the place where the recording electrode was positioned on the antenna.
Following SCHNEIDER et al. (1968), KAFKA determined first the amount of
radioactivity leaving the odour source (syringe) and second the amount of radio-
activity adsorbed on three parts of the antenna. Assuming homogeneous distribu-
tion of adsorbed molecules on the antenna, 190 molecules of caproic acid would
fall into the pit opening of 15!l2 with 3 sensory cells. Therefore about 60 molecules
elicit one nerve impulse in 20 % of the stimuli, which was the threshold response.
One may conclude that an average of 300 molecules of caproic acid is necessary
per nerve impulse.
Here the question has to be raised of whether the local density of adsorbed molecules may
vary due to complex aerodynamic conditions, perhaps induced by the recording electrode. This
systematic variance could be large since the threshold stimulus N transports 1.5 . 1012 molecules
per cm 2 or 2.25 . 105 molecules per 15 fl.2. Therefore about 1000 times more molecules would
enter the sensillum pit recorded from when the air stream passes the pit opening unhindered.
The adsorption quotient Qads of caproic acid in the locust antenna can be
approximately derived from the data given by KAFKA (1970) and was somewhat
olfactory thre8hold in insect8
Noutl Nads n Neffs Fa Feffs Neffs

h>1 h=l
Nads Neffs' n Nads' Qeff Nads' Feffs

NFoutl NoutI Qads - N - - -
outl Nads n Fa
molec. molec. molec. molec.
1.06 lOS 2.9 10' 0.27 0.75 32000 0.68

1.9 .103 520 0.27 0.75 32000 0.0122
3 . 1010e 8 .1070 0.0027 6.3e 15/3=5 63.5

3 '108e 8 lOse 0.00271 6.3e 15/3=5 0.635
5.4 .1012 2.7 . 1011 0.05 3.5 40/18=2.2 1.7.105

5.4 . 108 2.7' 107 0.051 3.5 40/18=2.2 17
d Response: about 5 impulses per stimulus.

e Values of that part of the antenna (6 mm long) to which the odor stream was
directed.
1 The values was taken from the experiments with HC-caproic acid.
smaller than found by KAISSLING in the honey bee with the same air stream
velocity (see p. 356).
This difference is probably due to the relatively small cross-section area of the airstream
used in the locust. The determination of Qads, however, requires an air stream cross section
larger than the outline area of the one antennal part used for the determination of N ads.
The threshold concentration of hexenal was found by means of a gas-chromato-
graph and by extrapolation over 3 orders of magnitude. Assuming adsorption
properties of hexenal similar to those of caproic acid (Qads = 0.0027), only 2 mole-
cules of hexenal would penetrate the pit opening directly on the average and
2000 in the (theoretical) maximum. The threshold response to hexenal of one
of the three cells per sensillum was one nerve impulse in 20 %ofthe measurements.
Therefore, the number of molecules per nerve impulse lies between 3 and 3000.
KAISSLING determined the threshold of the honey bee queen-substance receptor
to queen substance and caproic acid (1969 and unpublished). The calibration of
BOECKH (1967 a) was used for the threshold concentration of caproic acid (Table 5).
The Qads was found to vary strongly with c, v and t (see p. 356). The threshold concentra-
tion of queen substance was roughly calculated from the loss of efficiency of the odor source
under constant air flow conditions, this being checked against sources in still air by the cell
reaction itself. The effective surface area belonging to one receptor cell was estimated as 2.2. [1.2
which is 1/20 of the ring-shaped pore field (Fig. 11).
Assuming homogeneous distribution of the adsorbed molecules and a Qads
similar to that found with caproic acid one calculates Neffs = 17 molecules of
queen substance per 5 nerve impulses which was the threshold response. Since the
particle transfer at threshold is about 1 . 103 molecules per 2.2 [L2 one can conclude
that 3-200 molecules elicit one nerve impulse in the queen substance receptor. It
is not yet finally proved in the locust and the bee whether a single molecule is
enough to elicit a nerve impulse.
KAISSLING and PRIESNER (1970) determined the threshold of single receptor
cells in the male Bombyx, which reacts very sensitively to the female sex attractant
bombykol. This substance was analyzed as lO-trans, 12-cis-hexadecadien-l-ol
(BUTENANDT et al., 1959) and was synthetised in a tritium-labelled form by
KASANG (1968). The specific activity was 32 [LCi/[Lg and the minimum of detection
by a scintillation counter was below 109 molecules or 3 . 10- 7 [Lg of the labelled
compound.
The odor source was a piece of filter paper loaded with bombykol and placed into a glass
tube (stimulus cartridge) which was connected with the outlet or the air flow system. The
stimulus was an air puff of one sec directed either towards the antenna or into a second (filter)
cartridge containing a piece of cotton which adsorbs about 99% of the molecules leaving the
stimulus cartridge.
The adsorption quotient of the Bombyx antenna with respect to Bombykol
(Qads = 0.27) is much larger than the adsorption quotients calculated for the honey
bee and the locust antenna, which was theoretically expected from the arrange-
ment of 16000 long hair-like sensilla trichodea (see p. 355). Furthermore, we used
an effective fraction of adsorption Qeff = 0.75 for the threshold calculations in
Bombyx. This value was theoretically expected (ADAM and DELBRUCK, see p. 355)
and experimentally verified by STEIN BRECHT and KASANG (unpublished) who
measured separately the amounts of 3H-bombykol adsorbed on the hairs and on
the rest of the antenna.
The threshold concentration obtained with 10-4 [lg of bombykol on the odor
source was extrapolated from measurements with loads of 102 down to 10-2 [lg,
which show a release of odor molecules nearly proportional to the load. The number
of molecules adsorbed on the effective surface per stimulus and per receptor cell
was Neffs = 0.68. From 895 stimuli an average number of A. = 0.34 nerve impulses
above the spontaneous noise was elicited. Although this calculation reveals an
Bombyx mori
10'"'}Jg
lO'"'JIQ
10'"'}Jg
I
1O-3JJg
I, II IIII
Kl-2J1g
I Ii
11111 I"
III I I II
II
I I
10-1JIg
1 JIg
Fig. 37. Impulse response of a bombykol receptor of the male silkworm moth. AC-recording
(glass capillaries) from a sensillum trichodeum (scheme right above). With 10- 8 [.Lg
bombykol on the odor source, approximately 7 molecules per one sec stimulus duration are
adsorbed per one receptor cell (KAISSLING and PRIESNER, 1970)
average of 2 molecules per nerve impulse it can be proved that one molecule is
enough to elicit one nerve impulse and that the rest of the molecules gets lost on
the way from the adsorption place to the sensory cell.
At fir8t the distribution of the nerve impulses at different stimulus concentrations fits
nicely with a random distribution (Poisson distribution, see Fig. 38). Moreover the average
number of impulses per stimulus}. increases proportional to the stimulus intensity up to 10-3 (J-g
of bombykol on the odor source. This type of impulse distribution alone would not yet indicate
that a single molecule is enough for an impulse response. But this is highly probable since Neffs
and), are so close together. The two hit hypothesis, however, which says that at least two
molecules are necessary, is definitely excluded.
The Poisson equation was used in the following form:
k-l }.k. e-'<
P~k = 1-};
o
-k-'-'

(13)
P ~ k is the probability of the occurrence of k or more (= at least k) impulses per stimulus.

P~k is here expressed as the percentage of stimuli with ~ k impulses.
The final proof that the molecules do not act together at all in eliciting the impulse firing
comes from behavioral experiments. The behavioral response is significant with a stimulus of
25 fib. SClll!ory Physiology. Vol. IV/l
386 Ie-R. KAISSLING: Insect Olfaction
3 . 10- 6 !Lg or N ads = 520 molecules (Fig. 38). With Qeff = 75 % only 310 molecules fall on
the 25000 receptor cells of the sensitive type (80% of 32000 bombykol receptors). Accor-
ding to the Poisson equation only two of the cells receive double hits.
Linear extrapolation of the impulse number, however, reveals 160 impulses of the 25000
receptor cells. The stimulus concentration has to be 10 times higher in order to get 160 double
hits. The calculation of the theoretical maximum of adsorption (Qads' Qeff = 1) reveals a
5 times higher average molecule number per cell. One has to consider, however, that a con-
siderable part of the air stream will be deflected around the antenna, thus decreasing the Qads.
Bombyx mori
100 e % behavior /e-~'T7'~-5- - 0

% responses / :' f .-
/ i:,. : .
I '
" I
e / /V~3!
: : + :
" I
:: : c
50 e /: v~4:
/ / 'I +~ !
I
I % celts with
d !: v~51
:
I
e
. -: 0 ~ 1 impulses
,/ I +! A ~ 2 impulses
e / / : 0 ~10impulses
/~1 /~2 1;1 !~10
o 9 -----.. --~.-;/ ---..~-.---+!-",-/_/---"-----,,
-6 -5 -4 -3 -2 -1
10910 jJ9 Bombykol
Fig. 38. Evaluation of several 100 impulse recordings with different loads of the odor source
(see Fig. 37). The percentage of measurements with ~ 1 and ~ 2 impulses fits with
theoretical Poisson curves (hatched lines). At 1O- 3 !Lg several values are significantly
higher than the theoretical values (+). Only less than one percent of cells fire at the behavior
threshold. The spontaneous activity of the cells was subtracted (changed after KAISSLING
and PRIESNER, 1970)
Control experiments without bombykol odor showed a spontaneous activity of

0.085 impulses on the average per cell and per second. 1070 measurements of each
2 sec were evaluated. The distribution of impulse numbers was the following:
14.4 % of measurements with at least 1 impulse (14.4 %),
2.06 % of measurements with at least 2 impulses (1.14 %),
0.47 % of measurements with at least 3 impulses (0.06 %).
The experimental values are somewhat larger than the theoretical values (in
brackets) of a random (Poisson) distribution, which indicates a slight facilitation
of impulse firing by a preceding impulse. A similar effect is obvious at a stimulus
of 10-3 [Lg Bombykol which elicits significantly more impulses than expected from
the Poisson distribution (Fig. 38).
The significant behavioral response with 3 . 10-6 [Lg of Bombykol corresponds
to a significant signal to noise ratio of the nerve impulses. The 2 x 160 odor-
induced impulses of both antennae have to overcome a noiBc level of 2 x 1800 im-
pulses. The signal of 320 impulses lies just above the critical value of 3 . Vn
= 3 . V3600 = 180.
Specificity of Receptors 387
The entire threshold curve of the behavior reaction in Bombyx rises to maximum
within a hundredfold increase in stimulus strength (Fig. 14). Usually this type
of threshold curve is regarded as cumulative Gaussian distribution which has
the maximum at the 50 % point indicating the most probable position of the
threshold. The width of this type of curve indicates the variance of the threshold
due to variance of the stimulus, of the sensory response, and of the central data
processing leading to the motoric response. There may also be intraindividual
differences.
In Bomhyx it has been shown that variances in the stimulus and sensory responses are not
important. The signal to noise ratio of the sensory input becomes significant at a 10 times lower
stimulus intensity than needed for the 50% point of the behavior response. Therefore the
variance may be due mainly to the central processing which possibly varies inter- and intra-
individually. Fig. 39 shows a markedly non-linear relationship between the percentage of
receptor cells firing impulses and the percentage of behavior responses.
A marked non-linearity of the central data processing is also shown in the honey bee. Here
the behavior threshold has been found to be dependent on the number of sensory cells (Fig. 40).
The threshold does not rise very much on ablation of 7 of the 8 antennal segments supplied with
oHactory organs (DOSTAL, 1958). This indicates that the oHactory threshold lies far above the
1
critical signal to noise ratio of the sensory input, as described for Bomhyx.
100 + responses
---.-.-.
% Leptinotarsa
Bombyx mori
100 behavior I.
responses / ___ /.
50-" , , , I ' neutral
"10 //~
~~
100
% /.-.-.
sensitivity _ _ .

50//
rif
50
Apis mellifera
O~-r-r~~~r-~'--r-r-' O.'~'~'~'~'~I~~'~'~'-'I
o 50 "10 100 o 50 % 100
sensory cells with ~1 impulses number of sensRia
Fig. 39 Fig. 40
Fig. 39. Relationship between the sensory input and the motoric output (wing fluttering)
in Bombyx
Fig. 40. Relationship between the number of sensilla and sensitivity of the behavior
response in the honey bee (DOSTAL, 1958) and the potato beetle (SOHANZ, 1953), found by
ablation of antennal segments. The stimulus was the scent of potato plants in the beetle
and nerol in the bee (sensitivity = reciprocal of threshold concentration which is 3.23 . 109
molecules/cma at 100%)
G. Specificity of Receptors
Electrophysiologically, the specificity of a receptor cell is determined by the
stimulus-response curves of all effective substances. In addition to these charac-
teristics the time course of the excitation produced with similar stimuli (e.g.
25*
388 K.-E. KAISSLlNG: Insect Olfaction
square wave pulse) can be substance-specific (DETHIER and SCHOONHOVEN, 1969).

Ideally the receptor potential should be used in the investigation of specificity. But
in the case where it can originate from several simultaneously active cells the
activity of an individual cell can only be measured by means of the nerve impulses,
which usually differ in shape and amplitude from cell to cell (Fig. 22). In studying
specificity one has to consider adaptation, which may change the characteristics,
thereby distorting the specificity of the cell itself.
The effectiveness of a substance may be expressed by the reciprocal of its
threshold concentration. Another measure for the effectiveness of a compound is
the reciprocal of a concentration which elicits a half maximal cell response. Both
measures can give different results when the shape of the stimulus response curves
on a semilog scale differs depending from the compounds used, as is the case in
the Bombyx EAG (Figs. 42, 58) or in the impulse measurements from the locust
(KAFKA, 1970; Fig. 36).
The specificity of a receptor cell is completely described by its reaction spectrum
(= all effective compounds) together with the threshold concentrations provided
that the shape of the stimulus response curve is equal for all compounds in a
semilog plot.
To date, only about a dozen types of cells have been studied in insects. Their
specificity is only incompletely known in the sense of the postulates mentioned
above. Nevertheless, it appears that two opposing principles in the development
of specificity can be distinguished (SCHNEIDER et al., 1964). Most olfactory cells
found to date belong to the group of "specialists". These are cells with identical
specificity which occur in numbers on the antennae. To these "specialists" belong,
for example, sexual attractant receptors and possibly some food receptors and
also the CO 2 = and humidity receptors. Many different "specialists" can occur on the
same antenna and even belong to the same sensillum. Thus one finds with the same
electrode position in the honey bee pore plates, cells which respond to queen
substance and others responding to the scent of the Nassanoff gland (KAISSLING
and RENNER, 1968). As far as is known the odor spectra of both ofthese honey bee
"specialists" show no overlap (see p. 409).
In bees (LACHER, 1964), Antheraea (Figs. 22, 50) and lepidopterous larvae
(SCHOONHOVEN and DETHIER, 1966) cells have been found each of which show
different but overlapping odor spectra. These cells are termed "generalists" and
respond in these animals to flowery, aromatic and other odors.
On the one hand, an extreme "specialist" is obviously constructed for the
exclusive, and often the most sensitive, perception of a particular odor substance;
on the other hand the "generalists" as a group cover a large spectrum of substances
and may enable the animal to distinguish numerous odor qualities.
An intermediate cell type of "specialized generalists" (or as well "generalizing specialists")
seems to occur rather frequently. The same antenna has several groups of cells which differ
strongly between the groups but also to some degree within each group with respect to their
specificity (in mosquitoes: LACHER, 1967 b; in locusts: KAFKA, 1970; in honey bees: VAREscm,
1971, see p. 409).
1. Odor Specialists of High Specificity

For a study of receptor cell specificity, the "specialist" cells are of course
appropriate subjects. Since they often comprise a large proportion of the odor
receptors of the antenna, they can be studied not only by single cell recording but
also by the EAG. Here one has to be sure, however, that except for the studied speci-
alist no other cell type reacts to the compound used and contributes to the type
of measured response (EAG). The same condition restricts the use of behavioral
responses for studying receptor cell specificity. In some insects, however, a stereo-
typed behavioral response seems to be elicited olfactorily by a single type of receptor
cell. This type of behavioral response was above called a "one push button"
response in contrast to "multiple push button" responses which can be started
from different types of olfactory cells.
Bombyx mori
mV EAG-amplitude
'--~"
Bombyx mori -2
HH H /TC '"
100 responses - C = C - C = C . CHzOH ........
% t H t CT >~o
12 cis 10 trans / o_~ __ o- ....
TC-Bombykol ,/ CC,O-/-'
/ ' ,<::~~~.
-1
so
0
/ ' _0' .t/'

0-.-.- -/_;0-'
g~8~&~&
",,"" .: :-: : :. -,'/,--0
-6 -5 -I. -3 -2 -1 0 .1 -5 -4 -3 -2 -1 0 +1 .2 .3
log10 Jig Bombykol and isomers log10 Jig Bombykol (TCl and isomers
Fig 41 Fig. 42
Fig. 41. Behavior thresholds of male silkworm moths for bombykol (TC) and two of the three
isomers (CT, TT, cf. Fig. 42). Percentage of responses with different loads of the odor source.
The experimental arrangement was quite different from the one used by KAISSLING and
FRIESNER, 1970 (cf. Figs. 13, 14; SCHNEIDER et al., 1967)
Fig. 42. Maximum amplitude (quasi stationary) of the summated receptor potential (EAG)
of male silkworm moths as a function of the load of filterpapers with bombykol (TC = hexa-
deca-10 trans, 12.cisdien-1-ol) and its stereoisomers (CT, CC and TT) (SCHNEIDER et al., 1967)
Behavioral responses, EAG and single cell recordings have been used in
Bombyx mori and have shown the same results for the rank order of effectiveness
of bombykol and its three stereoisomers (Figs. 41, 42}. The behavioral tests,
however, showed a much greater difference between similarly effective concentra-
tions of bombykol and its isomers than the EAG. Thus, the 50 % behavior thresh-
olds diverge by 4-51og1o units (Fig. 41). The EAG curves indicate only a difference
by 1-21og1o units (Fig. 42), when the concentrations are compared at about half
maximal amplitude.
Here, one has to consider that the threshold concentrations for the behavior response lie
more than 4log1o units lower than those producing a haH maximal EAG (see below). The ratio
of effectiveness between bombykol and the isomers is obviously changed, together with the
absolute concentration range. Yet, there is no explanation for the relatively improved effect of
isomers at higher concentrations. Therefore one cannot determine whether the effectiveness of
these compounds is more adequately described by the behavior test or by the electrophysiologi-
cal measurement at higher concentrations.
390 Ko-E. KAISSLING: Insect Olfaction
The most peculiar feature of many "specialists" is their sharp specificity. Every
minute change in construction of the key molecules tested so far is followed by a
100 to lOOO times reduction in their effectiveness (Figs. 41, 42, 53). As in the case of
bombykol, 8tereoi8omer8 of other pheromones tested behaviorally have a strongly
reduced activity. Thus the ci8-form of the sex attractant trans-7-dodecen-1-yl
acetate (Argyroploce leucotreta, READ et al., 1968) is reported to be inactive. The
ci8-7-dodecen-1-yl acetate, however, is the sex attractant of Trichoplusiani (BER-
GER and CANERDAY, 1968). The trans-form is 300-2000 times less effective (Table 6).
Table 6. Relative sex attractant activity of several synthetic compounds to cabbage looper males
(Trichoplusia ni, a BERGER and CANERDAY, 1968; b TOBA et al., 1970)
Compound Relative loads of the odor sources
at the behavior threshold (recalculated)
a b
cis-7 -dodecenyl acetate 1 1
trans-7 -dodecenyl acetate 2000 300m
cis-7 -dodecenyl propionate 40000 >10000
cis-7-dodecenyl butyrate 40000 >10000
cis-7-tetradecenyl acetate 100000
cis-9-dodecenyl acetate 100000 3000m
cis-6-dodecenyl acetate inactive 300m
cis-5-dodecenyl acetate 100000 >10000 me
m producesmasking of the pheromone . e very attractive at low concentrations
Likewise the trans-ll-tetradecenyl acetate is "inactive up to 100 [lg" whereas

0.1 [lg of the ci8-isomer (sex attractant of the moths Argyrotaenia velutinana,
ROELOFS and ANN, 1968) "elicits maximum responses in the laboratory". In the
field no moth was attracted by the trans-isomer and 3000 by the attractant. The
trans-isomer even inhibits the effect of the attractant (Fig. 47, see p. 397).
Recently a very potent sex attractant was detected in the gypsy moth, Ci8-7,8-
epoxy-2-methyl octadecane (BIERL et al., 1970; gyptol was found to be inactive,
JACOBSON et al., 1970). The trans-form was only 10 times less active. Females of
the boll weevil (Anthonomus grandi8, TUMLINSON et al., 1969) are differently
attracted by the ci8-and the trans-form of a terpene aldehyde (Fig. 43). Also the
trail-following substance 3-ci8, 6-ci8, 8-tran8-dodeca-triene-1-o1 isolated both from
termites and wood infected with fungus was biologically more effective than the
8-ci8 isomer (MATSUMARA et al., 1968; see p. 393).
Even the artificial attractant tran8-trimedlure is uneffective to the medfly
(Certatiti8 capitata) if the Cl-atom is in the wrong stereochemical position (Fig. 43,
Mc GOVERN et al., 1966). In the bark beetle Dendroctonus brevicomi8 the aggregating
pheromone brevicomin is only active in the exo-form (Fig. 43, SILVERSTEIN et al.,
1968; VITE and PITMAN, 1969). In contrast the 9-oxo-ci8-2-decenoic acid inhibits
queen rearing in honey bees as well as the naturally occuring transform (PAIN et al.,
1962). But this effect is not yet finally proved to be olfactorily mediated by the
queen substance receptors on the bee's antennae.
Deuteration of the attractant c1te-lure (4-/p-hydroxyphenyl/-2-butanone acetate) did not
change the olfactory response of the melon fly Dacus cucurbitae (DOOLITTLE et al., 1968). This
contradicts WRIGHT'S theory that the far infrared spectrum (150-600 cm- ' ) is related to the
olfactory properties of molecules (WRIGHT, 1963, 1966).
The position 0/ donble bonds in the key molecule is very important. For example,
the cis-6 and the cis-5-dodecenyl acetate are behaviorally much less effective than
the naturally occurring cis-7 form (Table 6). The cis-9-tetradecenyl acetate even
inhibits the effect of the attractant cis-ll-tetradecenyl acetate (Fig. 47).
exo - Brevicomin Frontalin trans-Verbenol
+ + o o
2,3- Dihydro -7 -methyl-1H-pyrrolizin-l-one cI'-n-Hexa
decalactone
trans- Siglure o trans- Trimedlure o
Fig. 43. Ring compounds eliciting behavior responses in insects (+). Some synthetic analogs
of key compounds have no effect (0). 1. row: aggregating pheromones of bark beetles
(SILVERSTEIN et al., 1968; VITE and PITMAN, 1969); 2. row: male pheromone of the queen
butterfly (MEINWALD et al., 1969), queen substance of Vespa orientalis (bAN et al., 1969),
3. row: three compounds from orchids, which attract bumble bees (DODSON et al., 1969);
two pheromones of the boll weevil (TUMLINSON et al., 1969), 4. row: artificial attractants of
the mediterranean fruit fly (McGOVERN et al., 1966). In trans-trimedlure 1 of 4 positions of
the Cl-atom tested is ineffective (the dashed line mark two of the effective positions of the
Cl-atom)
Stmctnral isomers can also be sharply distinguished. Thus, the alarm reaction
oftwo genera of myrmicinae ants is elicited by the main component ofthe Dufour's
gland secretion, namely 4-methyl -3-heptanone, which is 103 -106 times more effec-
tive than all other isomers (Table 7). Several isomers of cis-7,8-epoxy-2-methyl
octadecane with shifted methyl and expoxy groups are 20 and more times less
effective in the gypsy moth (BIERL et al., 1970).
Homologs and analogs of the key molecule of pheromone receptors have

markedly reduced activities. Thus the chain length is important (Fig. 46; Table 6).
A methyl group can be essential as in 4-methyl-3-heptanone (Table 7) or in brevi-
comin and frontalin (Fig. 43) but not in the case of 2,3 dihydro-7-methyl pyr-
rolizinone (Fig. 43). A very marked effect is obtained by an exchange of functional
groups. Thus, 4-methyl-3-heptanol is practically ineffective in Atta texana (Table 7).
Table 7. Alarm response of ants (Myrmicinae) to carbonyl compounds (Atta texana, :M:OSER,
BROWNLEE and SILVERSTEIN, 1968. Pogonomyrmex barbatus. VICK et al., 1969)
Compound Alarm level

(cmS saturated air)
A. texana P. barbatus
3-:M:ethyl-2heptanone not tested

4 2 1 1
5 2 10
6 2 10 10
2 3 10 10
4 3 10-& 10- 2
5 3 10- 2
6 3 1 10
2 4 10
3 4 10-1 1
4.Methyl-3hexanone 10- 2 1
2-Pentanone 10
3 10
2-Hexanone 10
3 1
2-Heptanone 10- 2
3 10- 2
2-0ctanone 1 1
3 10- 2 1
2Nonanone 10 10
3 10- 1 10
4-Methyl3heptanol 1
6-Methyl-5-hepten-2one 10 10-1
2,4-Dimethyl-3-hexanone 10 10-1
Also, 9-hydroxy-decenoic acid affects single receptor cells of the drone honey bees
1000 times less than the queen substance (9-oxo-decenoic acid) (KAISSLING,
unpublished).
Recently BLUM et al. (1971) found that 19 alkenoic acids, closely related to 9-oxo-trans-2-
decenoic acid, did not attract swarming drones to a ballon. This shows that the drones are able
to distinguish between queen substance and its derivatives. It does, however, not mean that
the queen substance receptor cells were not stimulated by these compounds. Also caproic acid
failed to attract the drones although the receptor cells respond very well to physiological
concentration of this compound (Fig. 33; see also BOECKH et al., 1965). Nevertheless the bees
distinguish queen substance and caproic acid in behavior experiments (see p. 378 and 408).
This example shows that the behavioral response can be much more specific than the response
of the receptor cells which trigger this response. This seems possible in the bee since the specifi-
city of the queen substance receptor varies from cell to cell (VARESCHI, 1971: see p. 409).
Obviously, a mechanism of contrast increase occurs in the CNS of the honey bee.
Two key characters of the pheromone molecule seem to be responsible for its
fitting in with the receptor cells of the highly specific type: i) One (or two, e.g. in
queen substance) very special functional group(s) in a certain position of the
molecule. ii) A very definite stereochemical shape (including the chain lengths) of
the rest of the molecule. This seems to be true in bombykol (Fig. 46), cis-7-
dodecenyl acetate (Table 6), cis-ll-tetradecenyl acetate (Fig. 47), cis-7,8-epoxy-
2-methyl octadecene (BIERL et al., 1970) and 4-methyl-3-heptanone (Table 7).
Reticulitermes virginicus
Ho HO
V ~
HO
V ~
Ho
~
~2.10-a
b
~ 'I ~
HO
6 10- 2
10-
HO
~ ;,
10-2
HO
~
~
~
Fig. 44. The trail following pheromone of a termite 3-cis, 6-cis, 8-trans-dodecatriene-1-ol
(10- 8 ) and derivatives. Numbers indicate the threshold stimulus in [.Lg streaked across a
glass surface to make a 10 cm path in a 120 arc which is followed by the worker termite
0
(TAl et al., 1971)
A very striking example of pheromone stereospecifity was recently found in the termite
Retwulotermes virginwus (TAl et al., 1971, see Fig. 44) which follows a 10 cm path on a glass
surface marked by 10- 8 [.Lg of 3-cis, 6-cis, 8-trans dodecatriene-l-ol. The two cis-double bonds
seem to be most important for the effect whereas the 8,9-double bond can be absent but may
not be a cis-double bond. Interestingly the effectiveness is not abolished when the compound
contains a benzene ring as long as the geometry of the molecule is unchanged on its "convex"
side. One therefore may conclude that the molecule fits only with this side to a complementary
part of an acceptor molecule.
As shown above, highly specific pheromone receptors can be extremely sensitive,
i.e. they respond to a few molecules. In the extreme case a single one is sufficient
394 K.-E. KA!sSLING: Insect OHaction
for the sense cell to discharge. Such a highly specific effect of single molecules
presupposes molecular receptor units which are called "acceptors" (KAlSSLING, 1969)
since their molecular structure is unknown.
Analogous systems with similar high specificity and sensitivity are, for instance, the enzyme-
substrate reactions or, in the case of specificity, the antibody-antigen system. Therefore,
proteins or protein-like compounds may be responsible for the specificity of the pheromone
receptors and probably also other oHactory receptors. This implies that odor theories are
special cases of theories concerning the interaction of relatively small molecules with proteins
which are constituents of the cell membrane.
The small absolute amount of acceptor material available from a definite oHactory receptor
type may exclude the direct molecular analysis of the acceptor and of its site(s) for specific
adsorption. A detailed analysis of the receptor specificity, however, should give a comple-
mentary copy of the adsorption site on the acceptor with respect to the steric configuration
and the positioning of functional groups. It may be very worthwhile to study specialized receptor
cells_with different key molecules but overlapping side spectra (see p. 403).
Female pheromones
P. epithyrena
L.phaedusa
N.dione
N.krucki
N.cytherea
P. cynthia
D.albida
D.vacuna
c. promethea 0
~ E.calleta
.8 P. cecropia
g- P. gloveri
~ P. euryalis
~ S. pyri
o E.pyrelorum
:2: E. pavon; a
E.spini
C. andrei
Amylitta
A .paphia
Apernyi
Aroylei
Aselene
A. artemis
T. luna
Fig. 45. Interspecific effects of female sex attractants in saturniid moths. Selected from the
subfamily Saturniidae. Elaborated by EAG tests with male antennae exposed to the scent of
female abdominal glands. Filled circles: full effects equal to the effects of the conspecific
gland (in the diagonal line). Empty circles: no effect. Other symbols: weak effects. All
species with full interspecific effects belong to the same reaction group (PRIESNER, 1968, 1969)
Olfactory cells functioning as highly specific pheromone receptors are obviously

widely distributed in insects. As in Bombyx, it seems also possible in saturniids to
study the properties of single receptor cells by means of the EAG, because only
one type of receptor cell was found to be sensitive to the sex attractant (SCHNEI-
DER et al., 1964). These cells belong to the most numerous sensilla trichodea (Fig. 6a)
of the long type which is absent in the female (BoEcKH et al., 1960). SCHNEIDER
(1962) started the comparative study of interspecific effects in saturniids, using
in parallel the male's EAG and its behavioral response. The value ofthe behavioral
test was very limited because of the fluctuating reactivity of these animals. On the
other hand the EAG-test is less sensitive than the behavior test. Thus Bombyx
males showed behavior responses but no EAG to the saturniid glands.
FRIESNER (1968) has shown that there are at least 19 different female sex
attractants and male receptor types respectively in saturniid moths. Within a
group of systematically related species, each male antenna responded in the EAG
to female glands from other species as well as to those ofthe own species (Fig. 45).
Between these groups weaker or no effects were observed. The 19 reaction groups
found by comparing more than 100 out of the 1200 saturniid species correspond to
genera or groups of genera. Systematically related reaction groups obviously differ
very little in the chemical structure of the attractant since the male antennae also
respond weakly but significantly to female glands of related genera (Fig. 45),
Thresholds in 4 moth species for <16- <18 primary aliphatic alcohols

position and configu- Endromis Aglia
num- ration of double bonds Oeilephila tau
ber euph.
of C 12. 11. 10. 9. no 1~~1 >+3
16 cis trans 1 +3
16 trans cis 2
16 cis cis 3 +2
17 cis cis 4
16 rans trans 5 +1
16 = trans 6
16 trans 7 0
16 cis 8
18 cis 9 -1
18 cis cis 10 2
18 trans 11 -2
18 trans 12
( no. 1 = Bombykol)
-3
10910 jJg
Fig. 46. EAG effects of bombykol, its stereoisomers and analogs to Bombyx and three species
from different families of moths. Each line (right) represents one of the 12 compounds used
(see the numbers). The intersection of these lines with the verticals indicates the load of the
odor source eliciting a small but significant EAG (PRIESNER, 1969)
although the receptors are, presumably, highly specific to the pheromone of the
species, as in Bombyx. The receptor systems also seem to be related to one another
since the male antennae of different reaction groups have overlapping side spectra.
FRIESNER (1969) tested a series of bombykol-like synthetic substances by the EAG
on Bombyx and three species of saturniids. Several of these substances affected all
Table 8. Alarm pher01'lWnes identified in social insects. Comhined from CAVILL and ROBERTSON (1965) and BLUM (1969)
Alarm pheromone + identified compounds da;> --g ~

) ~ 0 ~ 0
~ a;> 0 ~ 0
other function (+ ) d. 0 ~
"d . ~ 09 <:D
a;> <6
~ ~ <; j
<6 M
~ '3 a;> "'" '"
.~
a;>
~.!!3 ..<;l ]< g.
iE ~
<:,)
;8 ... M ]< "d
~
~ a;> <6
~
0
l c<l ~ Z ..<;l
~ ~ ~ "'" to <6 ~
<6 ~ ~
.S "0" <6 <:,) ~
.S } g. ~ ]- d. ~
~
"S <; .,;, 0 ~
<6 p:: ~
~ a;>
~ 0 ~
s...
0
Genus rn ~ ;:;:.:
~ E-i ] .... d. Co)
~
c<l
1 Jl
A A
~
~ !1 ~
,~
ii
~ d. [:z;j Co)
"'~
ISOPTERA
Termitidae Amitermes I +
Drepanotermes I + r
HYMENOPTERA ~
Apinae Apis 3 + 0 Pi
Apis mellifera + (+) ~
Lestrimelitta I +
Trigona 2 ..L
I
+ ~
o
Ponerinae Paltothyreus 1 + +
Myrmicinae Myrmica 2 (+) ~
C
et-
Atta 7 (+) (+) +
Pogonomyrmex 6
o
+ s:
Crematogaster 1 +
Myrmicaria 1 + ~.
Dolichoderinae Azteca I +
Conomyrma p. ssp. + 0
Conomyrma p. ssp. + +
Dolichoderus I +
Dolichoderus 1 +
Forelius 1 +
lridomyrmex 4 + (+)
Iridomyrmex 1 +
Liometopum 1 +
Tapinoma I + +- (+)
Formicinae Acanthomyops 1 + (+)
Formica 3 +
Lasius 2 (+) +
4 species but each species in a different rank order (Fig. 46). This result thus
further exemplifies the remarkably high specificity of pheromone receptors.
A further example of the probable interaction of pheromones with highly
specific olfactory receptor cells was studied in social insects. A number of compounds
was identified chemically which elicit the stereotyped alarm response (Table 8). The
situation here is more complex than in the sex attractants of moths because
usually each species secretes several compounds which can be involved in the
alarm response (Table 4).
The alarm substance may additionally serve other functions; e.g., as defensive agent or
trail substance. The taxonomic correlations of alarm pheromones seem to be weaker than those
of the saturniid sex attractants (Table 8). Some substances (2-heptanone, citral) obviously are
of polyphyletic origin. In some cases different compounds are used as alarm releasers even in
the same genus. Most of these compounds have similar physical properties (CAVILL and
ROBERTSON, 1965; MASCHWITZ, 1966; BLUM, 1969).
In some cases the specificity of the behavioral response seems to be broader
than in the sex attractant response of moths. Thus, chemically rather different
compounds release the alarm response of Acanthomyop8 claviger (Table 4). Here, it
maybe true that several types of receptor cells trigger the same behavioral response,
which would be a multiple push button response. The broader specificity is
therefore also reflected in the very similar threshold concentrations of homologous
alkanes with 10 to 13 carbon atoms (Table 4).
The example of the 4-methyl-3-heptanone (see above, Table 7) suggests that highly
specific receptor cells can be involved also in the perception of alarm pheromones. In principle,
however, very specific behavioral responses can be multiple push button reponses, when
two or more types of receptor cells are involved with each broader specificity but overlapping
only with respect to the key molecule (see p. 392).
2. Masking and Enhancing

Concerning the highly specific action of most of the discussed pheromones it is of
great interest that sometimes a compound which is very similar to the pheromone
inhibits its behavioral effect. Even stereo-isomers admixed to a pheromone abolish
partially or completely its attractiveness. This "masking" of an odorant by another
volatile compound could be due to an antagonistic effect (inhibition) onto the
pheromone receptor cell. On the other hand, the pheromone and the inhibitory
compound could be detected by two different cell types, the inhibition taking
place in the central nervous system. Correspondingly also the synergism of odor
compounds can be explained.
In this context one has to remember that also the same compound can produce opposite
behavioral effects depending on the stimulus concentration (DETmER, 1963; MULLER, 1968,
see Fig. 15).
The most striking case of inhibition by a stereo-isomer is found in the moth
Argyrotaenia velutinana (ROELOFS and ARN, 1968). The sexual attractant cis-ll-
tetradecenyl acetate is not only inhibited by the trans-isomer but also by several
analogous compounds (see below). Mutual inhibition of the male response by the
sexual attractant of a related species seems to isolate sympatric species in moths
(ROELOFS and ARN, 1969; ROELOFS and COMEAU, 1970).
Recently it was discovered that cis-ll-tetradecenyl acetate is also the sex attractant of
the moth Ohoristoneura rosaceana (ROELOFS and TETTE, 1970) which is also inhibited by the
trans isomer. Since this species and Argyrotaenia velutinana (with the same pheromone, see
above) have overlapping seasonal and diurnal cycles and share the same host plant, "it is not
clear how specificity is effected."
Interestingly, dodecyl acetate (and other compounds, see below) drastically
increases the attractancy for A. velutinana males (Fig. 47) but decreases the effect
of the pheromone in Ohoristoneura. Possibly this way compounds secreted along
Synergists and inhibitors of 11cis-tetradecenyl acetate

I 10 I 11 12 13 14 15 16
A :.5cisV II
.l!! 7cis-V /'6"-/
.!!!
Pl
/';B rNBr

:
:
hOV
- Ai ; lolAiA ~Sy
(/)~ 4------~'-----~~~--------~'----(-~-(l-,~~~/-------------------------------
2 __,-_________/\/~:----~--------o~~:----------~------~-------
/ ~ ~ 5trlcisN /'Cf'A ~
CII 7cisj .1\:,0.. 71rlcisW 5cis'\lvv-
>
:;: /': I\.
$ /Vv 6cis'\lvv-
o 101/'if::. lOI/,o::.A... lol~ lol/'6'V'\ 7ciS'Vvv
CII
00-
lolNOy 9cis~::
li lolhS, lolAi,Sy

101~ lDI~
1
-"2 /~ prOPiir \
~ 1
.94 /V\. lol~
:0
:c
:
Atv
.f.
10l~ 101~ 10l//...
~
1IVv
11 cd
i formate/\( l"
]'ig. 47. Compounds influencing the attraction of the red banded leaf roller moth
Argyrot4enia velutinana to field traps. Numbers above indicate the length of the main
chain of the compounds all of which are acetates (exeptions are indicated). The dotted lines
label the ll-position. They erroneously label the 12-position in the two 9-ci8 compounds below.
Numbers at left are the factors by which the number of attracted males is changed in relation
to the test trap with the pheromone alone. The strongest effects lie above 10 times increase or
at a decrease to less than 1/10 of the numbers of moths caught with the pheromone alone
(ROELOFS and COMEAU, 1971)
with the sex attractant may modulate its effect thus improving reproductive
isolation (ROELOFS and TETTE, 1970). The opposite effects of dodecyl acetate in two
species and the equal effects of the pheromone cattraction) and its trans isomer
(masking) in the same two species make it very probable that the three compounds
act on two or even three different receptor cell types in each species (multiple
push-button response).
Very recently ROEEOFS and COMEAU (1971) found many synergists and inhibitors of
A. velutinana (Fig. 47). All compounds are structurally related to the attractant 1lci8
tetradecenyl acetate. Following properties characterize synergists and inhibitors:
i) The length of the straight chain moiety of the synergists is 11, 12, 14, of the inhibitors 10,
13, 14, 16.
ii) When one starts from saturated compounds, the synergistic effect is produced or
increased by Br, 0 or S within the chain in 11 or 12 position. Different from that, also the 5 cis-
or 7 cis-dodecenyl acetates are strong synergists. The synergistic effect is lost, when the acetate
moiety is replaced by a hydroxy group.
iii) Many of the inhibitory compounds are alcohols, formate or propionate. Nearly all
inhibitors have a cis- or trans-double bond in 9 or 11 position.
These findings lead to the assumption that the acceptor molecule has two or
even more sites of interaction with the pheromone molecule and that the synergists
and inhibitors interact only with some of the sites, either increasing or decreasing
the affinity of the acceptor to the attractant.
Still it has to be clarified whether all these effects occur on the same receptor
cell. All data of Fig. 47 are collected from field traps. The inhibitory effect can be
shown in the laboratory only after several minutes of exposure to 3 or 30 p,g of an
inhibitor (ll-trans tetradecenyl acetate). The number of animals responding to
0,3 p,g of the attractant is strongly reduced for at least several minutes. When
excitation and inhibition occurs at the same acceptor one would expect that the
inhibitor has to be present in markedly higher amounts than the exitatory com-
pounds in order to block a majority of the acceptors. The synergists did not act in
the laboratory.
JACOBSON (1969) reports masking of 10-propyl-trans-5,9-tridecadienylacetate ("propylure")
by 15% of its cis-isomer in the pink bollworm moth. This compound was resynthetized by
ElTER et al. (1967) and found to be ineffective, which may be due to the mixture of cis- and
trans-form in the synthetic product. Curiously enough, propylure only acts in laboratory tests
and not in the field. Propylure in mixture with an inactive female extract from which it had
been separated, however, was attractive in the field (JACOBSON et al., 1970). The activating
principle is claimed to be the mosquito repellent Deet (N,N-Diethyl-m-toluamide) which was
found in female extracts (JONES and JACOBSON, 1967) -an "ahnost incredible" finding. Recently
a new compound was discovered by empirical screening which was far more attractive than
propylure, especially to the (male) pink bollworm moth ("hexalure", cis-7-hexadecen-l-01
acetate, GREEN et al., 1969). The trans-form was not attractive.
In the EAG hexalure and propylure gave similar responses, the "wrong" stereo-isomers
were about 10 times less effective. No inhibition effects were found except whit Deet which
produces an EAG and strongly reduces the EAG-response to hexalure and propylure and
the isomers (KAJSSLING and JACOBSON, unpublished). These results suggest that several cell
types are involved in masking and enhancing observed in behavioral experiments. For
masking effects see alse RIDDIFORD (1967, in Telea polyphemus) and TOBA et al. (1970, in
Trichoplusia ni; Table 6).
Masking can be produced by chemically unrelated compounds as in male
Euglossinae bees which are attracted by orchid fragrances (Fig. 43). 1,S-cineole is
highly attractive to many species and can be inhibited by citronellal, benzyl
acetate or methylsalicylate. The latter two compounds are also produced by some
orchids and are attractive to other species of these bees (DODSON et al., 1969).
Female bark beetles (Dendroctonus pseudotsugae) produce a masking substance
which inhibits the aggregating pheromone (RUDINSKY, 1969). Verbenone (pro-
duced by males) but not trans-verbenol (from females) reduces the attraction of
males by frontalin in Dendroctonus frontalis (RENVICK and VITE, 1969). Some
terpenes produced by host plants are ineffective alone but enhance the effect of
brevicomin (enhanced by myrcene, BEDARD et al., 1969) and frontalin (enhanced
by a-pinene, RENVICK and VITE, 1969). PITMAN (1969) claims that 3-carene acti-
vates both attractants better than myrcene.
Synergistic effects as mentioned above in moths (Deet, dodecyl acetate) and in
species of Dendroctonus (terpenes) are claimed for several other terpenes (WOOD
et al., 1968; TUMLINSON et al., 1969; Fig. 43). Sometimes synergism may be a more
physical phenomenon. Some compounds (e.g. 2-Tridecanone produced by Lasius
umbratus, see BLUM, 1969) probably act as solvents or spreading agents duplicat-
ing the effect of the pheromone or its defensive power. On the effects of a spreading
agent (tridecane) see REMOLD (1963).
TraWl, trans-3,7-dimethyldeca-2,6-dien-l,1O-diol seems to function as a glue which st.icks
pheromone-containing dust particles to the female antenna of the queen butterfly (PLISKE and
EISNER, 1969; SCHNEIDER and SEmT, 1969).
3. Odor Specialists of Lower Specificity

Besides the highly specialised pheromone receptors another category of specializ-
ed cells have been found with a much broader and often slightly varying odor
spectrum. This is the case with a type of olfactory cell on the Locusta antennae
responding to green-odors which indicate food sources. These cells, which belong to
the sensilla coeloconica, were detected and studied electrophysiologically by
BOECKH (1967 a). Out of 60 tested compounds, hexenal and caproic acid were most
effective but also valeric and oenanthic acids were very effective. The threshold
concentrations of these compounds determined by a weighing method (see p. 383)
differ by a factor of 1 : 10 or 1 : 100, which is little in comparison to the difference
in effectiveness of the key molecules and their analogs on the pheromone receptors.
A 1000 fold increase of threshold concentration was found from hexenal to hexenol and
even more to hexanal which was nearly ineffective. Various substitutes abolish the effectiveness
of hexenal or caproic acid, for instance a NH2 group (amid of caproic acid or 6-amino-caproic
acid) or an esterisation (hexenyl acetate). Interestingly, "symmetrical" molecules have no
effect (adipic and mucic acid or 3-hexen-2.5-diol).
Recently KAFKA (1970) continued this work extending the list of compounds (380) and
controlling the stimulus intensity with improved methods (see p. 383). KAFKA measured the
stimulus-response curves of many single receptor cells for all effective substances. Not all of
these curves have the same shape on a logarithmic stimulus scale (Fig. 36). Thus, caproic
acid and hexenal were equally effective when compared at a moderate impulse response (Fig 48)
although hexenal usually had a 100 times lower threshold concentration than caproic acid
(see above). It is not yet clear whether this effect is due to physical properties of the stimulus
(e.g. higher degree of dissociation of caproic acid at low concentrations) or to the interaction of
the odor molecule with the receptor cell itself. Another complication is not yet understood:
Individual cells can differ considerably with respect to the rank order of effective compounds,
to the general sensitivity and also with respect to the shape and the general steepness of the
stimulus response curves (Fig. 36).
Fig. 48 shows the average effectiveness of Os-compounds with respect to a moderate
impulse response which is far above threshold. The most effective substances are hexenoic
acids which are at threshold similar effective as hexenal. Interestingly the 2 or 3 position and
the cis-trans-configuration of double bonds are almost equivalent for the effectiveness of these
compounds, which is in strong contrast to the relatively long chain pheromones of moths
(Figs. 41,42, 44,46, 47, Tables 6).
The effect of the double bond can be partially replaced by an oxo-group in 2-position of the
saturated acid. The addition of a methyl group at this position decreases the effectiveness but
this compound is still as effective as the hexenols. A more marked loss of effectiveness is obtain-
ed by changing the 2-oxo-group into a hydroxy-group. The difference between hexenal and
hexenol is here relatively small but much stronger at threshold according to BOECKH'S experi-
ments. To date it seems that the end-standing carbonyl or carboxyl group together with a
double bond in 2 or 3 positions or instead of this together with another oxo-group at C2, is the
essential functional configuration of a molecule exciting maximally the green-odor receptor of
the locust.
.All other substitutions tested so far reduce the effectiveness of the molecule by 3 or more
orders of magnitude. Here are included additional double bonds, oxo- and hydroxy-groups,
methyl, NH 2- or SH-groups but also halogens and ring structures. The hydrocarbon part of the
molecule is optimal with a chain length of 6 carbon atoms and without any substitution
(except for the keto group at C 2).
Locusta migratoria
OH
~O A/\/'OH
(7) (7)
t t OH c
~O ~O ~OH
5 4 6
cIt cit OH cit

~O
5
AAAo 4
/V\/'OH
6
OH
~O ~O /V'\/'OH
(9) 5 9"
OH
t t
~O ~O ~OH
7 4 8 9
OH
~O
6
/'VV
10
OH
~
8 Br
~O
7" OH
/"V"VI
n
Fig. 48. Compounds tested by BOECKH (1967) and :KAFKA (1970) on the green-odor receptor
of the locust. The numbers (KAlrxA, 1970) indicate the estimated loglo of molecule numbers
per sensillum which elicit 6 impulses per 200 msec during the maximum of frequency (0.
Fig. 36b, for the method of calculation soo p. 384). X no response with the concentration
indicated. Average values of many cells which strongly vary between each other (e.g. Fig. 36 b)
KAFKA suggests that two types of non-covalent binding forces are involved in
the interaction between the odor molecule and the acceptor: (i) dispersion forces
due to the hydrocarbon part ("substrate structure") of the odor molecule and
(ii) permanent dipole forces due to its functional groupings in the region of the
first C-atom ("active structure"). Both types of binding forces may cause an
26 Hb. Sensory Physiology, vol. IV!l
402 K.-E_ KA:rSSLING: Insect Olfaction
adsorption of the molecule on the acceptor_ The dipole properties of the "active
structure" may be more important for the "excitatory" effect to the acceptor
which may be a change of its conformation. It seems not unlikely that this part of
the odor molecule forms up hydrogen bridges to any acceptor materiaL It is also
an interesting question as to whether a single acceptor type is responsible for the
specificity of the cells reaction or if the molecule successively passes more than one
site which test for different properties. For instance a lipid phase could specifically
regulate the access of the molecule to the acceptor.
Considering this thorough investigation of single cell specificities one would like to have
finally answered the question of whether we deal with single molecule acceptor interactions or
if several molecules act together. The latter seems less probable but can not be excluded
(see p. 384).
On the other hand it would be most interesting to know more on the distribution of the
cell's specificity which varies very little in a single cell but considerably between cells. For
instance trans-2-hexenal was about 30 times more effective than tran8-2-hexenoic acid in one
case and about 3 times less effective in another one (Fig. 8a and b in KAFKA, 1970). This sug-
gests that the specificity of the green odor cells is not due to a single acceptor type alone.
Many authors considered the lipophil and hydrophil parts of odor molecules
but they did not yet discuss their properties with respect to specific bindings to
single molecular acceptors (see MONCRIEFF, 1967; STEINER, 1969; BEETS, 1970).
At least one example of chemosensitive mechanisms besides olfactory receptors should be
mentioned here. WIGGLESWORTH (1968) tested some 42 compounds for the juvenile hormone
(ROLLER et al., 1967) activity. He came to the conclusion that a proper balance between the
lipophil and hydrophil groups as well as geometrical isomerism but not particular chemical
groupings are important for the effect. The receptor sites for the hormone action could be
located on lipoprotein membranes. Part of the specificity "may rest upon the capacity for
gaining access to membranes of oriented lipids".
BLUM et al. (1966) came to the conclusion that "alarm-releasing activity is a
function ofthe presence of an electron-rich atom (oxygen) on a linear hydrocarbon"
in the ant Iridomyrmex pruino8U8. They tested about 50 ketones analog to the
natural pheromone 2-heptanone in field experiments and in the laboratory.
For stimulation they placed a piece of filter paper on a small platform 0.25 inch from one
of the nest outlets and applied measured amounts of the ketones. Then they counted the num-
ber of workers moving out of the nest after a 5-min exposure and the number of workers exhibit-
ing typical alarm behavior. According to these numbers each compound was ranked into
grades from 0 to 5. It has not been shown to what extent this rank order may be due to the
vapor pressure of the compounds used and what difference in threshold concentrations are
indicated by the five grades. The compounds of grades 5 and 4 reported by BLU1\! et al. (1965)
are shown on the left side of Fig. 49.
Recently A~iOORE et al. (1969) have continued this work on ants and found very
effective compounds which are chemically different but have nearly the same
stereochemical shape (n-butyl-acetate or 2-ethoxy-ethyl acetate). This is thought
to support the stereochemical theory of AMOORE (1963), AMOORE et al. (1969). But
even more appreciable changes of the shape of the hydrocarbon chain do not alter
the effectiveness of the molecule (Fig. 49, 6-methyl-5-hepten-2-one or 6.6-di-
methyl-3-heptanone). On the other hand the change of the oxo-group into a
hydroxy- or a halogen-group decreases the effectiveness of the compounds by
two grades (Fig. 49, on the right side). This decrease indicates the strong influence
of the functional group.
Interestingly enough the odor spectra of the green-odor cells of the locust (see
p. 401) overlap partially with the spectra of other insect receptors. Like the locust
cells, the queen substance receptor reacts also to caproic acid, hexenal and hexenol
and not to hexanal. Nevertheless, this cell type is specialized to 9-oxo-decenoic
acid since caproic acid is about 104 times less effective (KAISSLING and RENNER,
1968; KAIssLING, 1969; Fig. 33). The locust cells, however, are very sensitive to
'VVV
II
0 2 - Heptanone
5
\yovv
Iridomyrmex pruinosus
o n- Butyl acetate
5
,
~
o
~
3
II
0 3 - Heptanone H 2 - Heptanol
'VVV ,
oII , - Heptanone
~
o 2 - Octanone
, \yovAo~ ,
0 2 - Ethoxyethyl
acetate
~
II
, '\..AAA
T -_ _- ,
2
o 2 - Nonanone Br 2 - Bromooctane
yvy , /O~3
o 6 - Methyl- Methyl
~5-hePt:none sec-n - octyl ether
g 6,6 - Dimethyl-
3 - heptanone
Fig. 49. Compounds eliciting the alarm response of the ant Iridomyrmex pruin0.tU8.
2-heptanone is the natural alarm pheromone. Numbers indicate a rank order of compounds
determined by BLUM et al. (1966) (left side) and by AMOORE et al. (1969) (right side)
caproic acid (Fig. 48) and could not be excited by queen substance. Therefore,
besides some similarities, there should be a fundamental difference between the
acceptor systems of both cell types.
One immediately detects the identity of 9-oxo-trana-2-decenoic acid (queen substance) and
trana-2-hexenoic acid (most effective in the locust) with respect to the acid end of the molecule.
This suggests that the acceptor of the queen substance receptor may have two subsites one of
which is similar to the only ( 1) site of the acceptor in the locust. The functional significance of
the 9-oxo group of queen substance is evident since the molecule with a 9-hydroxy group is
1000 times leBS effective (KAisSLING, unpublished; BOECKH et al., 1965).
According to a note of KAFKA (1970) two or more functional groups (including
double bonds, e.g. in bombykol) of an odor molecule may increase the possible
26'
404 K.-E. K.uSSLING: Insect Olfaction
degree of specificity of the receptor cell. In fact, the queen substance receptor and
many other pheromone receptors are obviously much more specific than the green
odor receptor of the locust (p. 401). Correspondingly, pheromone molecules often
have several functional groups (including double bonds, see p. 390ft) The high
specificity to 4-methyl-3-heptanone seems to be an exception (Table 7). Here
probably the methyl group brings more specificity since 2-heptanone acts much
less specific (Fig. 49).
Furthermore the carrion receptors of Calliphora and Thanatophilus react as the locust cells
do to hexenol and hexenal, but in contrast to the locust cells are not sensitive to caproic acid
and are inhibited by butyric acid (BOECKH, 1967 a). Similarly, the carrion receptor of the
N ecrophorus beetle is inhibited by butyric acid, but excited by caproic acid. This cell type is
not only excited by lower fatty acids but also by amines (butylamin, trimethylamin, benzyl-
amin) and by benzylmercaptane. Ammonia and hydrogensulfide have no significant effect
(BOECKH, 1962). These examples show the very delicate differences in specificities of odor
receptors which make a general odor theory more doubtful.
In the case of the queen substance receptor it cannot be decided if its reactivity
to caproic acid and other compounds is only due to a poor selectivity of the mole-
cular acceptor or if it has a biological meaning, since we know that the honey bee
smells caproic acid. It may very well be possible, however, that a definite cell type
is specialized to several chemically unrelated compounds occurring in natural odor
mixtures, for instance in food odors (grass, carrion) but also among the pheromones.
Thus, the scent of the Nassanoff gland of worker bees contains geraniol,
geranic + neric acids and possibly citral (BOCH and SHEARER, 1962, 1964; BUTLER
and CALAM, 1968). KAISSLING and RENNER (1968) found an olfactory cell type
obviously specialized to this scent. It responds, however, not only to the substances
named but also to citronellol and also rather weakly to geranyl-acetate and linalool.
According to the list of effective compounds which differ in the number of double
bonds and the degree of oxidation of the functional group, this pheromone
receptor belongs to the broad spectrum specialists (see also p. 409).
The alarm response of the ant Acanthomyop8 is elicited by a number of chemi-
cally unrelated compounds which are secreted by two different glands (Table 4).
It would be interesting to know whether the same cell type responds to all of these
compounds or if the alarm response in this case is elicited by several different
types of receptor cells (multiple push-button response).
Complex odor mixtures are also produced by ants (remarkably overlapping in slave-
keepers and slaves, BERGSTROM and LOFQV1ST, 1968) by male bumble bees (main component
2.3-dihydrofarnesol, BERGSTROM et al., 1967), by solitary bees (macrocyclic lactones, ANDERS-
SON et al., 1966), by coreoid bugs (WATERHOUSE and GILBY, 1964), by the beetle Trogoderma
granarium (IKAN et al., 1969), by the boll weevil (TUMLINSON et al., 1969; Fig. 43), by Ips
con/usus (WOOD et al., 1968) and by many others (see BEROZA, 1970). A striking variety of
volatile compounds is found also in further species of ants (Lasius sp., BERGSTROM and LOF-
QVlST, 1970) and in bumble bees (Bombus sp. and Psithyrus 8p., KULLENBERG et al., 1970). The
secretion spectra of related 8pecies are different but overlapping. Interestingly some ant and
bumble bee compounds are similar or identical to those effective in moths (hexadecenol,
dodecyl acetate, (see p. 398) and other insects. The number of compounds appropriate for
pheromones seems to be limited since similar compounds are also used by beetles (14-methyl-
cis-8-hexadecen-l-01, RODIN et al., 1969) and termites (3-cis, 6-cis, 8-trans-dodecatriene-l-ol,
MATSUMARA et al., 1968, see p. 393).
4. Hyperpolarization and Inhibition

A peculiarity of olfactory cells is the fact that some compounds hyperpolarize
the receptor potential and consequently depress the impulse frequency which may
be spontaneous or elicited by a preceding or a simultaneous stimulus. For instance,
BOECKH (1962, 1967 a, 1969) found in the Necrophorus beetle and in flies that the
lower fatty acids (0 2 -04 ) depress and the higher fatty acids (05-0S) increase the
impulse frequency. In Antheraea the reaction to the female sex attractant can be
blocked by geraniol (SCHNEIDER et al., 1964).
It is an interesting question whether depolarization and hyperpolarization
are elicited by the same or by different specific acceptors. The same substance is
able to affect two cells even within the same olfactory hair in opposite directions.
Nevertheless another substance may excite both cells in the same direction
(Fig. 50). This variability as well as the curious correlation between the hair length
and the proportion between depolarizing and hyperpolarizing compounds both
found in Antheraea (SCHNEIDER et al., 1964) are easier to interprete with the
assumption of separate acceptors for the opposite responses of the receptor poten-
tial.
A variability like that of the "generalists" of Antheraea was found in the locust. Some of
the green-odor receptor cells (see p.400) are hyperpolarized by amines which depolarize
another cell type often found within the same sensillum coeloconicum (KAFKA, 1970). On the
other hand, this second cell type sometimes is blocked by compounds which stimulate the
green-odor receptor (e.g. hexenal) but also by others which have no exitatoryeffect (e.g.
hexanal). The stimulus concentration has to be higher for hyperpolar ization.
Depolarization and hyperpolarization ofthe receptor potential cause excitation
and inhibition of the nerve impulse activity. This electrical type of inhibition must
not be confused with the possible biochemical inhibition of the acceptor or of any
other system of the receptor membrane, which was a possible explanation of the
masking effects (see p. 399).
5. Odor generalists
Extremely varying specificity, including inhibitory effects, is the character-
istic property of the odor generalists (see p. 388). Their markedly overlapping
spectra, which vary from cell to cell (Fig. 50), raise the question whether several
types of specific acceptors exist, in different proportions in each receptor cell
(GESTELAND et al., 1963; SCHNEIDER et al., 1964). With a single acceptor type
in each cell the number of acceptor types would be enormous. The variability of
neighbouring cells within the same olfactory hair favours strongly the hypothesis
that the specific acceptors are located at the cell membrane (KAISSLING, 1969;
KAFKA, 1970).
The model of generalist cells offers an interesting sensory base for odor dis-
crimination. Each odor substance as well as each mixture of compounds is repre-
sented as a definite pattern of excited, unexcited and inhibited cells of the generalist
type (Fig. 50). A comparison of such patterns produced by odors which are
distinguished and those which are confused could show the manner in which the
ONS evaluates these patterns.
Generalist cells found by LACHER (1964) in worker and drone honey bees may
be involved in the well developed ability of odor discrimination as studied by
406 K-E. KAISSLING: Insect OHaction
Antheraea pernyi, sensilla basiconica. reaction spectra of single cells
38 4938 38 4949
Terpineo!
Isosalro!e
Phenyl Ethy! A!coho!
Cinnamaldehyde
Phenyl Ethyl Acetate
Geraniol
Benzyl Acetate
Nitrobenzene
Oil 01 Clove (Eugenol)
Cinnamic Alcohol
27 27 27
fmedium + strong frequency f medium 9strong inhibition f no reaction I no test
Fig. 50
Fig. 50. Reaction spectra of odor generalists
Q;
Apis mell ifera
QI
found by impulse recordings from sensilla
I Benzaldehyde
basiconica (Antheraea pernyi). Each vertical
Itrobenzene
line represents a single cell, cells from the
, 231 ~ 0 133
" /0 ~
same sensillum are indicated by the same
numbers. Horizontal rows of symbols re-
Q;
N...
o o present the pattern of excitation received by
~
I
Methyl
ON H2
anthranilate A'Y"
W p-Naphtho
the CNS characteristic for each odor substance
(from values of SCHNEIDER et al., 1964)
methyl ether
"-
Q;
II 1097 [.27
o
QI Isobutyl
benzoate
~
I
0
Isoamyl
OH salicylate
~
"
o~
1917 II vy 282
Q Q
o o
para- Cresyl meta - Cresyl
I methyl ether I methyl ether
/
/ 539 o 75

Q;
~ I
U_PhenYI
ethyl brom id
6, 599
~
~ I
~o
Phenyl
acetaldehyde
17 Fig. 51. Compounds partially confused by
honey bee workers (v. FRISCH, 1919). Six
vovv
II - I 908 ']("A( 29
experiments were performed. The left col-
o o umn shows the conditioning odors. The right
Isoamyl acetate 6 - Methyl - 5 - hepten column shows the most effective out of 3 - 6
2 - one additional compounds presented simultane-
ously.Numbers of attracted bees are indicated
Fig. 51 (see Table 9)
Table 9. Odor discrimi1UJtion by honey bee workers (v. Fru:SCH, 1919).4 odors are con/used with the conditioned odor"
"CI
ru :g
0 ~
test odors ~ i ~ -8 .c
bIl
~
se ~
:= .c ]> E .$ .8
I:: -! ~ ~ +>
0 ~ G)
~ -8 g.
fa -;3
-! a ~ a "; ..<;I :~
G)
~ ~ ~ i. "; ~
i15 ....
.c ~ ..c1 j III i- ., '9 "CI 0
~ 0
.c '" ~ '" -8
";
;::;p., ~ '" ~
~.6 ~
~ ~ ~
conditioned condor ~ ~ G)
~
t.g
III 1e ~ 0 ~ 0 ~ ~ a
1-1 p., e ~ c:6. ~ co
Z ~
i J.. a P:l
.e:A~ Z of
Nitrobenzene 61.2 35.3" 3.2 0.3 377
s
Methyl anthranilate 65.8 1.7 25.6" 1.1 0.7 1.9 3,2 1667
%
a
Isobutyl benzoate 68.6 4.0 10.3" 1.6 1.9 6.9 6.7 2839
para-Cresyl 83.6 0.9 11.6" 0.5 2.2 0.8 0.5 645
methyl ether
co-Phenyl ethyl bromid 2.0 92.4 0 2.6 1.7 0.5 0,8 648
Isoamyl acetate 93.0 0.4 3.0 1.6 0.2 1.8 979
f
conditioned odor conditioned odor odor-less 11.
Control: 57.4 37.8 1.3 1.1 1.9 0.5 0 371
Six experiments were performed (horizontal rows) with 4 to 6 compounds including the conditioned odor. The numbers show the percentage of bees
attracted to each odor in the test situation. Two places with the conditioned odor, together with five places without any odor were offered in control
experiments (see Fig. 51; evaluated from v. FRIsCH'S, data, 1919).

408 K.-E. KAISSLING: Insect OHaction
v. FRISCH (1919). v. FRISCH conditioned worker bees to one out of 4 to 7 compounds

using sugarwater as reward (see above). In the test situation (without reward) the
bees were strongly attracted by the conditioned odor. In four out of six experi-
ments, however, the bees significantly prefered also one of the additional com-
pounds presented (Table 9, Fig. 51). According to v. FRISCH, these compounds also
smell similar for man. It is still an open question, whether the same receptor cells
respond similarly to related odors or whether the two compounds may excite
separate receptor cells and the confusion takes places in the eNS.
Interestingly, the bees distinguished very well between two pairs of compounds (Fig. 51 and
Table 9, below) which have a very similar molecular shape in the sense of AMOORE (1963),
AMOORE et al. (1969). In the case of isoamyl acetate, a specialized cell type may be involved
since this compound is an alarm pheromone of the honey bee (BOCH et al, 1962, 1969; Table 8).
Recently it was shown by BrrTEL (1969) that worker bees distinguish between homologous
fatty acids (3-10 carbon atoms) and also between aldehydes.
V AREscm and KAISSLING (1970) made use of the proboscis reflex to condition
both worker honey bees and drones. Sometimes the bees appeared to be conditioned
after a single reward. The bees distinguished very well between queen substance
and caproic acid as well as between the scent of the Nassanoff gland and geraniol.
Apis mellifera
100-=============;:::-,
i .-,
"10 content of M in the mixture B + M

Fig. 52. Behavioral responses of honey bees to mixtures (B + M) of a conditioning odor
(B = Bromstyrol = co-phenyl ethylbromid) with another odorous compound (M = 6-
methyl-5-hepten-2-one). Two experimental series with different test situations: The bees
had the choice (i) between B + M and M (upper curve) and (ii) between B, B + M and M.
Values from v. FRISCH (1919)
Two types of receptor cells with no overlap of reaction spectra were found
electrophysiologically each of which responds to one of the two pheromones but
also to the distinguished compound (KAISSLING and RENNER, 1968). The question
arose how the distinction of two compounds is performed which act on the same
type of receptor cell.
This question could be solved recently by VAREscm (1971) who thoroughly
reinvestigated the whole problem of odor distinction in the honey bee combining
electrophysiological and behavioral studies. Contrary to LACHER (1964) there is a

distinct grouping among the reaction spectra of the olfactory cells. V ARESCHI could
separate at least seven cell types with no overlap of specificity, including the queen
substance receptor and the receptor type which is sensitive to the scent of the
Nassanoff gland (see above). Within each of the seven reaction groups, however,
considerable variation of specificity was found. This variability explains why the
bees (drones and workers) distinguished nearly all tested compounds behaviorally.
Only a few of many compounds were confused and they affected in each case
receptor cells of the same reaction group (see also p. 392).
Honey bees are also able to detect a definite compound out of a mixture of two
compounds. In v. FRISCH'S experiments they preferred a mixture containing only
5 % of the conditioned odor (bromstyrol) and 95 % of methylheptenone when it
was presented together with pure methylheptenone. If the animals have the choice
between the pure conditioned odor and the mixture of both substances, they reject
the mixture even if it contains only 10 % of the wrong substance (Fig. 52).
Cells with varying but not quite different odor spectra are found in mosquitoes (LACHER,
1967b). These respond to fatty acids, terpenes (often inhibiting), to leucin and several other
compounds. Cells of one sensillum type are excited by the mosquito repellent DEET (N,N-
Diethyl-m-toluamid), another cell type responded to sweat (LACHER, 1967b, 1969). In this
context it should be mentioned that mosquitoes are attracted also by amino acids, diphenoles
and steroids (ROESSLER, 1960).
6. CO 2 - and Humidity Receptors

The specificity of CO 2- and humidity receptors studied electrophysiologically
in honey bees (LACHER, 1964), caterpillars (DETHIER and SCHOONHOVEN, 1968),
mosquitoes (KELLOGG, 1969) and locusts (WALDOW, 1970) is not yet fully under-
stood. In comparison to the proper olfactory receptors, these cells work at much
higher (10 8 -lOlO times) stimulus concentrations. An increasing impulse number
was detected with steps from 0 %to about 2 % relative humidity and from 0.03 %
to 0.5 % CO 2 in the honey bee and 0.04 %to 0.05 % CO 2 in the mosquito. Because
of these relatively high stimulus concentrations the assumption of specific accep-
tors interacting with single molecules is not a necessity. The humidity receptors,
for instance, could register the expansion of a hygroscopic material. Interestingly,
the humidity receptors and the green-odor receptors of the locust (see p.400)
belong to the same morphological sensillum type but do not occur within the same
sensillum (Figs. 6c, 12; WALDOW, 1970).
The coeloconic humidity sensilla usually contain three receptor cells. One cell increases and
the other decreases its impulse frequency with rising humidity. A third cell responds only to
temperature.
Both humidity cells show a markedly phasic behavior with changes in humidity
(Fig. 30). The mirror image relation of reactions of both humidity cells suggests a
linkage mechanism between them. In neither cell is the impulse frequency clearly
correlated with the relative or with the absolute humidity. It is influenced in
addition by temperature, which drives the impulse frequency of both cells in the
same direction (WALDOW, 1970). It is astonishing that there seem to be no clear
differences in the morphology of these sensilla coeloconica corresponding to their
function as humidity or olfactory receptors.
H. The Sensory Transduction

A sequence of transducer processes occurs between the first contact of the odor
molecules with the antenna and the ensuing change in impulse firing rate. The
knowledge of these processes is, as yet, mainly hypothetical or based on conclusions
by analogy from other sense cells. Mter adsorption on the sensillum surface the
molecules have to be transported through the hair wall to the specific acceptors. An
interaction between the odor molecule and the acceptor (see above) results in a
change 01 conductance of the dendrite membrane in a manner as yet unknown. The
usual assumption is the conformational change of an acceptor protein induced by
the specific adsorption of a stimulus molecule, which locally "opens" the mem-
brane. The change in membrane permeability causes an ion flux across the mem-
brane and consequently the receptor potential. This graded potential spreads
electrotonically to more proximal parts of the receptor cell dendrite and generates
the nerve impulses. They probably originate proximally to the ciliary region. The
impulses are propagated along the axon directly into the eNS. Further mechanisms
may be connected with the transducer processes of chemoreceptors by which the
antenna rids itself of the effective odor substance after the stimulus.
Radioactively labelled bombykol is adsorbed on the antenna of Bombyx mori
(and those of other insects as well) as on other parts of the body. Only a neglegible
proportion evaporates back into the air space. Therefore one has to assume in-
activating mechanisms on the antenna to prevent continous stimulation of the
cells (see p. 420).
According to the calculations in Bombyx mori (p. 385, KAISSLING and
PRIESNER, 1970) about 50 % of the molecules adsorbed (at low odor concentrations)
at the hair surface affect the receptor cells. Presumably, the odor molecules enter
the olfactory hair by the way of the pore systems found in the wall of all olfactory
sensilla. Before entering the pores, the molecules have to be transported along the
cuticular surface. According to LOCKE (1965) a monomolecular lipid layer belongs
to the surface layers of the cuticle and also coats the cuticular pores. Within such
layers, molecules could be transported by 2-dimensional diffusion.
The molecules probably reach the receptor membrane directly through the pore
tubules without entering the sensillum liquor. This is strongly suggested since a
contact between the tubules and the receptor cell membrane is clearly shown in
several electronmicrographs (Fig. lOa, see p. 362) including Bombyx (STEINBRECHT,
1971). The assumption seems reasonable that the number of acceptors is not
smaller than the number of tubules (about 6000/cell in Bombyx, see Table 2). The
acceptors probably belong to the cell membrane since the same sensillum often
bears several receptor cells with different specificities (see p.405) which are
obviously genetically determined for each cell. All tubules of each sensillum,
however, are made by a single trichogen cell.
The reaction times of the bombykol receptors are sufficiently long to allow a
passive transport of the molecules from the hair surface to the nerve dendrite by
diffusion. ADAM and DELBRUCK (1968) assumed a two-dimensional diffusion from
an adsorption site anywhere on the sensillum surface to one of the pores by
developing the equation
(7)
The Sensory Transduction 411
where t is the average time for a particle to reach the pore from a distance b
(= 0.23 p,) which is about half the distance between two pores. D is the diffusion
coefficient, and y (= 0.8) corresponds to this special case of twodimensional dif
fusion to a target and depends on the ratio between the distance b and the diameter
a of the target. With the lowest reasonable estimate of D = 10- 8 cm2jsec one
obtains a maximum diffusion time of 80 msec from a site between two pores to the
pore opening. Considering one dimensional diffusion from the pore opening to the
dendrite surface one may use another equation derived by ADAM and DELBRUCK
(1968)
(14)
With a distance b = 0.4 P, one obtains about 50 msec as a maximum diffusion time.
Combining the two results one arrives at a total diffusion time ofless than 150 msec.
Bombyx mori
C 5
0 fl. _., . .'" .. _.,_ .
gt .JJ.~".nJl~,
, F I' r 9 I r i 9 I
10,5 "'In,'" A ~~
10" I0 I 1. ~ n.ll 1 2
o jJ ~~u~t~JIJ!J1 t'~ F i
1 2
30
20
10,3 10
O~~~~~~~~
2
30
10,2 20
10
o
2
40
30
10'\
20
....9
10
O~~~~~~~~
sec 2
Fig. 53. Black columns : Distribution of reaction times of the first nerve impulses of
bombykol receptor cells with one sec stimulus (KAISSI.JNG and PRtESNER, 1970). White
column: reaction times of the behavior response with 10 sec stimulus (KAIssLING, unpublished).
Loads of the odor source in Ilg bombykol
With low stimulus concentrations (10- 4 and 10-3 ftg), the nerve impulses ofthe
bombykol receptor cell elicited by single molecules have a reaction time of about
500 msec (200-1000) (Fig. 53). This is much more than needed for diffusion. But
one has to remember that part of this time is involved in the build up of the receptor
potential preceding the impulse, which has not been measured in single cell
recordings. This time comprises 100-200 msec if one compares the time course of
the EAG and the impulse frequency (KAISSLING, unpubl.). Therefore 150-250 msec
or more remain for the interaction of the molecule with the acceptor and for
its "response" which probably results in a local increase of membrane con-
ductance.
With strong stimuli (10-1 [.Lg, Fig. 53) the reaction time of the first nerve impulse decreases
to 150 (50--400) msec (KAtSSLlNG and PRIESNER, 1970). Contrary to situations of low stimulus
strength, the marked variance in the reaction times cannot be explained by the variance of
molecular hits, which is small here because of the high rate of molecules hitting the cell (700
per sec). Therefore the variance in reaction times depends alone on the statistical nature of the
transducer processes leading to impulse firing: diffusion, the interaction of the odor molecule
with the acceptor, and the build-up of the receptor potential.
The shortening of the average reaction time from 500 msec to 150 msec with strong stimuli
may mainly be due to a time overlap of the effects of many molecules. This overlap would
cause a faster build-up of the receptor potential and therefore an earlier impulse firing. In
addition, shorter diffusion and interaction times may be involved since with an increase in
molecule numbers the faster portion of events alone would be sufficiently large.
One of the goals in studying sensory physiology is to develop a complete model
of the receptor cell, which has to include the non-electrical as well as the electrical
steps of the transducer process.
[AS] ~ [A]+[S]
[AS] ~ [AJ+[P]
formation steady state decay
[AS]
t_
Fig. 54. Theoretical time course of AS or dPjdt with a square wave stimulus of S.
A = number of free acceptors, AS = number of occupied acceptors in the stationary
state. Tf= half time of the formation of AS following a square wave pulse stimulus of S.
Ta = half time of the decay of AS after the stimulus end. (KAISSLING, 1969)
The interaction of the stimulus molecules and the acceptors follows the mass
action law according to BEIDLER'S (1954) basic assumption in taste receptors. The
stimulus molecule S combines with the acceptor A to the complex A S which
decays into A and S or P. P symbolizes an odor molecule which is either chemi-
cally inactivated or otherwise prevented from combining again with the acceptor:
k k
A + S ....2-:-AS~A
kz
+ P. (15)
KAISSLING (1969) studied the kinetics of olfactory receptor potentials in the drone
honey bee and in Bombyx assuming that the receptor potential amplitude is pro-
portional to the number of occupied acceptors A S. Then the steady state amplitude
of the potential should be proportional to

AS 1
-- - -=:.,---- (16)
Atot K
S +1
when A tot is the total number of acceptors per cell and Kst = k2 t ks 1
is the
steady state constant. This hyperbolic function A 8 = f (8) is also called the adsorp-
tion isotherm (Fig. 55).
--[Ato~--------
[AS]
Bombyx mori
msec
[Atoll
----2----- 600
/e...
1./-1-(
500
-2 -1 400
log Kst +1 +2
log [5] 'd
300
r \. 1: = In 2
1-
d k2+k3
200
-1 t
100 ~ f
!~
e _ _ e .
0
C 106 107 10 8 109 1010 1011
-2 -1 +1 +2 Bombykol ~moleCUles]
log Kst
10g[S] cc air
Fig. 55 Fig. 56
Fig. 55. Terms of the kinetic model A + S !:; AS -+- A + P as a function of the concentra-
tion S. Atot= total number of acceptors, Kst= steady state constant. Kst= k2+ks For
kl
explanation of the symbols see Fig. 54 (KAIsSLlNG, 1969)
Fig. 56. The half times of the EAG-formation (T/) and decay (Ta) (KAISSLING, 1969). The
response to pure air (0) is a temperature effect. Ta rises dramatically at somewhat stronger
stimuli than used here (see Fig. 57)
With a square wave stimulus of odor concentration the potential should reach the steady
state amplitude with a half time Tf which decreases with increasing stimulus strength S. The
decrease half time Td should be constant (see Figs. 55, 56).
~_I_AS (17)
Ta - Atot
The total number of acceptors Atot is
Jeffs Kst . y
Atot = (k2 + ks) . c (18)
Jeffs ist the rate of molecules at the stimulus concentration c hitting the effective hair surface
per cell. y is the portion of Jeffs finally acting on the cell with low stimulus concentrations. Kst
is the stimulus concentration for a half maximal potential amplitude in the steady state. ks is
the turnover number of the acceptors, derived from the decay half time of the potential
Ta (assuming kg = 0):
(19)
The calculation of Atot revealed numbers which were of the same order of magnitude as the
number of pore tubuli in BCYi11lJyx and in the drone.
Nevertheless, the assumption oflinearity between AS and the receptor poten-
tial amplitude seems dubious. It may be more adequate to assume that each
occupied acceptor increases the conductance of the receptor cell by a constant
value L1Gacc '
LtG
AS = L1 Gacc (20)
Using a simple electrical circuit based on THURM's model of the receptor cell (see
p. 371) one finds the following relationship between the change of conductance
L1 G and the change of voltage L1 V (= receptor potential) across the receptor cell
membrane:
LtV 1
(21)
Lt Vmax 1
Go +
LtG +1
L1 Vmax is the maximum depolarisation with L1 G = 00. The membrane conduc-
tance of the outer segment of the dendrite is Go in the resting state. M is the sum
of all resistances in the circuit except l/Go. This equation shows the same type of
hyperbolic function as the mass action law (formula 16).
Combining formulae 16, 20 and 21 one finds again the same type of function:
LtV 1
(22)
Lt Vmax Kst +1
SW
Here L1 Vmax is the maximum depolarisation with S = 00. The constant W shows
the relationship between Kst and the stimulus concentration Sl/2 LlVmax which
causes a half-maximum receptor potential:
Kst=Sl ,W=Sl . ( A tot LtGacc1 +1 ) . (23)

"2 LlVmax -2 LlVmax G
o
+-
M
A similar equation was derived by MORrrA (1969) for taste receptors. According to this
equation the steady state constant Kst for the dissociation of the complex AS may be con-
siderably larger than calculated from BEIDLER'S equation. Correspondingly, the total number of
acceptors Atot is larger than calculated previously from Eq. (12) (which remains correct). It
should be mentioned that the calculation of Atot from Eq. (12) in BCYi11lJyx (KAISSLING, 1968)
was very preliminary since the EAG curve (Fig. 58) deviates strongly from the hyperbolic
function (Fig. 55) (see below).
Eq. (21-23) are thought to describe receptor cells with a sensitive (and stimu-
lated) patch of membrane which is small when compared with the total surface
area of the outer dendritic segment. This situation exists in taste hairs of insects.
The typical olfactory hair in insects, unlike taste hairs, has relatively long
and thin outer dendrites which are exposed to the stimulus on their whole length.
Therefore one has to take into account the decrease of the characteristic length t. of
the dendrite with increasing membrane conductance Ll G + Go of the stimulated

part of the dendrite according to the following equation:
A= L V +~ (Go G) . R (24)
where L is the length of the sensitive part of the outer dendritic segment and R (Q)
the sum of its internal and external resistances.
A shortening of the length-constant with increasing stimulus concentration
means that the propagation of the stimulus-induced ion currents to the spike
generating zone of the inner dendrite will be reduced. In this way the range of
stimuli over which the receptor responds could in principle be extended.
The change of length-constant can readily be shown to produce receptor-
potential-vs.-dendritic-conductance curves which are flatter than the hyperbolic
curves considered above. For example (KAISSLING and THORSON, 1971, in prepara-
tion) consider a long thin cylindric dendrite with length constant Al (cable 1), con-
nected to a cylinder (cable 2) of length-constant A2 representing the inner dendrite,
soma and axon. The outer dendrite is assumed to have zero current flow through
jts tip, and a membrane containing simply a variable conductance for an excitation
process with equilibrium potential zero. The latter corresponds to the receptor cell
model of THURM (1971) which has no ion pump in the outer dendritic segment
(see p. 371).
The membrane of cable 2, when the outer dendrite is unstimulated, has a
resting potential because of selective conductance to, say, potassium. To represent
the function of the accessory cells as proposed by THURM the extra-voltage and the
resistance Ra provided by this cell are included in the boundary condition matching
voltage and current at the external junction of the two cables. The relationship
between the change of potential Ll VjLl Vmax at the junction of the two cables (i.e.,
the maximum "generator potential") is then
t1 V = 1_
_1_ tanh
Ill' Al ,
(.)
Al,
+
(/2
1
Ai + Ra (25)
LI V max -1- tanh ( - L )
III Al Al
+ (12
1
-----:--_=_
A2 + Ra
where (!i = the sum of external and internal resistances per unit length (i = 1,2)
Ai = the length-constant of the ith cable in the resting state (r) and
during excitation (e)
L = length of external dendrite
Ra = the resistance between the outer sensillum liquor and the haemo-
lymph space across the auxiliary cells (Q).
If, for example, L < A, for the range of stimuli considered, the response is
similar to that of the hyperbolic function. If on the other hand L> 2A, the
hyperbolic tangent approaches unity so that the response can vary as IjA (i.e., as
the square root of the stimulus variable Ll G) over a considerable range of input.
The above relationship is of interest with respect to the response of Bombyx
olfactory cells, which have particularly thin dendrites, and response curves (for
both the EAG and single-receptor impulses) considerably flatter (Fig. 59) than the
hyperbolic function permits.
The stimulus-response curves plotted in double logarithmic graph in Fig. 59

are roughly straight lines over an appreciable range, and as such compatible with
STEVENS (1970) power-law descriptions. The physical model discussed above
may account for a variety of such curves with exponents between 0.5 and 1. It has
still to be clarified whether exponents below 0.5 and other deviations from the
theoretical curves (see Fig. 59; STEVENS, 1970) can be attributed to special
geometric conditions such as taper of dendrites or non-uniform distribution ofthe
stimulus intensity along the dendrite (BIEDERMAN-THORSON and THORSON 1971).
Finally, it seems important to emphasize that wherever large changes in con-
ductances are applied to fine dendrites - as perhaps in a number of situations in
the central nervous system - effects of changing length-constant upon the transfer
function of the system ought to be taken into account.
Exact data from single cells are available neither for the stimulus-response relation of the
receptor potential in Bombyx nor for the membrane and plasma resistances. Therefore the
following considerations ought to be considered preliminary. The aim is to estimate the minimal
change of conductance LI Gmin which produces a minimum receptor potential LI Vmin just
LI V .
eliciting nerve impulses. The EAG-curve shows that LI Vmill is about 7.5% for an average of
max
5 impulses per sec. The minimum depolarization eliciting a single impulse may be assumed to
be about 5 % of LI Vmax, taking into account the duration of the elementary receptor potential
which may be less than one sec. With Eq. 17 we calculate the length-constant AI' of the outer
dendrite of the bombykol receptor cell. We use a dendrite length of 100!1. with a diameter of
0.5!1. and a cross sectional area of the hair lumen twice that of the dendrite. The specific
internal and external resistance may be 50 Q. cm and the membrane resistance 105 Q. cm2
With these values we find AI, = llOO!1. for the unstimulated dendrite. This seems to be large
enough to guarantee the quasi-linear behavior of the receptor cell found up to 5 impulses per
L
sec (Figs. 37,38). Since,- is much smaller than unity
AI,
(26)
If we use this approximation in Eq. (25), derived for the distributed dendrite model, we find
LlVmin 1
LlVmax = 1 (27)
Go + --:----:--=-
(]2 A2 _+ R.
_ _---=c-.::.. _=_ +1
LlGmin
This equation corresponds to Eq. 21 derived for the single patch model.
. LI Vmin
WIth - - - = 5% we find
LI Vmax
(28)
Using the assumptions mentioned above, Go = 1.6 . 10-11 mho for the outer dendrite. The
specific membrane resistance may be 103 Q cm 2 for the auxiliary cells and for the inner
dendrite (cable 2) which has a diameter of about 1!1.. The specific plasma resistance may be
50 Q . cm. Therefore R. = 2 . lOS Q with 500!1. 2 surface of the auxiliary cells and (]2' A2
= 1.4 . lOS Q.
These approximations, used in Eq. (28), give a minimum increase of conductance
LI Gmin = 1.6 . 10-10 mho. This value corresponds roughly to recent estimates of the con-
ductance of a single "open" ion channel- i.e. about 4 . 10-10 mho. This is the value calculated
for sodium conductance in lobster axons (HILLE, 1970) and also in lipid bilayer membranes
modified by the bacterial protein ElM (BEAN et al., 1969). This estimate therefore shows that
a single acceptor alone may be able to produce large enough change of membrane conductance
to trigger a nerve impulse. HAGINS (1965) concluded that one ion channel opens per incident
photon in the squid retina. With the values used above, Eq. (25) does not exhibit the LI G V
behavior. However, we found that the curve shape is very sensitive to a change of (h, If (h is
increased by a factor of 6 (i.e. to 3 . 10 7 illft) a rather good description of the data from Bombyx
results, as shown in Fig. 59. Here we plotted the impulse numbers of the first second of responses
from Figs. 37 and 38. The impulse numbers of the bombykol receptor cell are rather propor-
tional to the EAG-amplitude between 10- 3 and 1 [Lg of bombykol on the odor source. The
increased value of (1, would be consistent with the assumption that the dendrite diameter is
underestimated in the electron-microscope because of artificial shrinking. Thus, the cor-
respondingly reduced extra-dendritic space would amount to an increased (11"
In the light of the behavior of the distributed dendrite model, it is interesting that shorter
olfactory sensilla often show branching of the outer dendrite. This would, of course, decrease
the length-constant of the dendritc and provide an appropriate working range of the sense cell.
Bombyx morl
Bombyx mon
E AG-ampl itude
lj.Jg mV
O~5s
ec100
mV 4
100j.Jg I ..... -I..
/_'\..v~ 3
1000j.Jg
2
/~-----
...,-......------
TC-Bombykol
Fig. 57
C -3 -2 -1 o ., .2 +3
loglo j.Jg Bombykol (TC) and

Fig. 58 related compounds
Fig. 57. Increased decay times of the EAG after strong stimulation. Sequence of 5 stimuli
(black bars). Loads of bombykol at the source in [Lg. H 2 -bombykol = lO-trans-hexadecen-l-ol
(KAISSLING, unpublished)
Fig. 58. Stationary EAG-amplitude with different compounds. The odor concentration was
checked with 3H-labelled bombykol and H 2 -bombykol (= Dihydrobombykol), and was found
to be approximately proportional to the load on the filter paper up to 100 [Lg. For the 1000 [Lg
stimulus a piece of cotton-wool was used instead of the filter paper. 16 = hexadecanol,
17 = heptadecanoic acid. There are no adaptation effects in these curves, as controlled by
test stimuli. (KAISSLING, unpublished)
Further morphological and electrophysiological work will be necessary to

validate the conclusions drawn from the proposed receptor cell model. Also the
model itself has to be refined. Main1.y it will be important to study the influence of
the stimulus-acceptor kinetics on the electrical model (Eq. 25). Adaptation experi-
ments prove this influence. Thus, it appears that the decay time of the slow
potential increases dramatically (Fig. 57) when obviously more molecules are loaded
onto the antenna than can be turned over by the acceptors. The same increase of
27 Hb. Sensory Physiology, Vol. IVjl
418 K.-R. KAISSLING: Insect Olfaction
the decay time and saturation effect is observed at much lower potential amplitudes
if one loads the antenna with similar numbers of molecules of a less effective odor
substance (e.g. TT-bombykol or dihydrobombykol) (Figs. 57, 58). Therefore the
saturation cannot be due to an adaptation of the membrane potential caused by a
too large amplitude of the potential. Obviously, the acceptors are fully occupied,
and moreover the accumulated active odor substance on the antenna continues to
stimulate (KAISSLING, unpublished).
1091 0 molecules bombykol /c e ll ' sec

-I 0 I 2 3 I.
2
. ..
/"----- .~ J:
.
2
",/;, .~
//
10 9 10
".
i m~ulses
cell sec ' 10 9 10
I
impulses

I
e I ,' cell sec
'.
j/
Bombyx I
I
I
Locusta
0
/
0 0
-J
2 3 I. 5 6 7
10910 molecules trans-2 - hexenal /c e ll sec
Fig. 59. Stimulus response curves of bombykol receptors and of a single green-odor receptor
of the locust (6) (from KAFKA, 1970, Fig. 6). Impulse numbers of the first second of
response. Dots: values calculated from Fig. 37 and 38. Circles: Average values from several
hundred measurements (calculated from KAISSLING and PRIESNER, 1970). Upper dashed line:
hyperbolic function (see Eq. (21)). Drawn line calculated from Eq. (25) (see text p. 415)
There may be several reasons for a weaker effect of analogs of the key compound
on the receptor potential. The shift of the stimulus response curve parallel to the
abscissa on a logarithmic scale may be due to (i) a lower affinity Kl = k ~k
st 2 3
between the "wrong" compound and the specific acceptors. The lower saturation
level of the stimulus response curve in the isomers could be explained (ii) by a
smaller number of acceptors fitting the wrong molecules. Another possibility (iii)
would be a lower affinity of the specific acceptors according to (i) combined with
the saturation of a preceding molecule transmission by an unspecific acceptor
system. It seems unlikely that (iv) an analogous molecule could produce a weaker
effect on the membrane conductance than the key molecule, since the acceptor may
act as an all-or-none mechanism. It may, however, be "open" for a shorter time
interval.
The prolonged decay times of the slow potential aftef strong stimulation cannot
be explained by the simple kinetic model (see Eq. 15). Obviously, the antenna
accumulates molecules which act on the acceptors (and the cell) after the end of
the stimulus. This can be taken into account by introducing a one-directional
step (1):
==
1. S gas -------> S ads ,
2. Sads +A A Sads -------> A + P.
Here Sgas is the stimulus concentration in air, Sads denotes the number of mole-
cules which are adsorbed at the antenna but not yet bound to the acceptor.
Bombyx moTi
100
I +n +U1
%
M
~
..s:
.,
I
u
'"
I
50
e
\e.~:___ -.----
fe~._e_ _e
~&
n chloroform
III methanol

"0
: I pentane
2::;)
V a
15x e\. _e_.____ e
0510 30 60 min 120
lax I+n+III
5x {exposure 2,5 min
a
0510 30 60 min 120
time between exposure and elution
Fig. 60. Fractionated elution of tritium-labelled bombykol after applying it to Bombyx

antennae. 6 probes of 40 antennae each, adsorbed 10'0 molecules per antenna during 2,5 min
exposure to the stimulus. After different waiting times each probe was successively washed
in pentane (I), chloroform (II) and methanol (III), 10 min in each solvent. The sum of the
eluted activities (ordinate) was always about 90% of the total activity adsorbed
(KAISSLING, unpublished)
Even this extended model seems to be too simple to describe the kinetics of the
slow potential because three processes Were found which probably influence the
kinetics of the potentials. The first process is the diffusion of the molecules from
the hairs to the branches and the stem of the antenna (STEINBRECHT and KASANG,
unpublished). The second process is a penetration of the odor molecule from an
outer lipophilic phase to an inner hydrophilic phase.
Several groups of antennae have been exposed to the same stimulus with 3H-bombykol.
After different waiting times each group was washed successively with pentane, chloroform
and methanol. By washing immediately after the exposure, most of the radioactivity was found
in the pentane fraction. With increasing waiting times the activity eluted with pentane decreas-
ed and increased in chloroform and methanol (Fig. 60). The sum of eluted activity remained
constant and was about 90% of the total amount of activity adsorbed on the antennae. This
percentage increases with the elution time (KASANG and KAISSLING, unpublished).
27*
420 K-E. KAISSLING: Insect Olfaction
This time-dependent reversal of elution behavior may indicate a penetration of

molecules from the outer lipophilic surface of the sensillum to its hydrophilic
interior. Presumably the hypothetical acceptors are not involved in this type of
penetration of the odor molecules since the reversal of elution occurs also with
stimuli much stronger than those which saturate the acceptor system as shown by
the potential kinetics.
b 3H-Bombykol b
[Butanol (NH3U rOiisoproPYl-]

start
l ether
start
l l
Ll
~~~e~~~
b a
c
R
----'
~ antenna c b a
~.~2hOO~
~ n legS c ba
JULJ5m~
Fig. 61. Thin layer chromatograms of pure 3H-bombykol (1. line) and of eluents of
bombykol-incubated antennae (2. and 3. line), legs (4. line) and of cotton wool (5. line).
(a) acidic fraction (b) alcohol peak (TC-bombykol ?), (c) ester fraction. Two systems of
solvents were used (see above) (KASANG, unpublished, see 1971)
The same phenomenon of penetration was found with 3H-bombykol and 3H-dihydro-
bombykol on male as well as on female antennae and in antennae of another lepidopterous
species which was not sensitive to bombykol. It did not, however, occur in the legs of Bombyx
which are covered with many large hair-like scales. Here the bulk of the radioactivity always
appeared within the pentane-fraction.
Another process found on the antenna was a partial chemical change ofbombykol
(KASANG, 1971). An analysis by thin-layer chromatography of the 3H-bombykol
from the antenna revealed a splitting of the previously uniform bombykol peak
into three peaks (Fig. 61). One of them retained the Rf-value of the bombykol, the
other two corresponded to an esteric and an acidic fraction. This metabolic change
of bombykol is due to enzymatic activities since the change can be blocked by
specific agents which inhibit enzymes or denatured proteins.
The Central Pathways of Olfactory Fibers 421
Several experimental results indicate that the specific acceptors are not involved in this
metabolism. (i) The metabolites are found also on female antennae (these being practically
insensitive to bombykol) as well as on the animal's legs. (ii) Dihydrobombykol is metabolized
to the same degree although the cells need 103 times higher threshold concentration than for
bombykol. (iii) The metabolism occurs within minutes whereas the cell response decays with a
half time less than a second (see Fig. 56).
The present state of the processes on the Bombyx antenna can be interpreted as
follows. After adsorption the odor molecules diffuse within an outer phase (PI> see
below) from which they directly reach the acceptors, diffusing along the tubules.
Simultaneously with this process, molecules penetrate from phase 1 into phase 2
(P2' see below) and are enzymatically metabolized (P', see below).
These latter two processes have a similar time constant. Nevertheless, both
processes are independent of one another since the metabolism occurs on other
tissue lacking penetration. The interaction of the molecules with the acceptors may
be followed by the same or by a separate mechanism of inactivation of the odor
molecule because one molecule elicits only one nerve impulse. This inactivation
could be a cis-trans isomeration or a removal of the molecule to a separate site
(P, see below).
The biological meaning of the enzymatic metabolism of bombykol may be to
inactivate the odor substance on the antenna and other parts of the body. This
process seems also to contribute to the receptor potential kinetics especially with
regard to the recovery after strong stimulation (KAISSLING, unpublished).
The kinetic model can be completed in the following way:
I} Adsorption Sgas --+ SPI :- -,. Ll G
2} Acceptor process Sp, + A --+ AS --+ A + P
3} Penetration SPI ~ SP2
4) Diffusion SP1.2 ~ SP1.2 (on ineffective surface)
5) Metabolism SP1.2 + E --+ E S --+ E + P' .
In some cases desorption of the stimulus from the antenna also has to be taken
into account, e.g. in the coeloconic sensilla of the locust (KAKFA, 1970) or in CO 2-
and humidity-receptors (Sgas ~ SpJ
Recently RIDDIFoRD (1970) found proteins on the antennae of saturniid moths.
Antennae of male and female moths were washed for half an hour with ringer
solution. Gel electrophoresis showed about a dozen protein bands. Animals with
washed antennae did not respond behaviorally to olfactory stimuli but recovered
after 3 or more hours. A second elution of the antennae revealed a complete protein
spectrum after 24 hours. It seems unclear whether these proteins contain acceptor
substance or enzymes.
No significant decrease of the EAG was observed in 5 antennae of Antherea and 11
antennae of Bombyx after washing up to 12 hours in ringer solution (KAISSLING, un-
published).
J. The Central Pathways of Olfactory Fibers

The first synaptic contacts between the axons of the sensory cells and secondary
neurons occur in the deutocerebrum. This region of the insect brain usually con-
sists of a central mass of nerve fibers, a region of glomeruli and a peripheral layer
422 K. -E. KAISSLING: Insect Olfaction
of perikarya of the secondary neurons (Fig_ 62). Synapses are restricted to the
glomeruli. (For details and references see GOLL, 1966; SCHURMANN and WECHSLER,
1968; BOECKH et al., 1970).
Another part of the deutocerebrum emits motor fibers running to the muscles
which move the antenna. The sensory and the so-called motor part of the deuto-
cerebrum are connected by nerve fibres. Secondary connections are also found to
the contralateral deutocerebrum as well as to higher centers of the CNS (cf. GOLL,
Fig. 62. Sagittal section of the deutocerebrum of a worker honey bee. The antennal nerve
coming from right below splits up in several branches. Right: the antennal lobe with a
central fiber mass and peripheral glomeruli. Left: dorsal lobe (so-called motor center).
Holmes silver stain. (By courtesy of A. PARETO, Seewiesen)
1966). GOLL also found interneurons between the glomeruli. BOECKH et al., (1969)
used a method of fiber degeneration (LAMP ARTER et al., 1967) and found sensory
fibers running from the antenna of Calliphora directly into the contralateral deuto-
cerebrum as well as fibers bypassing the deutocerebrum and connecting with
other parts of the CNS. YAMADA (1968) succeeded in extracellular recordings from
olfactorily activated single neurons in the deutocerebrum of the cockroach.
Recently very useful review articles on insect behavior with respect to chemicals were
published which are not yet dealt with in this manuscript (see BEROZA [ed.], 1970; JOHNSTON
et al. [ed.], 1970; and WOOD et al. [ed.], 1970). Sec also the interesting article of LEWIS (1970).
The author is indebted to D. SCHNEIDER and his collegues for discussion and E. PRIESNER
for many references to the literature of which not all could be mentioned. R. LOFTUS kindly
corrected parts of the manuscript. Eq. 25 was derived by J. THORSON. We thank M. BIEDER-
MAN-THORSON for help in computer programming. I thank I. BLOCK, G. SAMESREUTHER,
T. RIMBECK and M. LYSEN for technical assistance.
References 423
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Chapter 15
Olfaction in Birds*
By
BERNICE M. WENZEL, Los Angeles, California (USA)
With 7 Figures
Contents
Anatomy . . . . . . . 433
Electrophysiology. . . 435
Olfactory Perception . 439
Responses to Odors. 440
Learned Responses . 442
Feeding Behavior . 443
General Considerations. 444
Summary 446
References . . . . . . 446
If it were not for a long history of doubt concerning the existence and function-
ing of an olfactory system among birds, there would be no particular reason to
single out this class for discussion. The tone for one prevailing attitude about birds'
sense of smell was set more than a century ago by the advice of AUDUBON, the
American naturalist and painter of birds, who recommended that his readers
" ... abandon the deeply-rooted notion that this bird (the turkey vulture) possesses
the faculty of discovering, by his sense of smell, his prey at an immense distance ... "
(1826, p. 173). Although it became known that at least some forms had well-
developed olfactory epithelium, nerves, and bulbs, the ability to detect odors and
to respond differentially to them remained a highly controversial topic. Anecdotes
were exchanged in the literature to support each side, and contradictory results
were reported from experiments of variably skillful design (see SOUDEK, 1927;
STAGER, 1964; STRONG, 1911; and WALTER, 1943 for reviews), the sum of which
created a generally negative impression. This era of skepticism has been replaced
recently by a more positive one, for which the work of COBB (1960a) is proposed as
a suitable origin. He studied certain aspects of the comparative anatomy of the
avian brain and included a section on the olfactory bulbs that provided impetus for
a systematic consideration of the possibility of an olfactory system with different
degrees of function in different avian forms.
* Bibliographic assistance was received from the UCLA Brain Information Service which
is part of the Neurological Information Network of NINDS and is supported under Contract
DHEW PH-43-66-59. Financial support was provided by NSF grant GB 7677 to the author.
Anatomy 433
Greater understanding of olfaction in birds has begun to develop as more

appropriate questions have gradually been formulated to replace the vague query,
"Can birds smell 1" It is no more reasonable to expect that all members of the class
will have the same olfactory characteristics than it is to apply the same expectation
to all mammals. It is also very important to define what is meant by the ability to
smell. To AUDUBON and those who followed him, meaningful evidence was related
either to the effectiveness of odors in influencing feeding behavior or to an ability
to utilize odors as cues in discrimination learning tasks in the laboratory. Thanks
to modern electrophysiological methods, the range of meanings has been expanded.
At the same time it has been realized that stimulus perception can occur without
any necessary involvement of that stimulus in the regulation of the animal's
activities. KARE (1965, p.433) summarized this idea very well when he said,
" ... the presence of neuroanatomical structures suggests that olfactory informa-
tion can be transmitted even if it is not behaviorally meaningful."
The present review discusses the olfactory anatomy of birds, the evidence for
neural transmission of information about olfactory stimuli, and the ability of birds
to utilize such information both in guiding their natural behavior and in learning
to respond differentially to olfactory stimuli.
Anatomy
The only noncontroversial subject in avian olfaction at present is the anatomy
of the system. The existence of olfactory epithelium, olfactory nerves, and olfactory
bulbs in various species has been known for a long time. Beyond this simple
generality, however, variation is the rule, especially with regard to the relative
sizes of the several parts of the system in different species.
Variation extends to the nasal cavity as well. Entrance is through the paired
external nares located at the base of the bill, except in kiwis (Apteryx australis)
where they are at the tip. It is not known how much dynamic alteration of the
internal airway can occur, but the possibility for change of the external nares has
obviously been lost with their placement in the rigid bill. In general the nasal
cavity is well adapted both anatomically and physiologically to deliver a stream
of warm, moist air to the olfactory epithelium (BANG, 1960, 1964,1965,1966,1968;
KARE, 1965; PORTMANN, 1961). Typically, there are three successive chambers,
usually with conchae or turbinals, on each side of a median septum. Nasal glands
in the anterior chamber provide moisture and in marine-feeding forms they serve
also to secrete excess salt. Special adaptations with possible significance for olfac-
tion have been recognized recently. Marine, aquatic, and wading birds have
developed an anterior valve, which probably remains open during flight but closes
as soon as the bird enters sea water, thus preventing any destructive effects of the
water on the olfactory membrane (BANG, 1965). This structure has also been found
in the kiwi, a land bird with no known water-living ancestors (BANG, personal com-
munication). In certain species that have an especially prominent olfactory
epithelium, e.g., the turkey vulture (Cathartes aura), the Trinidad oilbird (Steat-
ornis caripensis) , and Laysan and black-footed albatrosses (Diomedea immutabilis
and D. nigripes), the anterior nasal chamber is especially well designed to promote
the passage of air to the olfactory receptor (BANG, 1960). In all birds, a portion of
28 Rb. Sensory Physiology, Vol. IV!l
434 B. M. WENZEL: Olfaction in Birds
the inspired air passes into the buccal cavity through the choana in the floor of the
middle chamber and then to the lungs for respiration while the remainder flows
into the third chamber, presumably to stimulate olfactory receptors.
The olfactory receptor cells are usually distributed along the surface of the
posterior concha, the nasal roof, and upper septum in epithelium with the typically
yellow pigmentation of the mammalian olfactory epithelium. The extent of the
epithelium differs among species but its general structure appears to be essentially
constant throughout the vertebrates (BROWN and BEIDLER, 1966; GRAZIADEI and
BANNISTER, 1967; STAGER, 1964). Three cell types are found, viz., receptors,
Sparrow Crow Porokeet Coot Loon Petrel
LAT. QfCjQ
VENT. ~
j---------I

H 1--1 I----i H 1-1
Fig.!. Anatomy of the olfactory bulb in six avian families. (From BANG and COBB, 1968)
sustentacular or supporting cells, and basal cells. The receptors are typical bipolar
ciliated neurons with one unique feature of fine structure that has been found in
both avian species studied so far but in no other vertebrate; specifically, a relatively
large number of microvilli on the terminal dendritic swelling (BROWN and BEIDLER,
1966; GRAZIADEI and BANNISTER, 1967). The effective receptor surface may be
increased significantly in this way, but inasmuch as the transduction process in
olfaction is not yet understood we can only speculate about the function of the
microvilli at the present time. No information exists about the number of receptor
cells but it varies considerably in correlation with epithelial area, and certain forms
probably possess as many cells as some of their macrosmatic neighbors in other
classes (BANG, 1960; STAGER, 1964). Axons of the receptor cells extend proximally
as olfactory nerve fibers to reach the olfactory bulbs located at the anterior pole of
the forebrain.
The size, configuration, and exact location of the olfactory bulbs is extremely
variable within the avian class (Fig. 1) ranging from the large and prominent bulbs
of the kiwi which extend directly anterior down to the minute, fused, and ventrally-
directed bulb in some of the passerine forms. Impressed with this diversity, Cobb
extended earlier work of others and attempted to systematize comparative data
on the relative size of the olfactory bulb. He chose the largest diameter of the
forebrain in each species as the equivalent base for a fraction with the diameter of
one olfactory bulb as the numerator. This ratio has now been calculated for 108 dif-
ferent species (BANG and COBB, 1968; COBB, 1960a, b). The values range from
37.0 for the snow petrel (Pagodroma nivea) to 3.0 for the black-capped chickadee
( Parus atricapillu8). The orders with the largest ratios are Apterygiformes (34.0),
Procellariiformes (29.0), Podicipediformes (24.5), Caprimulgiformes (23.8), and
Electrophysiology 435
Gruiformes (22.2). Those with the lowest ratios are Pelecanilormes (12.1), Piciformes
(10.0), Passeriformes (9.7), and Psittaciformes (8.0). In view of their observations,
BANG and COBB suggest that" ... in kiwis, in the tube-nosed marine birds, and in
at least one vulture, olfaction is of primary importance, and that most water birds,
marsh dwellers, and waders, and possibly echo-locating species, have a useful
olfactory sense. In other species it may be relatively unimportant" (1968, p. 60).
In all the forms examined, an approximation of the typical mammalian pattern
of internal bulbar anatomy has been found, consisting of a glomerular layer, mitral
cell layer, and an internal granular layer. The relative size of each layer and of its
cellular constituents, the details of cellular alignment, and the precise relationships
between adjacent layers vary somewhat from species to species, as does the posi-
tion of the ever-present olfactory ventricle (CRAIGIE, 1930, 1932, 1941; CROSBY and
HUMPHREY, 1939; HUBER and CROSBY, 1929). Very little is known about the
projections from the olfactory bulb. No experimental anatomical studies of this
area have been done in birds, but some descriptions are available based on exami-
nation of normal histological material (CRAIGIE, 1930, 1932, 1940, 1941; CROSBY and
HUMPHREY, 1939; HUBER and CROSBY, 1929; JONES and LEVI-MoNTALCINI, 1958).
All forms appear to possess a lateral olfactory tract as the main projection of the
olfactory bulb, a prepiriform area, and anterior olfactory nucleus. HUBER and
CROSBY (1929) point out that the amount of development of these secondary
olfactory centers is correlated with the size of the olfactory bulbs. CRAIGIE (1930)
also describes a huge olfacto-habenular tract and large habenula in the kiwi.
Even though limited in amount, all of the available evidence is in complete
agreement on the existence of an avian olfactory system with anatomical charac-
teristics predictably similar to those of amphibians, reptiles, and mammals.
Furthermore, the degree of development of the individual parts of the system is
highly correlated, suggesting the likelihood of a wide range of olfactory ability
among the various species with their diverse habitats and behavior patterns.
Electrophysiology
In light of the essential morphological identity between the olfactory system of
birds and that of other vertebrates, it is not surprising to find that the electrical
activity of different parts of the system is also essentially similar to that of other
forms. TUCKER (1965) provided the first evidence for electrophysiological adequacy
of the avian olfactory system when he recorded the action potential from the
olfactory nerve in 14 different species ranging from the house sparrow (Passer
domesticus), which is one of the least endowed on COBB'S list, to forms with much
larger central representation; viz., the muscovy duck (Oairina moschata) , the gray-
lag goose (Anser anser), and the black and turkey vultures (Ooragyps atratus and
Oathartes aura). Regardless of the species from which he recorded, his results were
unilorm in showing pronounced electrical activity in the nerve bundle correlated
with the presentation of various odors to the anesthetized bird. Fig. 2 shows the
results of presenting five different intensities of amyl acetate to four different birds
and, for comparison, to one macrosmatic mammal. The magnitude of the nerve
response is clearly related to the stimulus intensity and for some of the birds is
greater than that recorded from the olfactory nerve of the rat. He also recorded the
28
436 B. M. WENZEL: OHaction in Birds
electro-olfactogram (EOG), a large, slow, mucosal potential that occurs in response

to the onset of an olfactory stimulus. It has not been directly studied in this class,
so that no specific statements can be made about it but at least it is known to
resemble that recorded from other vertebrate forms.
The responses of single units in the olfactory nerve have been investigated to a
limited extent (SHIBUYA and TUCKER, 1967). The striking feature about these data
Sparrow Hawk
Black vu lture
... ' _.-J,'.i\~, I ' ..... . .)\\1..-............ I J!\
Ro t
""I h ' +e t t
...... ",..... ~, I. .., _,"'~'_._. .,. . . . . . . . .
"1-"1'
Fig. 2. Primary oHactory nerve responses to amyl acetate breathed in air at concentrations
expressed as exponential fractions of vapor saturation at 20 C. Odor was presented every
3 min for durations of 1 min in the first trace and 0.5 min in the others. (From TUOKER, 1965)
is the variety of response characteristics among the units studied. AB is usually the
case with sensory units, the thresholds vary from fiber to fiber for a given stimulus
compound as well as within a fiber for different compounds. In addition, some
fibers show an increasing response rate as a function of stimulus concentration
while others behave in just the opposite way. Some fibers even show both response
patterns depending on the stimulus compound used. Background activity in the
nerve tends to be very low. Spontaneously firing units can be found, however, and
they demonstrate familiar patterns of either increased or suppressed activity at
stimulus onset. No information about unit activities has been reported for any
other avian species, so that it is premature to speculate about possible mechanisms
of stimulus coding. Suffice it to say, the peripheral olfactory system is capable of a
response repertoire that is sufficiently varied to encode a number of stimulus
characteristics.
Just as the peripheral portions of the olfactory system show electrical responses
similar to those reported for members of neighboring classes, the behavior of the
Electrophysiology 437
central portion is also generally consistent as far as it has been studied (SIECK and
WENZEL, 1969). Recordings from macroelectrodes chronically implanted in the
olfactory bulbs of pigeons (Columba livia) have shown the characteristic wave
bursts associated with inspiration (Fig. 3) with frequencies of about 15-25 cps.
Absolute thresholds indicated by altered electrical activity approximate 10-8 to
10-10 molesjml for the compounds used as stimuli, viz., amyl acetate, methyl
A-I
'<'Y" ....."""""'Y.-v-~~"'\/\IVf-.-,.....~..--'_.h....,....."."""'....
1 - - -1 1100
1.0 sec /LV
C-42
\'I~~l'~'''I'o'I'\II~~~~
I---l 1100
O,Ssee IlV
0-2S
~~'~~~~~~~~~~~~~~
O
l---ls
, see 1100
P-13
~"""""'fI"r<,~~'''IV.''''''''''''~'~~WH-'r~~
O~ 1p.V
100
,I.,..~~~W )1 ~1'~'w:~,~~~1')\ ~I\\'i

I-----{ I 200
1.0 see /LV
Fig. 3. Intrinsic activity recorded from the olfactory bulb of five different species of birds, viz.,
albatross, chicken, duck, pigeon, and shearwater reading from top to bottom. All recordings
were bipolar between the two bulbs except for the albatross and pigeon in which both electrodes
were in one bulb
methacrylate, methyl salicylate, octanol, pyridine, and trimethylpentane. Inhala

tion of different stimulus compounds or of different concentrations of the same
compound is followed by changes in the electrical activity. For any given bird the
various patterns of change are correlated with the qualitative and quantitative
characteristics of the stimuli (Fig. 4). Another bird of the same species, however,
may show a different set of electrical responses to the same stimuli. This pronounc
ed individual variation appears to be unrelated to electrode placement and has not
yet been explained. Significantly, the changes do not appear either if the olfactory
nerve is sectioned bilaterally (Fig. 5) or if the bird breathes through its mouth. If
one nostril is plugged, stimulus-correlated activity disappears on that side.
Records have also been made from the olfactory bulbs of other species (Fig. 3),
representing both larger and smaller bulb-forebrain ratios than the pigeon (18.0),
including in ascending order of ratio the domestic chicken (Gallus gallus, 15.0),
mallard duck (Anas platyrhynchos, 19.0), black-vented shearwater (Puffinus puf-

finus opisthomelas, 29.0), and black-footed albatross (Diomedea nigripes, 29.0),
(SIECK, 1967; WENZEL and SIECK, in press). All of these forms show at least
some features of what can be called normal olfactory activity. There is no sug-
S3
vJt;...o(>/~~"''I;I'I'W~"'f'r'-'''r''('r''I~'''I>-. ~i'I~"""""'''''~r''!.''~,rI'W-~~
-8 _ 1100
AA 3.33'] 0 mImi 0.5sec /LV
~~\'n1:"~L~~'~'~~~iJ,~W'1I/~t 1~/~W~v+I/(.~,,~"fM'~'"
-10
MS 6.33,10
.NAJj. N~Jw,.,/'Jf'' 1,~~'~.nnllruw,~~..,..,wN#k-N~~

PVR 7,61,10 8
~~~~'I'#~J~"''''''~
TMP 1.87.107
Fig. 4. Olfactory bulb responses recorded through two electrodes in one bulb of pigeon to four
different odorants (amyl acetate, AA; methyl salicylate, MS; pyridine, Pyr; trimethylpentane,
TMP). Each trace starts just before the first inspiration that carried odorous molecules to the
olfactory receptors. (From SIECK and WENZEL, 1969)
31 TMP 9.63 10- 8 m/m!
Control Stimulus
N. Vseclion
~~~~~~;'\~~;M~~"'f\v.~)1.\/1,~~
N.l &"V sect ions f
Fig. 5. The effect of bilateral trigeminal (N. V) section and of combined trigeminal and olfac-
tory (N.L) sections on the response of the olfactory bulb to trimethylpentane. (From SIECK
and WENZEL, 1969)
Olfactory Perception 439
gestion from visual inspection of the records that an increase in the relative
size of the bulb is associated with different features in the electrical activity,
although finer-grain analysis may yet reveal correlated effects. Because of the
extremely small size and inaccessibility of the olfactory bulbs in forms with the
lowest ratios, it is very difficult to implant recording electrodes accurately and
no successful chronic preparations have been made.
Completed work has established the continuity of several avian orders with
other vertebrates in regard to gross electrical activity of the olfactory bulb. Far
too little has been done to permit any statements concerning regional differences
in activity within the bulbs, interactions between the bulbs, or coding mecha-
nisms within the bulbs. No unit recordings have been made from the olfactory bulb
of any bird nor has any type of recording been done from any downstream site
other than the prepiriform area in pigeon (SIECK and WENZEL, 1969). The latter
omission is not surprising in view of the fact that the projections from the avian
olfactory bulb have not yet been mapped experimentally. A great deal needs to be
done before the possible implications of bulbar development throughout Aves are
fully explored, a project, incidentally, that has yet to be undertaken in any other
order of vertebrates. Regardless of the many gaps in our knowledge, however, it is
perfectly clear that some birds, if not all, show the kind of activity in the olfactory
system that is seen in other vertebrates whose olfactory ability has never been
questioned.
Olfactory Perception
The steady trickle of reports concerned with olfactory perceptual ability among
birds that has continued since the time of AUDUBON has given us very contradic-
tory results. The accumulated anatomical and electrophysiological information
does provide confidence in the concept that birds perceive odors, and, therefore,
behavioral studies must be examined closely. In evaluating this literature it is
essential to remember that a great variety of procedures has been followed, so
great as to make it impossible to relate one set of experiments to another. At the
same time the number of experiments is still too small to permit identification of
the critical factors that would account for the differences in experimental results.
In addition to such obvious things as species, odorants used, and the number of
observations made, there have been profound differences in the types of behavior
observed and in the procedures of observation. The birds studied have run the
gamut of olfactory bulb size, of natural habitat, and of preferred food, whereas the
stimuli have been chosen from among both natural food materials and laboratory
chemicals.
As mentioned above, investigators have differed sharply in their definitions of
olfactory ability. Some have considered invalid anything but evidence for the use
of olfaction in obtaining food under natural circumstances; others have required
that the birds in their experiments learn to make specific responses in association
with the presentation of specific odors, while still others have accepted any con-
sistent alteration in any aspect of the bird's behavior as a function of odor pres-
entation. Clearly, these definitions are very different, representing a hierarchy of
ability such that tests at different levels could yield contradictory results. It is
possible that a bird might exhibit some consistent unlearned response in the pres-
ence of an odor, but find it very difficult, if not impossible, to learn to associate a
new response to an odor, even though it was capable of acquiring differential
responses to stimuli in another modality. Similarly, a bird might be able to form
such an association but never make use of olfaction in its regular feeding activities.
There is no basis on which to adopt anyone of the possible definitions as the only
satisfactory approach, for each of them is appropriate in testing the basic question
as to whether the olfactory system is functional. The last of the three definitions
above, i.e., consistent alteration in any aspect of behavior as a function of odor
presentation, answers the question most directly without either introducing ad-
ditional questions about learning ability or about the application of olfactory
information. All three facets are involved in the broad question of the mode of
functioning of the avian olfactory system provided that the first one is extended
to include any natural behavior rather than just feeding.
Responses to Odors
In a search for effector organs that show consistent unlearned responses to
odors, both respiratory rate and heart rate have been monitored. The rationale of
such experimentation is that the presentation of a new stimulus through any sen-
sory modality to an organism that is being tested in quiet conditions results in a
group of involuntary responses that usually includes a change in heart and
respiratory rates. Without raising the unanswerable question as to whether or not
such changes can be interpreted as conscious perception of the stimulus, the
parsimonious conclusion can be drawn that the olfactory system has functioned to
the point of transmitting information about the occurrence of a stimulus to those
portions of the central nervous system that mediate the observed responses.
Even in this type of experiment, the literature is in disagreement. WALTER
(1943) measured respiration in pigeons and found no changes in rate as a function
of the presentation of odors. On the other hand, both NEUHAUS (1963; FINK, 1965)
and WENZEL (1967) have reported positive results. NEUHAUS found that the pat-
tern of respiration of gray-lag geese changed reliably when a bottle of scatol
screened from view was opened under the bird's beak. No such consistent changes
occurred when control bottles of water were opened. Measurements of both heart
rate and respiration by WENZEL showed that the heart rates of pigeons increased
above the resting level in response to odorous stimuli delivered through an olfacto-
meter, and the amount of the increase was greater with more intense stimuli.
Respiratory rates also increased although not as regularly as heart rate, but the
dose-response effect on respiration was inverse with the stronger stimuli eliciting
smaller increases. This relationship probably indicates a mechanism for preventing
the inhalation of large amounts of an intense odor. The relationship between central
and peripheral responses is shown in Fig. 6. Comparison of the peripheral reactions
with the effects of control stimuli shows that the responses are of the same type as
those observed following a visual stimulus, and, in addition, they cannot be
attributed to possible nonolfactory cues associated with the presentation of odorous
stimuli. The ultimate control, that of sectioning the olfactory nerves, sharply
reduces the magnitude of the heart-rate response especially to the weaker odors.
This effect is shown in Fig. 7, which also shows the biphasic effect of interrupting
the appropriate portion of the trigeminal pathway; viz., a slight reduction in the
OHactory Perception 441
reactions to weaker odors but a greater reaction to the stronger ones, as if removal
of the trigeminal component of the sensation actually makes the remaining, com-
pletely olfactory, sensation more effective. The trigeminal nerve presumably
mediates the smaller responses to the more intense odors that the birds continue
Fig. 6. Bulbar electrical activity, EKG, and respiration from a pigeon in response to a IO-sec
presentation of amyl acetate at 8.76 x 10-8 moles/ml. The stimulus period is shown by the
signal marker in the second channel. (From WENZEL, 1967)
AMYl Ace TATe TRI Me THYL PENTANE

2~
w
~
...
0:
0:
10
'"w -~
=
0
170 241
~~ 161
w~ moles/ml mol.S/ml
<.0 0 PYRIDINe LIGHT
2:1:
~ - Z~ ~o
~ 20-. ~ 40 oS PRE- }
POS ,_ N I SECTION
~ I~ i JO
o PRE- }
::; 10, ZO o POST-
H :sz:: (OPHTHALMIC)
S[CT IO
10
O~ ........"-'---
617 76 1 10 4 .. 10- 7
moles 1m I
Fig. 7. The effect of bilateral trigeminal or oHactory nerve section on the heart rate responses
of pigeons to different intensities of three stimulus compounds and to a control stimulus of
light onset. The failure of one group to respond to trimethylpentane at 1.87 moles/ml before
oHactory nerve section is unexplained
to make after loss of the olfactory nerve; an observation that further supports the
groving conviction that very few, if any, odorants are "pure olfactory" stimulants.
Neither operation affects the response to the visual stimulus.
Records have also been obtained from shearwaters, mallard ducks, turkey
vulture, chickens, Amazon parrot (Amazona ochrocephala), black-footed albatross,
raven (Corvus corax), canaries (Serinus canarias), and kiwi. In all but canary,
raven, and parrot, the presentation of odorous stimuli is associated with some type
of consistent change in one or both of the indices. Apparently, species differ in the
nature of their reactions to new stimuli presented under these conditions. The
pigeon, for example, shows more consistent changes in heart rate than in respiratory
rate, but the opposite is true for the shearwater. In the case of the one kiwi studied,
no change in heart rate occurred to auditory, olfactory, or visual stimuli, but
characteristic alterations in the pattern of respiration took place, which were
accompanied by a desynchronization of the brain electrical activity.
In view of the information above, it is difficult to account for Walter's failure
to record any consistent respiratory changes from his pigeons in response to either
amyl acetate or pyridine, the two odorants he presented, even though his birds did
react to needle pricks on their wings. His failure could be attributable to a combi-
nation of effects, including the lower reliability of the respiratory response in the
pigeon compared with heart rate changes and certain procedural differences in his
experiment, such as the use of some odorous materials in the construction of his
olfactometer, the omission of an exhaust other than diffusion from the bird's
enclosure, and placement of the olfactometer outlet behind the bird's head rather
than in the vicinity of the nostrils.
The balance of all of the evidence in this area to date justifies the conclusion
that perhaps the majority of avian species are capable of perceiving odorous
stimuli in the minimal sense of that concept.
Learned Responses
To complement the data in the preceding section, some convincing evidence is
available for the ability of certain birds to learn to use olfactory stimuli as the basis
for differentiated responses. To review the history of research on this topic (cf.
WALTER, 1943) is interesting but confusing because the reported experiments
represent such a spectrum of methods, species, and scientific adequacy that nothing
but contradictory results could be expected.
Two recent experiments with pigeons have included such stringent controls
that their positive results appear to be conclusive. In one of them (MICHELSEN,
1959), two pigeons were trained by an operant procedure to respond differentially
to an airstream that had been bubbled either through distilled water or through
one of two odorants, viz., sec-butyl acetate or isooctane (more generally called
trimethylpentane). Mean performance during test sessions was maintained at
80-95 % accuracy. This level dropped to 50-60 %, however, when one or the
other of two control conditions was introduced in which both the background and
stimulus air streams were bubbled through either distilled water or an odorant so
that no olfactory differential occurred during "stimulus" presentations. Thus, if
the bird had been responding to possible auditory or pressure differences between
the two lines or to a humidity difference, accuracy would not have fallen close to
Olfactory Perception 443
the chance level when the odor difference was removed. COBB (1960a) reports that
the effect of cutting the olfactory nerves in these two pigeons was to reduce per-
formance even lower than that reached in the control tests.
With an entirely different type of operant procedure known as conditioned sup-
pression, HENTON et al. (1966) trained three pigeons to discriminate between an
airstream that had bubbled through amyl acetate and one that had bubbled
through distilled water. The birds first learned to peck continuously on a response
key for food as reinforcement. At intervals during test sessions, odor was added for
30 sec to the air supplying the chamber. After 30 sec the bird received a brief
electrical shock through electrodes implanted around the pubis bone. The effect of
repeated presentations of odor and shock was to reduce the amount of pecking
during the odor, which served as a warning signal for the approaching shock. The
index of suppression is the difference in response rate between the 30-sec odor period
and the immediately preceding 30 sec expressed as a ratio of the latter value. The
birds reached mean suppression ratios of 0.90 or better in regular sessions. They
failed to suppress during control periods when pure air rather than odorized air was
added to the airstream, as well as after olfactory nerve section. Following nerve
section, they could be retrained only at higher levels of intensity than those at
which they had been performing reliably before surgery. This result is consistent
with the effect shown in Fig. 7 for the heart-rate response in pigeons after olfactory
nerve section and must have depended on trigeminal activation. The pigeon's dif-
ference threshold for amyl acetate measured by the same technique lies between
0.57 and 0.71 (SHUMAKE et al., 1969).
In another fairly recent experiment (HAMRUM, 1953), bobwhite quail
(Colinus virginianus virginianus) were reportedly trained to use the scent of
coumarin as a discriminative stimulus for choosing a feeder. Some of the important
controls are described but others that seem necessary are not mentioned.
Even though a number of investigators have had no success with learning
experiments (e. g., CALVIN et al., 1957 ; WALTER, 1943), the positive results obtained
in well-conducted experiments put the burden of proof on the others. At present it
is reasonable to assume that members of many avian species are capable of forming
associations between olfactory stimuli and specific responses provided that the
training conditions are optimal, as with well-developed operant procedures. For
the pigeon such associations are much more difficult to establish than comparable
ones involving visual stimuli, but this might not be the case with birds that have a
larger proportion of central olfactory tissue. The situation is reminiscent of spectral
sensitivity in cats, which was demonstrated at the neurophysiological level but
could not be shown behaviorally until very specially contrived experiments were
done and unusually large amounts of training given. Olfactory discriminations
hardly appear to be favorite responses of pigeons but the fact that they can be
forced to occur is indicative of a basic perceptual ability.
Feeding Behavior
Finally, a body of literature deals with birds' use of odors in locating and
identifying sources of food under natural conditions. The inclusion of suitable con-
trols in some cases has provided some fairly conclusive evidence. Two outstanding
candidates for this type of behavior on the basis of anatomical evidence and pre-
444 B. 1\1. WENZEL: Olfaction in Birds
vious anecdotal reports are the turkey vulture and the kiwi, and both have been
shown to behave in this way (STAGER, 1964; WENZEL, 1968 in press).
After extensive field work with a number of Old and New World vultures,
STAGER has concluded that the turkey vulture definitely possesses an olfactory
food-locating mechanism and the king vulture (Sarcoramphu8 papa) may also have
this characteristic. In his field work with the turkey vulture, he released the scent
of ethyl mercaptan from concealed sources at the bottom of canyons during the
early morning when air currents lifted the fumes as the birds began their daily
foraging expeditions. The vultures were seen to collect in the vicinity of the odorous
updrafts and to follow them into the canyons although no visual cues were present.
The kiwi was able to identify which of three containers from which it was
accustomed to feed every night contained food on test nights when the other con-
tainers were filled only with dirt. In order to reach the food, the birds were forced
to break through stiff netting that covered each container and made gustatory and
tactile discriminations impossible. They did this with 100 % accuracy; i.e., they
always broke into the baited dish and never broke into a decoy dish, even when a
layer of dirt covered the screening over each dish. In different versions of this
experiment, kiwis regularly showed themselves to be able to utilize olfactory cues
at a rather high level of effectiveness. The experiments were done in a bird reserve
and not under totally natural conditions, so that it cannot be said that kiwis
regularly locate their food by smelling but the evidence strongly suggests that they
are capable of doing so.
STAGER (1967) has also described the behavior of two African indicators or
honey guides (Indicator indicator and I. minor), which are attracted by the smell
of a burning beeswax candle. These birds normally feed on the wax of beehives.
STAGER'S limited initial observations support the conclusion that these birds do
indeed respond to an odorous cue, and he has found that they have a very large
olfactory tubercle. Additional candidates include the Trinidad oilbird, a nocturnal
fruit eater (SNOW, 1961), and the tubenosed birds who feed on odorous marine
forms and who collect rapidly in the vicinity of heated animal fat distributed on
the surface of the sea.
Numerous anecdotes can be quoted about apparent responses of many different
types of birds to odor cues; each one can be matched by both anecdotal material
and controlled observations that show just the opposite. In this type of behavior
above all, species differences are undoubtedly paramount. It now appears reason-
able to expect to find that certain species from a wide range of families have
developed a reliance on the olfactory system for at least a part of their food-getting
activities; however, most species seem not to have done so.
General Considerations
Anatomical, electrophysiological, and behavioral evidence is gradually accumu-
lating in support of the concept of avian olfaction. The compelling question for
many has become, "What use is made of the information 1" The ethology of
olfaction is largely unexplored among all vertebrates. Broadly speaking, there are
three logical outcomes of effective stimulation of the olfactory pathway: infor-
mation is provided for recognition or guidance; the input may have affective value
General Considerations 445
only as is often the case with man; or the process may be so vestigial as to have no
significance at all. Usually, only the first is implicit in discussions of this subject,
but there is no legitimate reason to exclude the second and third.
Consideration of the possible applications of olfactory stimuli in the species-
specific behavior of birds has barely begun. Higher vertebrates carryon a great
variety of activities in the course of a year, and certain avian species may have
developed reliance on odor cues for different aspects of some of them, e.g., mate
selection, breeding, care of young, identification of young, maintenance of territory,
homing, migration, etc. The strong musky smell of tubenosed birds and of their
nesting sites has often been mentioned, and in this connection the nocturnal hom-
ing of the shearwater becomes especially interesting. Many excellent studies have
established the importance of visual stimuli for specific birds in such responses as
mate selection, brooding, and pecking. THORPE (1968) has recently emphasized the
significance of auditory patterns among song birds for recognition of individuals.
These observations do not eliminate a role for olfaction. The concept of chemical
communication or pheromones among birds is completely unstudied. It is not
necessary to go as far as the medieval belief that female partridges were so aroused
by wind blowing toward them from males that they could become pregnant
(WHITE, 1960) to give some credence to the general idea. An interesting form of
behavior called anting has been observed, which is defined as the application of
foreign substances to the plumage and possibly to the skin as well, often with
accompanying excited moments that may reach a level describable as ecstasy
(CHISHOLM, 1959; WHITAKER, 1957). The motivation for such activity is not yet
understood, but odor attraction has been included among the various suggestions
because strong-smelling materials are typically used. The fact that the great
majority of species that practice anting are passerines or near relatives with very
small amounts of olfactory tissue argues against this explanation. It might be an
example of an affective role for odors, as may another observation that bobwhite
quail in captivity developed pronounced preferences for one of a pair of feeders
under circumstances that led to the conclusion that they were selecting on the basis
of odors (FRINGS and BOYD, 1952). Throughout the literature on this topic, there
runs the theme of a progressive decline in the use and development of the olfactory
system as new avian species have evolved. In recent forms there appears to be an
increasing reliance on visual and auditory cues with a resulting diminution in the
olfactory system as other parts of the brain enlarge. This trend should be kept in
mind when considering the possible role of olfaction.
Finally, the olfactory system has been credited with a nonspecific function that
helps to maintain normal adaptive behavior (WENZEL and SALZMAN, 1968; WENZEL
et al., 1969). When the primary olfactory pathway of pigeons is interrupted by
transecting the nerves or destroying the bulbs, the birds are slower to adjust to a
new situation and show other anomalies of orienting and adapting to nonolfactory
stimuli. Observations of changes in affective behavior of rodents (VERGNES and
KARLI, 1963; UEKI and SUGANO, 1965) and fish (ATEMA et al., 1969) following
olfactory bulb ablation may be related to the results obtained with pigeons. In
addition to their possible cue function, therefore, odorous stimuli may also be serv-
ing to maintain some necessary level or pattern of activity in the olfactory bulbs
and associated structures. A suggested hypothesis proposes that the olfactory
system in the course of evolution has acquired some general significance, quite apart
from a specific sensory function, that preserves its value for animals who may place
little reliance on it for smelling (of. HERRICK, 1933). Its close anatomical relations
with the limbic system originally named the rhinencephalon is provocative in this
connection. A full understanding of the phenomenon awaits more extensive
investigation.
Summary
Evidence has been presented for the existence of an olfactory epithelium,
olfactory nerve, olfactory bulb, and secondary olfactory areas in a great variety
of avian species, representing the entire range from the oldest orders to the newest.
In general the older forms have larger olfactory systems than are found in those
that have developed most recently, but some olfactory tissue is present in all.
Studies of the electrical activity of both peripheral and central portions of the
system in several species have shown it to be similar to that recorded from reptilian
and mammalian forms. Therefore, the class Aves appears to be continuous with
Reptilia and Mammalia in terms of the anatomical and electrophysiological
characteristics of olfaction.
Behaviorally, it can be said that olfactory perception as indicated by in-
voluntary response of organs such as the heart occurs in many birds. Those with
smaller olfactory systems may be exceptions. Pigeons (Oolumba livia) have been
successfully trained in rigorous recent experiments to perform different responses
to different odors, thus establishing their ability both to perceive and to discriminate
odors. Section of the olfactory nerves abolishes this performance as well as the
perceptual indicators mentioned above. Olfaction has convincingly been implicated
in food location by recent studies of two species, the kiwi (Apteryx australis) and
the turkey vulture (Oathartes aura). Careful investigation of possible additional
applications of olfactory information to behavioral regulation in birds has yet to be
done but should be considered more promising than formerly, in light of all of the
positive evidence already accumulated on other aspects of their olfactory system.
Some support has also been provided for the hypothesis that the olfactory
system serves a general function quite apart from its transmission of olfactory in-
formation, which may involve primarily participation in the activity of the limbic
system. If this proposed role for the olfactory system proves to be a valid one, it
could help to account for the preservation of the system in species that may have
little other use for it.
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WHITE, T. H.: The Bestiary, A Book of Beasts. New York: Capricorn Books, Putnam 1960.
Chapter 16
The Use of Ionizing Rays as a Mammalian

Olfactory Stimulus
By
JOHN GARCIA, Stony Brook, New York (USA)
and ROBERT A. KOELLING, Los Angeles, California (USA)
With 7 Figures
Contents
A. Historical Perspective . . . . . 449
B. General Consideration of the Physical Stimulus . 450
C. Early Studies of Arousal to the X-Ray Stimulus . 452
D. Detection of X-Ray Signals by Rats, Monkeys, and Pigeons . 454
E. Response of Olfactory Bulb Neurons to Ionizing Rays 456
F. Chemical Mediation of X-Ray Stimulus 459
G. Radiation-Induced Aversions 460
H. Summary and Conclusions. 462
References . . . . . . . . . . 462
A. Historical Perspective
Originally labelled "X" for unknown, ionizing rays were often described as an
energy form without stimulus properties for living organisms, presumably because
of their position on the electromagnetic spectrum far beyond the violet end of that
narrow segment visible to the human eye. However, since the announcement of
ROENTGEN'S discovery of X-rays in 1895, and discovery of radium by the CURIES
several years later, a wide variety of biological observations have revealed that
this energy form has a host of stimulating properties. The visibility of the rays
was soon discovered and then rediscovered repeatedly in the fact of widespread
belief in the invisibility of the mysterious "X" -rays. The extreme radiosensitivity
of the human eye is illustrated by a recent report describing perception of single
cosmic meson particles by dark adapted subjects (D' ARCY and PORTER, 1962).
Early investigators, including WILHELM ROENTGEN, soon learned that responses
to X-rays could be elicited in a wide variety of species under certain conditions.
LIPETZ (1955, 1960) in an historical review of the X-ray and radium phosphenes
cites early observations of positively phototropic insects and crustaceans approach-
ing the source of ionizing rays. Others reported that water fleas swim away from
29 Hb. Sensory Physiology. Vol. IV,!
450 J. GARCIA et al.: The Use of Ionizing Rays as a Mammalian OUactory Stimulus
the source and snails retracted their horns (BAYLOR and SMITH, 1958). LEVY (1967)
has recently tabulated studies reporting neural activation and behavioral arousal
in a broad phylogenetic array including planaria, insects, crabs, frogs, reptiles and
mammals. Most of these effects were apparently mediated by the visual system,
but decades ago the chemoreceptors were believed to be equally sensitive to
ionization (HUG, 1958). Verbal responses from human subjects substantiated these
notions. Patients sensed a luminous greenish flow when stimulated with less than
one roentgen under conditions of visual dark adaptation (NEWELL and BORLEY,
1941; GODFREY and SHENK, 1945). Others reported smarting sensations of the skin
and discriminative odor upon exposure to scatter from high intensity cathode rays
(ROBBINS et al., 1946).
Paradoxically, ionizing radiations have proven to be such an ubiquitous
stimulus in the laboratory that the precise nature of the physiological mechanisms
of stimulation have remained obscure to this day. Unlike mechanical, thermal,
acoustical, and luminous stimuli traditionally employed in the psychophysiological
laboratory, ionizing radiations have the capacity to penetrate the living organism
and deliver energy throughout all the tissues and fluids of the body with temporal
and spatial uniformity. However, the neural and behavioral responses are quite
specific, depending upon the site and sequalae of energy absorption, and upon the
experimental test imposed upon the organism. Thus ionizing rays fit the classical
stimulus doctrine of specific energies of nerves promulgated by JOHANNES MULLER
and HERMANN VON HELMHOLTZ.
The diverse effects of ionizing radiation upon neurophysiological function and
behavior have been the subject of several volumes recently (VAN CLEAVE, 1963;
HALEY and SNIDER, 1964; KmELDORF and HUNT, 1965). It is our purpose to
discuss specifically the use of radiation as an immediate stimulus for laboratory
mammals and as a technique for the study of olfactory function. First, let us
briefly examine the physical properties of the stimulus.
B. General Consideration of the Physical Stimulus

Sources of ionizing radiation are traditionally divided into two classes:
I} electromagnetic or photic radiations which include some ultraviolet rays, X-rays
and y-rays, and 2) corpuscular emission of subatomic particles such as delivered by
cyclotrons. Perhaps more important for our consideration are two other factors,
namely the penetrating power of the photon, or particle, and the degree of ioni-
zation which it produces along its track. Penetration and ionization determine the
scope of the physicochemical changes in tissue for both forms of energy.
X-rays from low voltage sources of less than 50 kv have low penetration and
are referred to as solt X-rays. Such X-rays expend most of their photic energy in
matter by photoelectrical absorption; a process in which an electron is removed
from an atom, disrupting its electrostatic equilibrium and leaving that atom in a
positive ionized state. The ejected electron, or photoelectron, is left in a negative
ionized state. X-rays from sources of over 200 kv employing thin filters of copper
and aluminum to block low voltage rays are referred to as hard and penetrate
large animals effectively. Scattering absorption becomes important with hard X-rays.
A photon may give up only part of its energy by scattering, a process in which the
General Consideration of the Physical Stimulus 451
photon mayor may not cause an atom to eject an electron, but travels on reduced
energy in a different direction. This photon may produce further ionization or
scatter. Scattered X-rays are extremely important in radiobiological experimental
arrangements because they may increase considerably the total energy absorbed
by animals placed in the beam.
Two other effects deserve mention as possible stimuli. When an ejected electron
is replaced by another electron from within the atom or from outside the atom,
fluorescent radiation is released which is characteristic of that particular atom and
that particular electron. Photons of extremely high energy, greater than 1000 kv,
produce effects described as pair formation forming electron and positron pairs.
X-rays of such high energy sources are not routinely available for biological
research, but the more common radioisotope cobalt 60 sources release y-rays of
1.17 and 1.33 mev energy. The radioisotope cesium-137 yields y-rays of 0.662 mev
y-rays, like X-rays can be considered electromagnetic waves with shorter wave
lengths than ordinary X-rays, released during radioactive disintegration.
Corpuscular emissions also vary in their capacity to produce ionization and to
penetrate matter. IX-particles are the nuclei of helium atoms and produce high
ionization. They have such low penetration that they are absorbed by a fraction of
a millimeter of soft tissue. Thus they are useful for limiting radiation to surfaces
and membranes . .8-particles, fast moving negative electrons, are more flexible.
They produce much less ionization but vary in their penetration depending upon
their energy. Neutrons and protons, basic constituents of atomic nuclei with great
mass compared to electrons, can be ejected with considerable energy from atomic
reactors or cyclotrons to interact with matter to make it radioactive in turn.
Proton beams have tremendous utility since they can be readily "focused" into a
narrow beam and thus utilized for precise stimulation or surgery. Deuterons,
nuclei of deuterium, consisting of a proton and a neutron are also available from
accelerators. Finally mesons, heavy particles of cosmic origin continuously
bombarding the earth, add to "background" or ambient radiation to which all
organisms are continuously exposed. Charged heavy particles have a unique and
useful property for biological research, referred to as the Bragg Peak Ionization.
They tend to have high homogeneous energy and their tracks in tissue end abruptly
and uniformly, delivering most of their energy deep in the tissue rather than at the
surface.
Gamma- and X-rays are measured in roentgens (r) based upon the electrostatic
charge due to ionization in one cubic centimeter of dry air at zero degrees centigrade
and 760 mm of mercury pressure. This represents the formation of 2.1 x 109 ion
pairs or the absorption of 87.6 ergs of energy per gram of air. The roentgen is by
definition not applicable to particulate radiation. The appropriate unit for parti-
culate radiation and for mixed sources is the radiological absorbed dose (rad) which
represents 100 ergs of energy absorbed regardless of the source or the absorbing
matter. Since one rad is the approximate energy absorbed when soft tissue is
exposed to one r of hard X-ray, the two units are approximately equivalent for the
majority of radiation studies discussed here.
Photons and particles interact not only with the tissues of the experimental
animal but the air it breathes, producing ozone and similar odorants. The energy is
scattered and degraded by dense metals of the apparatus and may cause fluorescence
29*
452 J. GARCIA et al.: The Use of Ionizing Rays as a Mammalian OHactory Stimulus
in many materials surrounding the animal. Moreover, noisy machinery is often

associated with generation of ionizing radiation or with moving shields and opening
shutters associated with isotope emitters. Any of these events can alert the animal
through ordinary sensory processes and thus obscure the stimulating action of the
radiant energy itself unless such artifacts are carefully controlled.
In biological tissue absorption of ionizing rays has direct and indirect effects.
Direct effects result frOIp. the hit or the initial transfer of energy to a target molecule
and are usually described as an exponential function of radiation dose. The target
molecule may disassociate directly affecting cellular metabolism, reproduction, and
viability. In areated fluids peroxides are formed, organic substances show exidation,
gaseous hydrogen and oxygen are released. These initial events lead to complex
indirect effects depending upon the organs and tissues involved which may be more
important than the direct effects. Let us now decsribe the initial stimulating effects
of ionizing rays.
c. Early Studies of Arousal to the X-Ray Stimulus

The general stimulating properties of ionizing rays are extremely simple to
demonstrate. It is only necessary to habituate an animal to the radiation facility
until it will rest tranquilly there, then rays of sufficient strength will arouse the
animal from this drowsy state immediately. Machinery and generators associated
with radiation sources produce acoustical and electrical noise creating practical
problems. In the initial studies the animals were placed in acoustically and electri-
cally shielded boxes. Radiation machinery was usually maintained in constant
operation during experimental sessions to eliminate noises coincident with the
onset of exposure. Shields or shutters were devised to expose the animal silently.
As a final precaution, sham exposures were instituted. In this control procedure,
all the machinery required to expose the animal is operated without the radiation
actually impinging on the animal. This can be accomplished by the use of auxiliary
shielding, or by interrupting the power of the X-ray tube.
Under conditions such as these, HUNT and KIMELDORF (1962, 1964) visually
observed behavioral arousal through the leaded glass window of the X-ray room
at the onset of an exposure to hard X-rays at 1.9 r per second. In addition they
recorded an increased heart rate oscillographically through electrodes clipped to
the rat's skin. Bilaterally ophthalmectomized rats, as well as intact animals,
responded within 12 seconds, indicating that the retina was not the essential site
of action.
TSYPIN and GRIGOR'YEV (1960) recorded electroencephalographic changes in
rabbits to five-minute exposures to y-rays ranging from 0.013 to 7.5 r per second.
A change from high voltage slow waves to low voltage fast waves was evident at
the lowest levels of radiation. A number of investigators in other laboratories using
chronically indwelling electrodes in the skull of laboratory animals obtained
similar results (GRIGOR'YEV and TSYPIN, 1958; GARCIA et al., 1963; LIVANOV,
1962; COOPER and KIMELDORF, 1964). Fig. 1 illustrates the prompt arousal
response of a laboratory rat sleeping in a shielded chamber to the onset of a brief
exposure to hard (250 kv filtered) X-rays delivered at the rate of 2.5 r per second
(HULL et al., 1965). Electroencephalographic changes and motor movements
Early Studies of Arousal to the X-Ray Stimulus 453
indicate arousal in approximately one second_ This animal did not arouse to a
sham exposure_
When animals are allowed to return to sleep after each arousal and repeatedly
awakened with brief exposures, they will soon cease to respond behaviorally to
radiation. Fig. 2 illustrates the number of rats responding to repeated 10-second
exposures of 250 kv filtered X-rays at a dose rate of 2.0 r per second (GARCIA et al.,
MOTOR ACTIVITY
t X-RAY t ~
2 sec
Fig. 1. Electroencephalographic and motor (arousal) responses to the onset of X-ray exposure
in a sleepy rat. (Courtesy of HULL et al., Nature, 1965)
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Fig. 2. Habituation of the electroencephalographic (arousal) responses to repeated lO-second

exposures of X-rays. (Courtesy of GARCIA et al., Science, 1963)
1963). Mter the fourth exposure all animals gave up responding. When they were
returned to the X-ray room four days later over half of the animals awoke for the
first exposure, but all animals remained asleep for the next three exposures. This
pattern is characteristic of responses to repeated mild stimulation via the peri-
pheral head receptors. Animals will arouse and orient to novel odors, sights, and
sounds, but will soon habituate if these stimuli do not signal events of consequence
to the animal.
In contrast to the hypothesis of peripheral receptor action of the radiation
stimulus, several alternative central mechanisms were postulated based upon the
454 J. GARCIA et al.: The Use of Ionizing Rays as a Mammalian Olfactory Stimulus
pervasive penetrating nature of this unique stimulus. An early hypothesis sug-

gested that energy delivered directly to the peripheral and central ganglia might
account for cardiovascular changes observed in irradiated animals. Increased
electrical activity in sympathetic and intestinal nerves recorded in rabbits exposed
to y-rays supported this notion (TOYAMA, 1933). General changes in cellular mem-
brane permeability and the release of biogenic amines seemed to be indicated as
well (BRINKMAN, 1962; BACQ and ALEXANDER, 1961). Similarly, the ionizing rays
could be acting on synaptic and ganglionic sites throughout the central nervous
system causing minute alterations, which could in turn be amplified by the
reticular activating system to produce arousal similar to the spontaneous awaken-
ing at the end of sleep (HUNT and KrnELDORF, 1964). Indubitably, widespread
events such as these occur in the irradiated animals and may be involved in the
radiation-induced aversions discussed below, but subsequent research has indicated
that immediate arousal was mediated by peripheral receptor action.
D. Detection of X-Ray Signals by Rats, Monkeys, and Pigeons

Rapid habituation of animals' responses to X-ray as a novel arousing stimulus
makes the procedure unsuitable for repeated testing to determine thresholds, and
for repeated probing to locate the "radiation receptor." Therefore, conditioning
procedures yielding more stable response measures were devised in later studies of
the X-ray stimulus (GARCIA et al., 1962). Brief exposures were used as signals to
warn animals of a subsequent electrocutaneous shock to the paws. After such
training the X-ray becomes associated with shock and elicits defensive reactions in
anticipation of shock. The X-ray signal can then be used to interrupt an ongoing
behavior such as lever pressing or licking at a water spout.
Fig. 3 illustrates the type of record obtained with a rat responding to a 0.10 sec-
ond exposure of hard X-rays at 0.20 r per second (GARCIA et al., 1964a). These
brief exposures were obtained with the aid of a slotted lead disc revolving between
the X-ray tube and the animal's compartment. Duration of the exposure was con-
trolled by adjusting the width of a slot and the speed of the revolving disc. In the
first two trials the animal scarcely hesitates to the brief exposure until the foot
shock, following a second or two later, disrupts his licking at the water spout. In
the last two trials, the X-ray signals disrupt licking in the absence of shock,
indicating that conditioning or learning has occurred. Similar records indicated
that rats appear to respond within 0.2 seconds to the onset and end of exposure.
Moreover, rats could detect exposures of less than 0.02 seconds duration totaling
less than 10 milliroentgens.
These conditioning procedures were employed in a search for the "radiation
receptor" (GARCIA et al., 1964 b). In order to reach a water spout, thirsty rats were
required to enter a narrow compartment and thrust their heads through a hole into
a confined space which fixed the position of the head relative to a hard X-ray beam.
Preliminary experiments indicated that shielding the head of the animal would
block detection of exposure even though the remainder of the rat's body was
exposed to large doses of radiation. In subsequent experiments, a narrow (0.45 cm)
beam was aimed at various points in the animal's head. Fig. 4 illustrates the results.
Tests with vertical and horizontal beams indicated that the area of highest sensi-
Detection of X-Ray Signals by Rats, Monkeys, and Pigeons 455
-------------~.~______=__----....X-RAY (O.2rlsec/o.l sec

-----------------------------------_~r-----SHOCK
~,..",...,"""'">rV"""',.."...,.rvv_v~J"'V"'.,....,."""''''''''~v_v_v_v----UCKING RESPONSE
Trial I
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Fig. 3. Conditioned suppression of licking by thirsty rats to an X-ray signal followed by foot
shock. (Courtesy of GARCIA et al., Bol. Inst. Estud. Med. BioI. Mex., 1964)
-----Whole Head-- --------
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Fig. 4. Relative sensitivity of the head to X-ray signals centering on the oHactory area.
(Courtesy of GARCIA et al., Science, 1964)
tivity was in the vicinity of the olfactory bulbs and the cribriform plate. Irradiation
of the nasal air passages anterior and posterior to the olfactory area produced no
more detection than one would expect due to scattered radiation reaching the
olfactory receptors.
HULL and his associates (1965) demonstrated that surgical ablation of the
olfactory bulbs abolished the arousal response to X-ray. Ablation ofan equivalent
amount of tissue from the anterior portion of the cerebral hemispheres did not put
456 J. GARCIAetaI.: The Use of Ionizing Rays as a Mammalian OHactory Stimulus
an end to the response. Furthermore, rats with chronic tracheotomies which pre-
vented nasal inhalation aroused promptly to X-ray indicating that sniffing
irradiated air was not required. These results were supported and extended by
others demonstrating that removal of the olfactory bulbs abolished conditioned
responses to X-ray signals as well (COOPER and KrMELDORF, 1965; BRUST-CAR-
MONA et aZ., 1966; DINC and SMITH, 1966).
TAYLOR, SMITH, and HATFIELD (1967); TAYLOR et aZ., (1968) employed a similar
conditioning technique to test X.ray detection in rhesus monkeys. A 15-second
exposure to hard X-rays terminated in a shock to the foot of the monkey who was
pressing a lever for food pellets. Monkeys exhibited clear detection in about 25 X -ray
shock training trials. No loss of detection capacity was observed when the dose
rate was decreased from 0.63 r per second to 0.03 r per second. Some monkeys
detected dose rates as low as 0.008 r per second. These data closely agree with data
obtained by MORRIS (1966) who tested rats and found no essential loss in detection
when dose rate was reduced from 0.05 r per second to 0.04 r per second. Monkeys
detected hard (300 kv filtered) and softer (80 kv) X-rays as well. Complete bilateral
sectioning of the olfactory tracts did away with the detection response; however, if
as little as 20 % of the fibers of a single tract remained intact, animals maintained
their capacity to detect the ionizing rays.
SMITH and TUCKER (1969) trained the pigeon to detect X-rays, choosing this
subject because of the difference in the olfactory anatomy of the bird. Unlike the
rat, a space of several millimeters separates the olfactory receptors from the
olfactory bulb on birds, making surgical section of the primary nerves relatively
simple. Pigeons required much more training and higher doses to detect X-rays
than mammals. Only half of the pigeons acquired the capacity to detect after one
hundred X-ray shock-training trials. Virtually all rats learn to detect with less than
half that amount of training. Those birds that learned to respond lost the ability
after the olfactory nerves were sectioned. Sham operations where nerves were
exposed but not severed did not impair detection. These data indicate that an
intact olfactory nerve is required for X-ray detection in the pigeon, although this
bird is able to discriminate odors, presumably via the trigeminal system, after the
olfactory nerves have been sectioned (HENTON et al., 1970).
To summarize, the behavioral studies employing X-ray as a signal demonstrated
conclusively that ionizing radiations are a potent olfactory stimulus for both
macrosmatic and microsmatic mammals, and for birds as well. In addition, the
conditioned responses indicated that the ionizing rays had to be directed at the
olfactory receptors to be effective as a signal.
E. Response of Olfactory Bulb Neurons to Ionizing Rays

A series of investigations of the responses of single neurons in the olfactory bulb
to ionizing rays amplified and refined the conclusions reached on the basis of the
arousal and detection studies. COOPER and KrMELDORF (1966a) used extra-cellular
microelectrodes to study these units in anesthetized rats. Stainless steel electrodes
with tips approximately 0.5 to 2.0 microns in diameter were advanced through the
bulb until a unit was obtained whose spike amplitude remained stable for at least
five minutes. Then the whole animal was exposed for 1 to 5 seconds duration to
Response of Olfactory Bulb Neurons to Ionizing Rays 457
hard X-rays ranging from 1.5 to 2.0 r per second. Approximately 15 percent of the
units examined responded, usually with an increased frequency, to the very onset
of the dose. The responses observed resembled those illustrated in Fig. 5. Responsive
cells were found in the mitral, granular, plexiform and glomerular layers of the
olfactory bulbs. No responses were observed when the area posterior to the bulb
was irradiated, or the anterior (5 to 8 mm) muzzle of the animal was irradiated.
~d, ,! ,1'!!.~'\!IW~~~~~'!i
1 sec
Fig. 5. Response of an olfactory bulb neuron to beta irradiation of the olfactory epithelium in
the rabbit. (Courtesy of COOPER, Am. J. Physiol., 1968)
Responses were abolished by perfusion of the nasal cavities of tracheotomized rats

with ethyl alcohol. Similar microelectrode recordings were made in the olfactory
bulbs of cats, rabbits, and dogs (COOPER and KrMELDORF, 1966b). In all three
species brief exposures produced increases in the firing rates of some cells, but in a
few cells decreases were noted.
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Fig. 6. Response rate of olfactory bulb neurons as a function of X-ray dose rate in the rat.
Each point is a raw datum obtained from one of six units. (Courtesy of COOPER, Am. J.
Physiol., 1968)
COOPER (1968) studied the responses of olfactory bulb neurons in anesthetized

rats as a function of intensity (dose rate) and duration of the ionizing rays. He used
a fast shutter mechanism operated by a solenoid which permitted exposures of
millisecond duration. Fig. 6 shows the relationship between the firing rate of
olfactory bulb neurons and X-ray intensity for an exposure of two seconds. The
458 J. GARCIA et aI.: The Use of Ionizing Rays as a Mammalian OUactory Stimulus
firing rate is linearly related to the logarithm of the dose rate, a relationship
classically referred to as the Weber-Fechner function in sensory psycho-
physiology. These data agree remarkably well with the behavioral data of
BUCHWALD and associates (BUCHWALD et al., 1964; GARCIA et al., 1964c) which
indicated that the probability of arousal to X-ray and detection of an X-ray signal
were also functions of the logarithm ofthe dose rate. Fig. 7 indicates the relationship
between duration and intensity of the X-ray stimulus. The product of stimulus
intensity and duration is approximately a constant, a relationship classically
known as the Bunsen-Roscoe sensory function.
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Fig. 7. Minimum duration of X-ray exposure to produce threshold responses as a function of

dose rate in the rat. Each point is a raw datum obtained from one of two units. (Courtesy of
COOPER, Am. J. Physiol., 1968)
The effects of various gases in the nasal passages on the olfactory neuron's
sensitivity have been studied in an attempt to elucidate receptor function. Tests
were made in anesthetized rats breathing through a tracheal tube which allowed
air, oxygen, argon and nitrogen to be freely perfused through the nasal cavities
(COOPER et al., 1966). Argon and nitrogen perfusion depressed or abolished the
response to radiation within five minutes in some units. Some neurons responded
during perfusion of all four gases indicating that the activity was not wholly
dependent upon any particular gas in the nasal cavity. However, in a later study
the responses of bulb neurons to X-ray were examined under various concen-
trations of oxygen (COOPER, 1969). Responses were stable until oxygen concen-
tration was reduced to 2% then fell off precipitously, indicating that oxygen
in the nasal passages potentiated the response.
In summary, the research on the olfactory bulb neurons substantiated the
notion that ionizing radiation was stimulating animals via the olfactory system.
Furthermore, this evidence revealed that only a smaH segment of the population
of bulb neurons responded to the rays, indicating a rather specific effect within the
olfactory system. An olfactory receptor action requiring oxygen was implicated,
suggesting chemical mediation of the stimulus.
Chemical Mediation of X-Ray Stimulus 459
F. Chemical Mediation of X Ray Stimulus

The hypothesis that ionizing rays were producing a direct effect upon the
olfactory receptors was challenged by studies which indicated that X-ray detection
was indirectly mediated by an airborne odorant produced by the action of radiation
on the air inhaled by the animaL GASTEIGER and HELLING (1966) used
the technique of masking to test the hypothesis that ozone produced in air could
act as the odorant signaling the animals of the presence of radiation through
ordinary receptor processes. Rats were conditioned by a 10-second exposure to hard
X-rays at 0.20 r per second, followed immediately by a shock to the paws. Licking
at the water spout was the response measured. An ozonizer, estimated to produce
from 5 - 8 mg/hr, supplied the masking ozone. At this concentration ozone smells
very pungent and is aversive to humans; X-ray machines do not produce such
harsh and repulsive odors at levels of operation used in studies of arousal and
detection. Nevertheless, the data clearly indicated that ozone disrupted the rat's
conditioned response to X-rays. Other strong odors such as wintergreen did not do
so. Furthermore, these high levels of ozone apparently did not disrupt the rat's
ability to smell out bits of apple, indicating that the ozone was not producing a
general impairment of the rat's ability to smell.
Other studies (HELLING and GASTEIGER, 1967) tested the similarity of just
detectable levels of ozone to X-ray by the use of generalization or transfer tests.
Rats were conditioned to stop licking at a water spout whenever an odorant was
introduced into the airstream which they inhaled. The gaseous odorant was either
introduced into the nasal passages through cannulae chronically implanted in the
frontal sinuses of the animals, or injected into the air near the water spout. Shock
punishment was administered for licking during olfactory stimulation. Conditioned
responses to ozone at minimal concentration transferred to 10 seconds of X-ray.
Conditioned responses to pineapple odor (ethyl-butyrate) did not. The rats were
acting as if ozone and X-ray smelled alike.
Let us now consider all the evidence bearing on the hypothesis that the animals
are sniffing an odorant produced in air by radiation. First, rats in conditioning
experiments appear to be able to suppress licking within 200 milliseconds of the
onset of exposure, and resume licking within one second after the end of the
exposure. In rooms where X-ray machines are in constant operation and no
attempt is made to control the atmosphere, an exposure as brief as 17 milliseconds
has served as a signal to a behaving animal and briefer exposures have stimulated
single neurons (GARCIA et al., 1964a; COOPER, 1968).
Second, rats are able to discriminate a double-pulse from a single-pulse of
X-rays (GARCIA et al., 1969). Thirsty animals were trained to inhibit licking at a
water spout during a 1.0 second pulse of hard X-rays at 0.4 r per second. Shock to
the paws was administered for failure to do so. Then a double-pulse (two 0.25 second
pulses separated by 0.50 seconds) was randomly distributed among the single-
pulses but never followed by shock. In the beginning the rats inhibited licking to
the double-pulse, as well as the single-pulse. With continued practice they learned
the discrimination, inhibiting licking only to the single-pulse and drinking con-
tinuously during the double-pulse. Examination of the individual records indicated
that the animals were actually discriminating the stimulus duration of the first
460 J. GARCIA et al.: The Use of Ionizing Rays as a Mammalian OUactory Stimulus
pulse. This seems to be a discrimination too fine to depend upon odorants wafted
to the nostrils in an uncontrolled atmosphere.
Third, COOPER (1968) in a preliminary attempt succeeded in driving an olfactory
bulb neuron of a rat with flickering X-ray. The unit showed discrete responses to
stimuli up to a frequency of 7 pulses (0.050 seconds) per second before fusion
occurred. He also surgically exposed the olfactory bulb and epithelium of an-
esthetized rabbits, and stimulated olfactory bulb neurons with p-rays from stron-
tium-yttrium medical application. These soft rays can be blocked by three milli-
meters of paper. Neurons responded if, and only if, the p-rays were focused directly
upon the olfactory epithelium (see Fig. 5). This evidence seems to indicate that
ionizing radiation is acting directly upon the olfactory receptor or its immediate
vicinity.
Fourth, all attempts to stimulate animals with irradiated air have thus far
proved negative. Radiation of the nasal air passages is not effective as a conditioned
signal for the behaving animal (GARCIA et al., 1964 b). Room air irradiated at 2.0 r
per second and drawn into the nasal passages with a pump will not stimulate bulb
neurons sensitive to X-rays when the olfactory areas are shielded (COOPER, 1969).
Furthermore, X-rays stimulate both intact animals (HULL et al., 1965) and neurons
(COOPER et al., 1966) when animals are breathing through tracheal tubes, hence
sniffing by the animal is not a requirement. Moreover, irradiation of the posterior
part of the head and the thorax and all the air therein with high intensity X-rays
(0.4 r per second) does not stimulate the behaving animal although it is pre-
sumably exhaling irradiated air (GARCIA et al., 1964b).
However, GASTEIGER1 points out that ozone and other ozone-like gases are
highly reactive, and that water is an effective scavenger of such products. Therefore
irradiation of the nasal air passages and lungs may not be effective, because any
ozone-like gas must pass unchanged through a water vapor barrier in order to
stimulate the olfactory mucosa. His preliminary tests support this contention.
Detection of low intensity hard X-rays (less than 0.017 r per second) by conditioned
rats is more effective in low humidity (less than 30%) than in high humidity
(more than 80%). Detection of higher intensity X-rays is unaffected, pre-
sumably because they generate more ozone-like gases. This evidence supports the
hypothesis that an ozone-like product may be involved in X-ray detection;
however, it appears to be so unstable in a humid nasal passage that only those
chemical events generated within millimeters of the olfactory surfaces serve as
effective stimuli. In the final analysis this line of evidence and argument agrees
with the previous contention that the initial stimulating event occurs on or near
the olfactory mucosa.
G. Radiation-Induced Aversions
Ionizing radiation produces aversive reactions in animals to foods and liquids
consumed in conjunction with exposure. Discussion of this phenomena may appear
digressive since these food aversions are not dependent upon irradiation of the
olfactory system; however, the research on aversions preceded and is confounded
1 The personal communication by EDGAR L. GASTEIGER discussing this difficult problem is
gratefully acknowledged.
Radiation-Induced Aversions 461
with research on olfactory effects in the early literature (HUNT and KIMELDORF,
1962; BUCHWALD et al., 1964; GARCIA et al., 1955, 1960, 1964c). Furthermore, the
aversive reactions of the animals are directed primarily at olfactory and gustatory
stimuli and therefore provide another behavioral tool to study the chemical senses.
An exposure to sublethal doses of radiation is usually followed by a symptom-
free period lasting perhaps several hours, before radiation illness is manifested.
Humans complain of nausea, monkeys vomit, and rats become flaccid and listless
(KIMELDORF and HUNT, 1965; HUNTER et al., 1957; COURT-BROWN and MAHLER,
1954). If the animal is presented with a distinctive flavor or odor before the onset
of radiation illness, it will later display a reduction in preference for that flavor or
odor which is evident for weeks after a single exposure totaling 10 to 50 r (GARCIA
et al., 1955, 1960). These aversive effects can be distinguished from the olfactory
effects on functional and anatomical grounds. First, the aversive effect results
from the cumulative late effects of exposure while the olfactory response coincides
with exposure. Second, the aversive response, unlike the olfactory response, is
independent of dose rate and is a function of total dose absorbed. A dose of 50 r
will produce a similar aversion whether it is accumulated in minutes or hours.
Third, exposure of the abdomen with the head shielded is much more effective than
exposure of the head with the body shielded in producing aversive reactions
(BUCHWALD et al., 1964; GARCIA et al., 1964c; GARCIA and KIMELDORF, 1960).
Radiation-induced aversions can be mimicked by allowing the animal to con-
sume a distinctive food and then injecting it with an emetic drug, a toxin, or
probably any agent which produces a viscerally referred malaise (GARCIA and
KOELLING, 1967). Aversions have been produced by transfusions of whole blood
and by injections of serum from irradiated donors (HUNT et al., 1965; GARCIA et al.,
1967) ; therefore, it is likely that these responses are estabUshed by some circulating
by-product of energy absorption. The initial energetic events may be similar in
both olfactory and aversive effects but the behavioral consequences are different
because physiological substrates and afferent systems involved are different.
Strong aversions to the smell and taste of food develop even when illness is
delayed for hours after consumption (GARCIA et al., 1966; REVUSKY, 1968; SMITH
and ROLL, 1967). Avoidance reactions do not develop for visual, auditory, or
tactual stimuli associated with the food (GARCIA and KOELLING, 1966; GARCIA
et al., 1968). The rapid and specific association of gustatory stimuli with internal
visceral feedback is mediated anatomically by the convergence of their respective
afferents to the nucleus of fasciculus solitarius, and reflects evolutionary speciali-
zation of feeding control mechanisms (GARCIA and ERVIN, 1968).
This system provides an excellent preparation for neural and behavioral
analysis of the chemical senses. For example, NACHMAN (1963) studied the neural
coding of taste preferences. He gave rats a toxic solution of lithium chloride and
tested generalization of the aversion to other salts of equal molarity. The degree of
generalization to the other salts reflected the degree of similarity of their afferent
volleys recorded from the chorda tympani. TAPPER and HALPERN (1968) used
ionizing rays to produce an aversion to a sweet solution and tested its generali-
zation to other sweet solutions. BRAUN (1969) has initiated a program to test the
stereochemical hypothesis of olfaction. He induces an aversion for one odor by
following it with an injection of a noxious drug. He then tests odorants of similar
462 J. GARcuetal.: The Use of Ionizing Rays as a Mammalian OHactory Stimulus
molecular structure by presenting them into the air sniffed by the animal while it
is drinking. Aversive odors, like aversive flavors, inhibit drinking. The stable and
long lasting aversions induced by radiation and toxins made it the method of
choice in these studies.
H. Summary and Conclusions

Ionizing radiations provide an effective tool for stimulating the olfactory
system. With penetrating rays it is possible to deliver a discrete olfactory pulse of
a few milliseconds duration to a freely moving animal. It appears feasible to
present olfactory pulses at approximately five per second and to use these pulses as
time-locked stimuli to explore the projections of the olfactory system in the same
manner as the visual system has been studied. With the nasal cavities opened in an
anesthetized animal it would be relatively simple to use a small beta source with
its soft rays and a slotted spinning disc to produce accurate trains of olfactory
stimuli. The initial stimulating event appears to be a "hit" of a photon (or particle)
in the thin atmospheric layer near the o1factory mucosa or in the mucus sheath,
but hits on membranes or other cellular components of receptor units cannot be
discounted. The product of the hit appears to have an extremely short life. It may
be a member of the "ozone family" or a gaseous oxide, but aqueous radicals and
peroxides cannot be discounted, nor can photochemical effects such as fluorescence.
Perhaps anyone of a number of such events is sufficient to stimulate the olfactory
receptor. The final answer awaits a more complete understanding of olfactory
receptor mechanisms.
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Inc. 1963.
Chapter 17
OHaction and Nutrition

By
J. LEMAGNEN, Paris (France)
With 11 Figures
Contents
A. The ChemoSensory Analysis of Foods 466
B. Food Flavors and Food Intake in Man 467
C. Innate Responses in Animal Species . 468
D. Food Odors in Palatability Responses and their Adjustment . 469
E. Learning of the Determining Effect of Associated Odors on Food Selection: its Role
in Regulatory Mechanisms . . . . . . . . . . . . . . 471
F. Neural Mechanisms of the Olfactory Control of Feeding. 475
References . . . . . . . . . . . . . . . . . . . . . . 479
The most characteristic feature of animal life is that animals must actively
seek and select their foods in their natural environment. A natural product is con-
sidered as a food of a particular species when it is currently selected and effectively
eaten by members of the species and when, in addition, it corresponds to some of
its nutritive requirements. Thus, to be a food, this natural product must possess
two different series of biochemical properties. These properties act successively, as
sources of information for the C.N.S., in the control system regulating food intake.
In a first step, at the entry of the alimentary canal, the food is already controlled
through its stimulating activity upon the various sensory systems level. As a
result of this oral sensory appraisal, the food is either accepted or rejected and,
when accepted, is eaten in definite amounts. This sensory activity to foods is a
critical determinant of innate or acquired feeding responses, insuring an oral
selection and a metering of intakes. Through the second step of action of foods in
the feeding process, these orally determined responses to food are "regulated."
At the post-absorptive and systemic level, food as a nutrient acts as a metabolic
signal upon regulatory centers and, through positive and negative feed-back
mechanisms, "modulates" oral feeding responses.
Among the sensory activities elicited by food involved in this oral control
system and in this regulated response to food, the important role of olfaction and
more generally of chemical senses is a common concept. This concept is mainly due
to our subjective experience. In man the role of flavors in food acceptance and in a
30 nb. Sensory PhysioJogY7 Vol. IVjl
466 J. LE...l\UGNEN: Olfaction and Nutrition
more or less deficient regulation of food intake is obvious. It certainly represents,

in our species, the more prominent remaining function of the olfactory apparatus.
Nevertheless, even in man, and most of all in various animal species, the exact
role of the chemical senses and the neurophysiological mechanisms underlying
their contribution in the regulation of food intake, had to be investigated and
verified. These investigations in mammalian species and the present knowledge in
this field will be reviewed in this chapter.
A. The Cherno-Sensory Analysis of Foods

First, some comments are necessary concerning the chemo-sensory mechanisms
in olfaction and in taste that insure the peculiar and respective adaption of these
two systems to feeding control.
In order to permit the building up and the regulation of adjusted feeding
responses, the oral analysis of foods and the informations transmitted from this
level must be chemical in nature. Further, this information will be effective in
guiding adjusted responses in as much as this analysis will be biochemical, the
food being evaluated in the mouth as a metabolite. This biochemical differen-
tiation is partly performed by taste. A separate segregation of afferent patterns of
discharges (based upon some incompletely known properties of receptors) allows
roughly the discrimination of four biochemical functions. Sweet, salty, acid and
bitter compounds correspond to the carbohydrate, saline, acid and deleterious or
toxic substances. Innate responses of attractiveness and repellency are based upon
this biochemical discrimination. But this gustatory repertoire is limited and the
biochemical identification is only approximative. Erroneous responses, for instance
to non-nutritive sweet substances or to the toxic salty LiCI, are possible for the
reason that various substances of the same taste quality are undiscriminable.
This fine individual analysis of chemical stimuli, lacking in taste, is yielded by
the olfactory discrimination. By means of olfactory analysis, each food product
can be identified. This identification can drive specific differentiated responses.
This gain in chemical selectivity, as compared to taste, is accompanied by a
decrease in the possibility of analysis of common biological functions. However,
the question may be raised of the possible presence of undifferentiated and innate
responses to classes of odors corresponding to alimentary or non-alimentary,
protein or lipid materials. One of the causes of this inability of olfaction to provide
a basis for responses to metabolic properties is that, in contrast to taste, the in-
volved olfactory stimuli in the natural foods are not nutritive components, but
generally are associated impurities. As shown by gas-liquid chromatography, these
associated odorous fractions which constitute food aromas are very numerous.
Thus, olfactory directed responses are necessarily differentiated responses to
various associated signals of nutritive components.
Another fundamental difference between the role of the two chemosensory
systems in the sensory control of feeding responses is that the gustatory analysis is
performed on the food already accepted and taken in the mouth. Taste, as a
contact chemoreception, may not be involved; olfaction, as a telechemoreccption,
is involved in the food seeking and in the earlier food selection. Behavioral olfactory
data in mammals (VON BEKESY, 1964) and their neurophysiological correlates
Food Flavors and Food Intake in Man 467
(LEVETEAU and MAC LEon, 1968) have proved that an accurate binarinal localiza-
tion of external sources of odors is available and is used in the orientation towards
foods.
Finally, the neuroanatomy of the olfactory system and particularly its central
connections (see MAc LEon's chapter in this volume) favors the concept of an
olfactory contribution in nutrition. The close relationship between specific olfac-
tory projections and both rhinencephalic and diencephalic areas involved in the
regulation of food intake, represents the structural basis of the interactions between
olfactory afferents and the feeding responses described below.
B. Food Flavors and Food Intake in Man

Natural or added food aromas in association with other flavor components are
clearly the direct cause of human acceptance, rejection and preferences of foods
and, in some extents, of food consumption. This effect of food odors as a critical
determinant of human feeding behavior has not been extensively investigated.
The presence of innate responses is plausible, but has not been decisively
proved. Respiratory responsiveness to odors has been tested in neonates (ENGEN
et al., 1963-1965). Sucking responses are modified by odor stimulations (JENSEN,
1932; DISHER, 1934). The relation of such responses to feeding control is not
ascertained. Whether the general pleasantness in adulthood of vegetal and floral
odors and the unpleasantness of putrid odors are related to innate feeding
responses may be asked. Studies on the development of odor preferences and their
comparison between children and adults seem to prove the contrary (KNEIP,
MORGAN, and YOUNG, 1931; FOSTER, 1950; STEIN and OTTENBERG, 1958). This
slow development does not exclude, however, congenitally determined responses
and may be the result of neural maturation. The relation of these clear cut odor
preferences to feeding is therefore stilI doubtful. Pleasant floral odors added to
food may cause food aversion: the very unpleasant and putrid odors of fermented
cheese or fish, for example, may be the basis of food diIection. By contrast, the
acquisition and changes of food preferences in relation to feeding experience are
evident in man. The role of cultural and familial influences do not deny but rather
confirm these acquired responses. Individual food preferences or aversions are
commonly observed in relation to gastric or hepatic diseases or to food allergy.
Changes of flavor preferences or aversions after experience of starvation have also
been reported (BERGER and LEMAGNEN, 1957-1960). This field is still open to
further investigations.
At a given time in the dietary history of a particular subject, the differential
effect of food flavors in directing feeding activity has been recently tested. The
electromyographic recording of chewing and swallowing movements of human
subjects during test-meals has shown that feeding reflexes are effectively influenced
by food flavors. Twelve different flavors added to pieces of bread of a constant size
and presented in separate and standardized test-meals induced different amounts
eaten until the feeling of satiety was attained. This difference in meal size was
significantly correlated with different characteristics of feeding reflexes. The higher
was the flavoured food in the hierarchy of palatability, the shorter was the time
of chewing before swallowing of each mouthful. Hunger and palatability acted in
80
468 J. LEMAGNEN: Olfaction and Nutrition
the same direction upon this objective response and seemed substitutive in their
similar effect as feeding stimulants (PIERSON and LEMAGNEN, 1969).
Two phenomena, whose equivalents have to be found in animals, must still
be noted in man. According to our common subjective experience, food odors are
pleasant when we are hungry and these odors reinforce the feeling of hunger. The
same odors become unpleasant and may elicit nausea when we are satiated. Non
alimentary odors, generally fecaloid and putrid odors, induce nausea in hungry or
satiated subjects. Many proposed classifications of odor qualities have used this
characteristic feature with the class of "sickening" odors. The neural mechanism of
these effects is unknown. However, a slight and inconsistant effect of offensive
smell on gastric contractions has been observed (GINSBERG et al., 1948).
C. Innate Responses in Animal Species

A difference of odor preferences corresponding to the general pattern of food
selection has not been studied in herbivorous, carnivorous and omnivorous mam-
malian species. In kittens, rodents and chiefly in wild and laboratory rats the pre-
sence of innate feeding responses based upon food odors (the maternal milk flavor
for example) is only suspected. After the removal of olfactory bulbs, kittens become
unable to find the maternal nipple and they apparently lose the sucking response
(KOVACH and KLING, 1967). After weaning, the differential palatability of solid
foods by mammals may be in some cases based upon congenitally determined
preferences for food odors. The giant ant eater seems able to find its specific food
by smell (McADAM and WAY, 1967). Deer-mice select their natural foods more
by olfactory than visual cues (HOWARD, MARSH, and COLE, 1968). Wild rodents in
their biotope are dependent on smell in their food seeking of seeds and nuts
(SVERICENKO, 1954; RAD'KO, 1957). Food preferences by naive wild and domesti-
cated rats possibly based upon innate responses to food odors, have been tested.
It has been shown, by detailed study, that rats exhibit such preferences among
unfamiliar foods: whole wheat preferred to grains and to white wheat, horse liver
preferred to grains, and so on (BARNETT and SPENCER, 1953 a, b; BARNETT, 1956).
These immediate preferences are interpreted by the authors as innate responses.
Through a typical sampling behavior, rats modify rapidly these immediate
responses. As a result of this experience, earlier familiar foods when nutritionally
effective are preferred to new ones. At a given time in adulthood, rats as suggested
by other investigations (STEINBRECHER, 1962), exhibit a pattern of odor pre-
ferences in accordance to their acquired food preferences in their natural environ-
ment. In these conditions the identification of innate responses is always difficult.
In contrast to taste preferences, olfactory preferences and their possible
relations to feeding have not yet been systematically explored in laboratory
animals. Variations of olfactory preferences with increasing concentrations, com-
parable to the well known preference-aversion function of NaCI solutions, are not
known. It should be noted that various preferences or aversions exhibited by rats
towards sapid solutions, commonly attributed to gustatory stimulation, are in fact
due to olfaction. The response of rats in a choice between ethyl alcohol solutions
versus water and its variation with concentrations is deeply changed by the
removal of olfactory bulbs (KAHN and STELLAR, 1960). Similarly, the rejection of
Food Odors in Palatability Responses and their Adjustment 469
a bitter solution may be due, not to the taste, but to the odor of the solution
(GESELL and FISHER, 1968). Responses to sugar and NaCI solutions may be also
influenced by olfactory stimulating effects of the solutions (MILLER and ERICKSON,
1966).
However, the procedure of pure odors added to natural or synthetic diets
provides conclusive evidences that odors elicit typical variations of feeding
responses in naive animals. Various odorous substances added in minute amount
to the familiar diet of rats induce preference or aversion in a choice situation with
the unflavoured diet (SCOTT and QUINT, 1946a; ADOLPH, 1947; LEMAGNEN, 1956a
and others). For example, in a choice between two flavoured forms of the same
synthetic diet, one flavoured by Citral, the other by Eucalyptol, rats massively
preferred the Citral form. On the first days of alternate presentations of the two
flavoured forms, intake of the Citral meal was higher than of the Eucalyptol one
(LEMAGNEN, 1959a). But, except for offensive smells at high concentrations, these
naive responses to flavoured foods are transient. After a few days of continued
alternation of presentations of the same food flavoured either by Citral or Eucalyp-
tol, the preference, then tested in single or two recipients, had disappeared (Fig. 1).
After 12 days of ad libitum intake, the difference of intake due to various added
odors was no longer observed in SCOTT and QUINT'S experiment.
It has been argued from this transient effect of added odors in inducing
calorically unadjusted responses that food odors do not play any role in the current
and adjusted control of feeding. The contrary has been clearly demonstrated
leading to the conclusion that food odors, even artificially added to the diet, may
be in learned responses the effective basis of oral sensory control of food intake.
11
A : Diet flavoured by Citra I
B : Diet flavoured by Eucalyptol
I
Initial 0 6 Days 12 Final
Choice Choice
Fig. 1. Final no differentiation of two differently flavored forms of a diet, compared to the
initial preference for one of the two forms, as a result of repeated alternate free consumption
D. Food Odors in Palatability Responses and their Adjustment

When a pure odor is added to the familiar diet without a concomitant change
of its caloric density, a deficit or an excess of the short term or daily intake occurs,
as shown above, in rats. The rapid disappearance of this so called "palatability
response" is a common caloric adjustment which does not exclude the further con-
tribution of the flavour as an oral determinant of the new adjusted feeding response.
In an ad libitum feeding situation, the occurrence of this transient palatability

response and of its adjustment has been studied in detail. A graphic and con-
tinuous recording of the free food intake by rats of a maintenance diet permits to
follow their spontaneous feeding pattern: meal size and frequency of meals. When
this maintenance diet was suddenly modified by addition of 10 % corn oil and 15 %
cellulose, this new flavoured diet (unchanged in its caloric density) induced a
significant increase of the daily intake. This increase was generally due to an
elevation of both meal sizes and meal frequency. After a few days the previous meal
size and frequency, and therefore, the daily intake were restored (LEMAGNEN,
DEVOS, and TALLON, unpublished data). That such olfactory cues added to the
diet remain active in the sensory control of adjusted intake was shown by the
observation that the removal of the added odor after some weeks induced a new
unadjusted excess of the daily intake (LEMAGNEN, 1956a).
The role of flavour in the regulatory control of meal size has been demonstrated
in another experiment. Rats were trained for 32 days to eat, in a two hour morning
presentation of food, the same synthetic diet flavoured either by citral, eucalyptol,
benzyl acetate or by benzaldehyde. Then the four differently flavoured forms of
the same diet were presented successively in the same morning meal, 30 minutes
each. A dramatic overeating, up to 170 % increase, occurs as a result of this
sequence of flavored diet in the same meal (Fig. 2). In a comparable experiment,
20 r- 26
9 1 2 9 A B
22
1St--
18
10 :2 CO B
~~ ~
11.

5
~~~~ 10
u
~r;, & 6
D C C B C D B D A B B
B C B B D 0 C D D B A Fig. 3
A c D B B D A D B B C
C C A B A 0 D D C B D Fig. 2
Fig. 2. 1. Mean meal sizes during the training period. 2. Intakes compared in meals with
successive or constant flavored forms of the diet. A succession of four familiar flavored forms
during the meal induces hyperphagia
Fig. 3. A: mean intake in meals with one flavored diet. B: mean intake on 6 successive days in
meals giving a permanent choice of three flavored forms of the diet. - A slight and transient
increase of the meal size is induced by the choice
the simultaneous presentation of the flavoured diets throughout the two hour meal
leads also to a significant, though lower, increase of the total intake. By a daily
repetition of this self-selection of these flavoured forms of the same diet in the
meal, this increase disappears after the 7th day (Fig. 3) (LEMAGNEN, 1956b, 1960).
Learning of the Determining Effect of Associated Odors on Food Selection 471
Thus, as a result of prior association to feeding, the added odor acquires a

determining effect upon oral eating reflexes. This effect and its consequence on
amount eaten is specific for each particular odor. The successive or simultaneous
effects of various odors are partly additive. This acquisition of a determining effect
of a particular odor upon intake by its previous association to the familiar food is
accompanied by an acquisition of an odor preference. This preference is displayed
by the reinforcing property of such odors in an instrumental learning. Rats can be
trained to press a lever to obtain a puff of odorized air (LONG and TAPP, 1967).
When two levers were available in the test cage, one delivering amyl-acetate, the
other pure air, naive rats learned to press the levers and both deprived and satiated
rats obtained on the 1st day of testing an equal self-stimulation by the odor. This
preference for self-stimulation with amyl-acetate disappeared the following days
of testing. When one of the levers delivered the odor of the maintenance diet, only
hungry rats exhibited a sustained preference for the self stimulation by the food
odor versus pure air.
E. Learning of the Determining Effect of Associated Odors

on Food Selection: its Role in Regulatory Mechanisms
HARRIS et al., 1933, later confirmed by SCOTT and QUINT, 1946b, SCOTT and
VERNEY, 1949, have demonstrated decisively that a "specific appetite," that is a
food selection and quantitative intake adjusted to a specific nutritional need, may
be determined only by a learned response to olfactory cues.
Thiamine-deficient rats given the choice between the same diet in two cups,
one containing, the other lacking the minute required quantity of the vitamine,
selected the correct one only when an odorous substance had been added to it. The
association between the beneficial effect of the ingested thiamine with the olfactory
stimulation received while eating the correct food, is a basis for the acquisition of
a learned regulatory response. The fact that this response is really and exclusively
determined by the added odor is clearly proved. When the correct response was
obtained, the move of the added odor to the deficient diet led rats to prefer this
diet. This mistake was corrected later and rats after a relearning preferred again
the beneficial unflavoured diet.
Comparable results and conclusions have been reached with toxic foods. The
bait shyness or toxicophobia is a learned response. After this learning the avoidance
of the poisoning food is based upon the oral chemosensory control of that food
(RzOSKA, 1953; NACHMAN, 1963; PAIN and BOOTH, 1968) (Fig. 4).
In contrast to the rule of immediate contiguity between the conditioned and
unconditioned stimulus in classical conditioning, a latency of several hours between
the oral intake and the occurrence of the postingestive reinforcing effect has been
shown still effective to induce the learned response. This alimentary learning which
appears as a fundamental phenomenon in the regulation of food intake, seems to
be a particular type of learning or conditioning (GARCIA et al., 1967; NACHMAN,
1969, unpublished data).
The same process and the same acquired effect of food odors are demonstrated
in caloric appetite, that is in food selection and in quantitative intake adjusted to
caloric requirements of the body. Normal rats given a choice between two samples
of the same stock diet differing in their respective caloric density by addition of
inert material (cellulose), eat after several days of habituation approximatively the
same amount of calories from the two diets. Expressed in weight, the low caloric
diet is preferred (KHAmy et al., 1963; LEMAGNEN, unpublished data). The classical
adjustment on diluted foods in single presentation proves in the same way. Like
the above specific appetites, differential caloric appetites may be driven by
olfactory discrimination of two foods.
7.---------~------------------,
a , i /'
: "f\:-:}/ /~
4~ !\ /
3I I
!

2.----------r------------------,
...(; 1
- Poison
u ---- Saline
VI 0
c0 Injection
.~ -1 day
...
~
-2
-3
C
,
1 2 14 15
Day number
Fig. 4. A and B: daily glucose intakes in successive experiments with four out of eight rats
given 60 mgfkg cyclophosphoamide in the first and the other four 100 mgfkg in the second
(continuous lines), with saline injected conversely (broken lines). Open circles: days when the
glucose was odorized - lavender in A, citral in B. C: mean odor aversion score differences
between poison. and saline-associated odorized glucose intakes (from PAIN and BOOTH, 1968)
Rats trained to eat in two daily meals were given alternatively two differently
flavoured forms A and B of the same diet (Fig. 5). In a group the free meal of the
form A was followed by a subcutaneous injection of a glucose solution at a dosage
of 25 % of the calories eaten in the just preceding meal. The consumption of the
form B was followed by a control injection of isotonic saline. In another group the
intake of the form B was supplemented by the 25 % supply of calories from the
glucose injection and form A by the saline solution. After three weeks of this
treatment, injections were discontinued and a test choice was offered between the
two flavoured forms of the diet. In this choice, rats of the two groups showed a very
high preference for the previously nonsupplemented diet: form B in the first group,
form A in the second. Various controls of this procedure have confirmed that this
Learning of the Determining Effect of Associated Odors on Food Selection 473
fallacious caloric appetite is orally controled by chemosensory activity. The absence

of odor labelled forms or the removal of the olfactory bulbs prevent the acquisition
and exhibition of the differential appetite (LEMAGNEN, 1959b, 1969).
o 2 weeks 3
Means of the two groups
--~-.
:-c... ->: ... .. ..x
B
1--
o 2 weeks 3
Group 2
previously supplemented
10
g
~~
!.---
x..---.-..
"'X
~
)(-"' -X---x
A
5-
o 2 weeks 3
Group 1
Fig. 5. Progressive acquisition and final exhibition of a discriminative appetite by injecting

glucose solution after each successive intake of one of the two flavoured forms of the diet
Cho i ce
9.2
Fig. 6. Discrimination by: 1. odors, 2. textures, 3. brightness, 4. positions, 5. without stimuli,

of 2 forms of a diet after the learning of the differential appetite by d-amphetamine previously
added to one of the two forms. Olfactory discrimination permits the exhibition of a maximal
difference in the choice of intake
Amphetamine added to the diet substituted for the post-prandial adminis-

tration of glucose solution induces the same effect (Fig. 6). By this procedure the
respective ability of various sensory cues to serve as food discriminating stimuli
could be tested. Four pairs of two forms of a diet, one form containing D-Amphet.
amine, were tested in four groups of rats. In these pairs the two forms were
discriminable respectively by olfactory, spatial, texture and brightness cues without
any change in the nutritive properties. In this condition the maximal response of
selection of the form previously lacking Amphetamine was obtained in the final
choice with the odorised pair. The ratio of the intake of the two forms was respec-
tively: 3 - 1,57 - 1,74 - 2 (LEMAGNEN, 1959a).
The acquisition of food preferences based upon odor preferences in relation to
the dietary experience has been verified in another experiment.
40 days of agt' 60 days of ogt' 120 days of age

100 r-
%
d' 50 -
?; I~
I
'"
a ~ 2 3 2 3
~ 2 3
100 ,--
%
r- diet flavoured by citral
I
.,natural diet
/.
~ 50 l-
~
~
a ~ 2 3
/
2
,
3
f'?
l2
2 3
Fig. 7. 1. Controls. 2. Youngs from lactating mothers injected with citral during 10 days.
3. Youngs from lactating mothers injected with citral during 30 days. At 60 days of age,
youngs reared with the maternal milk flavored by citral prefer a solid food flavored by citral
Mother lactating rats were injected daily with 2 mg/kg of Citral (Fig. 7). This
odorous material excreted in their milk provided youngs from birth to weanling
with a citral flavoured milk. After weanling they were maintained on a regular
stock diet and their response to a choice between an unfamiliar synthetic diet and
the same flavoured by Citral was tested at 40, 60, and 120 days of age. Compared
to controls, rats (particularly females) exhibited a preference for the diet flavoured
with the odor of the maternal milk. This preferential response, maximal at 60 days,
had disappeared at 120 days of age (LEMAGNEN and TALLON, 1968a).
The results of TAPP et ai., mentioned above, have shown that such food pre-
ference induced by association of an odor to the maintenance familiar diet, leads
also to an odor preference tested by a positive reinforcement of olfactory self-
stimulation in hungry animals.
This last finding is instructive in view of the elucidation of neurophysiological
mechanisms underlying this learned olfactory control and regulation of feeding
responses.
Neural Mechanisms of the Olfactory Control of Feeding 475
F. Neural Mechanisms of the Olfactory Control of Feeding

a) As a result of innate responses modified by experience, the odor of foods
contributes to the current oral sensory control of food intake. This control promotes
a regulation because metabolic signals act in modulating these immediate responses
to food. These metabolic influences are both current and delayed. The onset and
the size of the oral feeding response are dependent on the present state of food
deprivation or hunger. On the other hand postabsorptive repleting effects reinforce
and regulate the metering action of this oral control.
16 normol rolS. Mean ealing role during Ihe meal

70
Deprivotion time
cg/mn oh 24 h 48 h
1\"." ""Obi""
60
RI
I~\ diet
SO

\
~ \ ~.
40 I'/~\~
~
.II \ -. \
'\
? \,..0
30

~/mean
, ,
20
'.
R3/
'{
'.
3
Fig. 8. Up to 3 or 4 grams consumed, the eating rate increased. The initial speed is correlated
with the palatability of the diet, only when rats are moderately hungry. cg/mn: Cent./min .
- - high palatability diet; 0 mean; low
The neural mechanisms underlying the effect of sensory input in this regulatory
system of food intake have been partly elucidated. A variety of evidences exists
demonstrating that separate mechanisms are involved in the regulatory control of
the meal size and of the onset of meals by which their frequency is determined
(LEMAGNEN and TALLON, 1966). Whether oral sensory afferents contribute to meal
size by stimulating or inhibiting feeding reflexes is discussed. Recent data suggest
a facilitatory effect at the beginning ofthemeal. When the rate of eating was recorded
in rats it was observed that, up to three or four grams consumed, the rate was
positively accelerated. When the previous deprivation was minimal at the beginn-
ing of the meal (2 or 3 hours) both initial and maximal rates were very dependent
on the palatability of the diet (Fig. 8). This dependency on the level of palatability
was not observed in meals initiated after fourty eight hours of previous deprivation
(LEMAGNEN and DEVOS, unpublished data). This finding supports other data sug-
gesting that the differences of intake dependent on palatability are larger in
moderate than in acute hunger. It suggests also that, at the beginning of the meal,
the sensory action of food is involved in a positive feed-back mechanism stimulat-
ing the feeding activity. Inhibiting effects probably due to the distension of
stomach are responsible of the following decreasing rate of eating, up to the end of
the meal. But a positive correlation between the maximal rate and the total
amount eaten in the meal confirms that palatability, its level and the duration of
its stimulating effect, are a critical determinant of the meal size.
This oro-pharyngeal control of food intake is dispensable. Rats can be trained
to press a bar to feed themselvesintragastrieally (EpSTEIN and TEITELBAUM, 1962).
They can be trained also to move their head in the suitable position to cut an
infra-red beam and thus to drive the injection of glucose solution through their co-
dal vein (COPPOCK and CHAMBERS, 1954). But dispensability is not a proof of no
physiological usefulness. In fact, when trained to feed themselves intragastrically,
normal and hyperphagic rats are "energized" in pressing the bar by simultaneous
oral stimulation (MAe GINTY et al., 1965).
The onset of meals and therefore their daily frequency in ad libitum feeding
situation has been shown to be directly dependent on hunger or metabolic signals
(LEMAGNEN and TALLON, 1968b). Whether more or less palatable foods affect the
critical threshold of this onset is still unclear.
The neural pathways and regulatory centers involved in this sensory and
metabolic control of feeding have been explored. The primary role of ventromedial
and lateral hypothalamic regulatory centres is wellknown. Their responsiveness to
various metabolites has been tested. Some evidences exist that the activity of
these hypothalamic centres is also influenced by afferents coming from the
alimentary tract. Through the vagus, distension of the stomach affects activity in
the V.M.H. (ANAND and PlLLAr, 1967). The stimulating action of oral afferents
upon the lateral feeding centre might be suspected. A first evidence of this effect
has been recently obtained (OOMURA et al., 1967).
In cats bearing electrodes chronically implanted in L.R. and V.M.H. areas,
nasal stimulation by the odor of the food elicited a change of the E.E.G. recording
responses of the two sites. In the L.H. a typical arousal (fast waves - slow
amplitude) occurred. The same pattern was observed when animals, free of their
movements, search for their foods. After prolonged smelling like after feeding,
L.H. activity returned to the slow waves high amplitude pattern. Responses in
V.M.H. were less consistent. In single units explored by bipolar electrodes in the
same L.H. site, the blowing up in the nose of vapor of acetic acid increased three
times the basal firing rate of the responsive units after a latency of 10 seconds
(Figs. 9 and 10).
The behavior of rats after hypothalamic lesions displays the correlation and the
respective role of sensory and metabolic signals in the regulation of intake.
After V.M.H. lesion, the responsiveness of hypothalamic centres to metabolic
informations is impaired. As a consequence, hyperphagic rats exhibit an over-
reactivity to sensory properties of foods and they no longer adjust their altered
feeding responses (TEITELBAUM, 1955; KHAmy et al., 1963; Me GINTY et al., 1965;
GRAFF and STELLAR, 1962). The removal of olfactory bulbs added to the V.M.H.
lesion exaggerates and prolongs the dynamic phase of hyperphagia (LARUE and
LEMAGNEN, 1968). After L.H. lesion the response to food deprivation is lost and
rats become aphagic. In their later recovery they eat only high palatable diets
Neural Mechanisms of the Olfactory Control of Feeding 477
which appear then as substitutes for metabolic signals in eliciting feeding (TEITEL-
BAUM and STELLAR, 1954; TEITELBAUM and EpSTEIN, 1962).
The study of the rewarding properties of L.R. area displayed by intracranial
seli-stimulation provides a model of this oral afferent control of feeding activity.
Rats allowed to press a lever to obtain electrical stimulation of the L.R. feeding
centre learn readily to press the lever. The rate of seli-stimulation increases with
LH "'''''''{''VV'~~~
Smell
Fig. 9. Simultaneous EEG records from VMH and LH during olfactory stimulation in a
chronic animal. Broken horizontal bar at right, one sec. During sniffing of food, high voltage
slow waves in LH change into low voltage fast moves and vice versa in VMH. When odor
stimulation was terminated, EEG returned to the previous pattern (from OOMURA et al., 1967)
10,-------------------------------~
1\
~ }\'."\/I \\.
~V
I ../\1
5
l
\ /\ .'. \ ..1 L.H,
\ V V'
j
o 20 30 sec
Fig. 10. Acute experiment. SUD in LH. Blowing acetid acid vapor (indicated by arrows) upon
the nasal mucosa increased the frequency (from OOMURA et al., 1967)
food deprivation or hunger and is inhibited by various actions inhibiting also

feeding such as fullness of the stomach, gastric preload and so on (ROE BEL, 1968).
Lateral stimulation is thus rewarding when the animal is hungry and it loses this
rewarding property when it is satiated. This finding supports the hypothesis that
oral afferents, and among them oliactory ones, act to initiate, to sustain and to
stop feeding activity as does L.R. electrical stimulation in inducing seli-stimulatory
behavior. Each specific food stimulus, through innate response or learning, possesses
its particular rewarding effect on oral eating. This rewarding effect in which the lateral
feeding centre should be involved is effective only when the animal is hungry and is
gradually inhibited by post-ingestive effects of foods. Thus rats would eat to
stimulate themselves through oral sensory pathways like they press the lever to
stimulate electrically their hypothalamic feeding centre.
b) Afferents in the various sensory systems are affected through centrifugal
pathways by motivation and elicited responses. At the various levels of sensory
systems from peripheral organs to cortical specific projections, inhibitory and

facilitatory networks act to modulate the sensory input.
Such a modulation in relation to neuroendocrine events has been proved for the
first time in olfaction. In women the olfactory threshold to various odors, and
particularly to the musky odor of the c 15 lactone "Exaltolide," varies throughout
the menstrual cycle (Fig. 11). A dramatic peak of sensitivity is observed at the time
of ovulation and reaches a minimum during the menstruation (LEMAGNEN, 1950).
Taking account of the presence in human male urine of a steroid musk (Androstan)
(BROOKSBANK and HASLEWOOD, 1961), it is thought that this neuroendocrine
dependent variation of the sensitivity for a specific odor is a vestigial aspect in man
of the relationship between olfacto-hypothalamic interaction involved in sexual
behavior and hormone release.
110- 8 , - - - - - - - - - - - - - - - - - - - - - - - - ,
1. 0- 9 - ........--;~.-.-
1. 0- 10 _
_--......,.,.-_.7'/---~
1.10-" -
1.10- 12 -
.10- 13 -
\. I
I
. a-It u _ _ _~V.:. i_ _ _ _ _ _ _ _ _ _ :...._J
menstruat . 24 hours menstruation
Fig. 11. Fluctuation of olfactory threshold to Exaltolide throughout the woman menstrual
cycle. A peak of sensitivity is observed at the time of ovulation. In dashed lines: maximal and
variant responses of some individual subjects. The units represent the dilution in water of a
saturated alcohol solution of Exaltolide sniffed by the experimental subjects
The presence of such a centrifugal modulation of olfactory afferents in relation

to feeding might be suspected. Since 1947 GOETZL et al. claim that olfactory thresh-
old varies in a constant relation to hunger and satiety in human subjects. Using the
contested olfactometric technique of ELSBERG, they found a rise of the sensitivity
to the odor of coffee before lunch and a decrease after the meal correlated with the
feeling of satiety. The omission of the principal meal led to a progressive and con-
tinuous lowering of the threshold. The authors proposed to use the level of olfactory
acuity as a measure of the sensations of hunger or satiety. These results were con-
firmed later by the same investigators using a greater number of subjects (GOETzL
et al., 1950) and by others (HAMMER, 1951).
The above concept was denied by JANOVITZ and GROSSMAN (1949) and ZIL-
STORFF-PEDERSEN (1955) although the same Elsberg's technique was used in their
experiments. A more recent revision of these controversal data with the use of
improved procedures, has shown that this lowering of olfactory threshold under the
influence of hunger does exist in some individuals, but this fluctuation is slight and
is entirely absent in other subjects (FURCHTGOTT and FRIEDMAN, 1960; BERG et al.,
1963). However this question remains open to discussion and to further investiga-
tions. Alimentary and non alimentary odors have not been compared. This
decrease of sensory threshold by hunger in man might be only an effect of a non-
References 479
specific central arousal. Performances in attention tests and problem solving have
been shown improved by hunger (CHAMPION and FIELD, 1963). According to
SMOLIC (1953), nausea induced by apomorphine injection should also decrease
olfactory threshold in man. The wellknown hypersensitivity to odors of pregnant
women could be the result of this phenomenon which has to be verified and studied
further.
The inverse effect, namely, hunger enhancement byfood odors, is substantiated by
some experimental data. Nonalimentary odors in the environment induce hyper-
phagia in rats (DIGIESI et al., 1963). Food odors have been claimed to influence
blood glucose level in man (BASSI and PASCUCCI, 1942; CANIGGIA and BROGI, 1947)
and in dog (REID, 1943).
Electrophysiological investigations in laboratory animals are necessary to
provide conclusive evidence of the existence of such phenomena and to confirm the
role played by efferent centrifugal fibers in their mechanism at various levels. The
peripheral level seems to be excluded. A modification of peripheral olfactory
thresholds under the influence of electrical stimulation of sympathetic ganglia has
been demonstrated (TUCKER, 1963). But it is unlikely that specific changes of
sensitivity in relation to metabolic state and to motivation might be due to a
centrifugal transformation of the discharge pattern elicited at this receptor level.
In gustatory systems, the peripheral electrophysiological threshold is unchanged
after Insuline administration or induced salt depletion leading to a change of
preferential responses respectively to glucose and to saline solutions (PFAFFMANN
and BARE, 1950). At the bulbar and particularly at the mitral cell level, this
modulation of afferent patterns as a result of various factors affecting behavioral
responses and among them learning is more probable. A first evidence of such a
centrifugal modulation of afferent pattern at this level under the influence of
learning has been recently obtained (MOULTON, 1968a, b).
Further investigations in this line will be probably rewarding to clarify the
exact contribution of olfactory messages and of their individual structures in the
control system of feeding behavior.
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Author Index
Page numbers in italics refer to the bibliography
Abbatt,J.D., see Hunter,C. Amoore,J.E. 236,242,245, Arnstein, C. 28, 55

G. 461,464 246, 249, 251, 252, 253, Arvanitaki, A., Chalazonitis,
Abel,M.S., see Goetzl,F.R. 254, 255, 255, 261, 262, N. 69,71
478, 480 276, 282, 283, 285, 287, - Takeuchi, H., Chalazoni
Adam,G., Delbruck,M. 354, 298, 306, 307, 308, 309, tis,N. 133, 146, 148
355, 360, 364, 384, 410, 316, 345, 346, 347, 348, Ash,K. O. 249,255,334,348
411,423 402,408,423 Atema,J., Todd,J., Bardach,
Adam,N.K. 340, 347 - Johnston,J.W., Rubin,M. J.E. 445, 446
- Jessop,G. 339,347 348 Atz,J.W. 2,7,8,23
Adams,J.R., see Dethier, V. - Palmieri,G., Wanke,E. Aub, J., see Robbins, L. 450,
254, 255, 309, 317, 345, 464
G. 360,424
348 Audubon,J.J. 432,433,446
Adams,R.M., see Dodson,
- - - Blum,M.S. 402, Aufsess,A. V. 367,423
C.H. 391,399,424
403, 408, 423 Axtell,R.C., see Dubose, W.
Adey, W.R. 194,201 - Venstrom,D. 242, 242,
Adolph,E.F. 469, 479 P. 360,425
247, 255, 287, 317, 340,
Adrian,E.D. 65, 71, 104, 345, 346, 348
108, 116, 122, 128, 147, Babin,R., see Guillot,)!.
- - Davis,A.R. 247,248, 302,319
148, 154, 177, 198, 201, 251, 255, 309, 317
206, 208, 212, 215, 264, Babuchin,A. 28, 55
- see Venstrom,D. 248, Bach.y.Rita,G., see Buch
265, 280, 281, 305, 316 256
- Matthews, R. 124, 128 wald,N.A. 458,461,462
Anand,B.K., Pillai,R.V. - see Garcia,J. 452,453,
Ahokas, A. J., see Goetzl, F. R. 476, 479
478,480 463
Anderson,P. 169,178
Ai,N., see Shibuya, T. 142, Backman,E.L. 117, 128
Andersson, C. 0., Bergstrom,
Bacq,Z.M., Alexander,P.
150 G., Kullenberg, B., Stall.
454,462
Akert, K., see Benjamin, R.M. bergStenhagen,S. 404,
423 Baginsky,B. 77,81,93
193,201
Balboni,G.C. 32,55
- see Boeckh,J. 372, 422, Andres, K. H. 31,40,53,55,
424 83, 86, 87, 88, 93, 93, 186, Balucani,D., see BuuHoi,N.
201 P. 292,317
- see Lamparter,H.E. 422, Bang, B. G. 17, 18, 23, 60,
426 Anthony,J., see Millot,J. 9,
24 61, 72, 433, 434, 446, 447
Albritton, P. F., see Wenzel, B. - Bang,F.B. 30,55,67,72
M. 445,448 Anton,E., see Braun,J.von
301,317 - Cobb,S. 434,435,447
Alexander,P., see Bacq,Z.M. Bang,F.B., see Bang,B.G.
Aoki,K., Takagi,S.F. 133,
454, 462 30,55,67, 72
148
Allen,W.F. 167,168,169, - see Takagi,S.F. 92,94 Bannister,L.H. 32, 38, 50,
170,177,178 Aplin,R.T., Birch,M. 353, 55,160,178
Allison,A.C. 28, 29, 55, 61, 423 - see Graziadei,P. 32, 56,
71, 91, 93, 160, 178, 194, Apotheker,D., see Rubin,M. 434,447
201 298, 320 Baradi,A.F., Bourne,G.H.
- Warwick,R. T. 187,197, Arai, T., see Takagi,S.F. no, 29, 55, 61, 72, 334, 348
201,264,316 131 Barber,S.B. 141,148
Altner,H., Boeckh,J. 142, Ardouin,P., Maillet,M. 29, Barbier,M., see Pain,J. 390,
148 55,61,71 427
- Muller, W. 38, 50, 55, Arn, H., see Roelofs, W. L. Bardach,J.E., see Atema,J.
154,178 390, 397, 428 445,446
31"
484 Author Index
Bare,J.K., see Pfafi"mann,C. Beidler,L.M., Tucker,D. Beroza,M., see BierI,B.A.

479,481 134, 148, 165, 178, 258, 390, 391, 393, 423
Barnett,S.A. 468,465 278, 317, 336, 346, 347, - see Doolittle, R E. 344,
- Spencer,M.M. 468,479 348 349, 390, 425
Bartell, R J., Shorey, H. H. - see Brown,H.E. 32, 55, - see Jacobson,M. 304,
378,423 434,447 319
Bassi,M., Pascucci,F. 479, - see Moulton,D.G. 29, - see McGovern,T.P. 390,
479 57, 114, 130, 264, 320 391, 427
Bate-Smith, B. C., see Harper, - see Tucker,D. 171, 181 - s. Valega, T.M. 353, 430
R 153, 179, 237, 243, Bekesy,G.von 170, 178,
Bertau,M. 13, 23
252, 256 200, 201, 223, 244, 330,
Bertmar,G. 8,9, 12,21,23,
Baumann,F. 162, 178 348,466,479
175,178
Baumgarten, R. von, Green, Bell,E. T. 81, 93
Bellairs, A. d' A., Boyd, J. D. Bessey,O.A., see Wolbach,S.
J.D., Mancia,M. 188, B. 66,67,73
191,201 13, 23, 163, 164, 178
Bellas, T.E., see Silverstein, Biedenbach,M.A., Stevens,
- see Mancia,M. 186, 198,
R.M. 390,391,429 C.F. 193, 199, 201
203
Baylor,E.R, Smith,F.E. Bellavita, V., see Buu-Hoi,N. Biedermann-Thorson,M., see
P. 292,317 Beroza,M. 416,422,423
450,462
Benjamin,RM. 163, 178 Bienfang,R. 237,242
Beams,H.W., see Slifer,E.H.
- Akert,K. 193, 201 BierI,B.A., Beroza,M., Col-
360, 362, 429
Bennett,M.H. 69, 72, 200, lier,C. W. 390, 391, 393,
Bean,RC., Shephard, W.C.,
201 423
Clan, H., Eichner,J. 417,
Berg,H.W., Pangborn,RM., Biffini 76
423 Roessler,E.B., Webb,A. Birch,M., see Aplin,R.T.
Beasley,A.B., see Kuhlen- D. 478,479 353,423
beck,H. 163,179 Berger,P., LeMagnen,J. 467, Bittel, H. 408, 423
Beck,A. 96, 103,128 479 - Boch,R., Doolittle,R.E.,
Beck, L. H., see Miles, W. R - see LeMagnen,J. 481 Tribble,M. T., Traynlam,
101,129 Berger,RS., Canerday,T.D. J.G. 423
Becker,J., see Ohlofi",G. 293, 390, 423 Blakeslee, A. F. 246, 255
320 Berglund, B., see Ekman,G. Block,B.C., see Schneider,D.
Beckmann,R., see Bute- 221, 222, 224, 243 365, 389, 429
nandt,A. 384,424 Berglund, U., see Ekman, G. Block,J. 422
Bedard,W.D., Tilden,P.E., 221, 222, 224, 243 Bloom,G. 28,44,55
Wood,D.L., Silverstein, Bergmann,E.D., see Ikan,R. - Engstrom, H. 28, 55
R.M., Brownlee,R.G., 391, 404, 425
Rodin,J.O. 399, 423 Bloom, W., Fawcett, D. W. 55
Bergstrom, G., Kullenberg, Blum,H.F. 64, 72
- see Wood,D.L. 400,404, B., Stallberg-Stenhagen, Blum,M.S. 353,396,397,
431 S., Stenhagen, E. 404,
400,423
Beer,G.R.de 10,23 423
Beets,M.G.J. 118,128,196, - Warter,S.L., Traynham,
- Lofqvist,J. 404,423
242, 242, 257, 262, 266, - see Andersson, C. O. 404, J.G. 392, 402, 403, 423
267, 276, 288, 291, 292, 423 - see Amoore,J.E. 402,
294, 295, 296, 297, 300, - see Kullenberg,B. 404, 403, 408, 423
317, 340, 344, 345, 348, 426 Boch,R., Shearer,D.A. 404,
402,423 Bernard,R.A., Halpern,B.P. 423
- Theimer,E.T. 296, 309, 66,72 - - Petrasovits, A. 408,
310,317 Bernhard,C.G. 97, 98,128 424
Beevor,P.S., see Moorhouse, - Granit,R, Skoglund,C.R. - - Stone, B. C. 424
J.E. 427 97, 128 - see Bittel, H. 423
Beidler,L.M. 147, 148, 165, Be~oza, M. 423 Boeckh,J. 107, 128, 141,
178, 212, 215, 219, 242, Beroza,M., Green,N. 353, 148, 272, 276, 277, 278,
265, 301, 317, 412, 414, 404, 423 317, 369, 370, 373, 374,
423 - Thorson,J., Biedermann- 375, 377, 379, 382, 384,
- Smallman,R.L. 87, 88, Thorson,M. 416, 422, 400, 401, 404, 405, 422,
93 423 424
Author Index 485
Boeckh,J., Kaissling,K.E., Boyd,W.A., see Frings,H. Buchwald,N.A., see Gareia,

Schneider,D. 140, 141, 445, 447 J. 452, 453, 454, 455,
149, 272, 276, 307, 317, Branson, B. A. 5, 23 458, 459, 460, 461, 463
326, 339, 348, 353, 354, Braun,J.J. 461, 462 - see Hull,C.D. 452, 453,
373, 392, 395, 403, 424 Braun,J. von, Anton,E. 301, 455, 460, 463
- Priesner,E., Schneider,D. 317 Bullock, T.H. 147, 149
98, 107, 115, 128 - Haensel, W. 301, 317 Burghardt,G.M. 163,178
- Sandri,C., Akert,K. 372, - Kaiser, W. 301, 317 - see Sheffield,L.P. 164,
332,424 - Kroper,H.H. 300,317 180
- Sass, H., Wharton,D.R.A. - Teuifert, W. 301, 317 Burkholder, W. E., see Rodin,
372, 422, 424 Brebion,G., see Gavaudan,P. J.O. 428
- see Altner,H. 142,148 267, 270, 318 Burne, R. H. 5, 7, 23
- see Schneider,D. 135, Brewer,E.D., see Elsberg,C. But, V. I., Klimova-Cherka-
150, 365, 389, 429 A. 168,179 sova, V.I. 165, 178
Bohme, C., see Riemschnei- Briggs, M. H., Duncan, R. B. Butenandt,A., Beckmann, R.,
der,R. 428 62, 68, 72, 264, 317, 333, Stamm,D., Hecker,E.
Boekenoogen,H.A., see Ru- 334, 348 384, 424
zicka, L. 298, 320 - see Duncan, R. B. 60, 62, Butler,C.G. 352,353,424
Boydanovsky,D., see Pain,J. 66,67,72,246,255 - Calam,D.H. 404,424
390, 427 Brightman,M. W., see Reese, - Paton,N. 424
Bogert,C.M. 163,178 T.S. 185 - Simpson,J. 424
Bojsen-Moller,F. 152, 162, Brink,A.S. 16,23 Buu-Hoi,N.P., Bellavita, V.,
171,178 Brinkman, R. 454, 462 Ricci,A., Balucaoni,D.
Brogi,G., see Caniggia,A. 292,317
Boness,M., see Eiter,K. 425
479,480 Byzow,A.L., Flerova,G.J.
Bonner,J., Varner,J.E. 70,
Broman,I. 10, 23, 161, 162,
72 112, 128, 137, 149
164, 175,178
Booth,D.A., see Pain,J.F.
Bronshtein,A.A. 40, 42, 55
471,472,481 Cain,W.S. 217,218,220,
- Ivanov,V.P. 44,55 221, 222, 223, 224, 242
Borg, G., Diamant,H., Strom,
Brooksbank,B.W.L., Hasle- - Engen, T. 219, 224, 226,
L., Zotterman, Y. 124, wood,G.A.D. 478,480
125, 128 242
Brown,C.W. 163,178 - see Engen, T. 218, 233,
Borley, W.E., see Newell,R. Brown,H.E., Beidler,L.M.
R. 450,464 234,243
32, 55, 434, 447
Bornemissza,G.F. 352, 424 Cajal,S.R. 28,56, 184, 186,
Brown,K.S., Robinette,R.R.
Bortolami,R., see Manni,E. 187, 188, 189, 190, 191,
250, 255
172,179 192, 194, 201
Brown,P.K., see Wald,P.
Bosack, T.N., see Engen, T. 330, 350 Calam,D.H., see Butler,C.G.
226,243 404, 424
Browne,L.E., see Silverstein,
Bosley,J.J., see Stone,H. R.M. 390,391,429 Callens,M. 195, 200, 202
235, 236, 244 Brownlee,L.E., see Wood,D. Calvin, A.D., Williams,C.M.,
Bossert,W.H., Wilson,E.O. L. 400, 404, 431 Westmoreland,N. 443,
366,424 Brownlee,R.C., see Moser, 447
- see Wilson,E.P. 380, J.C. 379,392,427 Camp,C.L. 16,23
431 Brownlee,R.G., see Bedard, Campbell, B. 68, 72
Bourne,G.H., see Barady,A. W.D. 399,423 Canerday, T.D., see Berger,
F. 29, 55, 61, 72, 334, - see Silverstein, R. M. 390, R. S. 390, 423
348 391, 429 Caniggia,A., Brogi,G. 479,
- see Shantaveerappa,T.R. Bruce,H.M. 194, 199,201 480
186,204 Bruner,H.L. 175,178 Carey,J.H. 154,178
Bournot,K., see Doll,W. Brunn, A., von 28, 56 Carpenter,M.S. 289, 292,
263,302,318 Brust-Carmona, H., Kas- 295,317
Bowman,J.P., Combs,C.M. przak, H., Gasteiger, E. L. Carregal,E.J.A., see Stone,
172,178 456, 462 H. 170,181
Boyd,J.D., see Bellairs,A. Buchwald,N.A., Garcia,J., Carroll,H.W., see Hunt,E.L.
d'A. 13, 23, 163, 164, Feder,B.H., Bach-y-Rita, 461, 463
178 G. 458, 461, 462 Case,J. 141, 149
486 Author Index
Castronovo,A., see Grassi, V. Comeau, A., see Roelofs, W.L. Davies,J. T. 261, 307, 311,
28,56 397, 398, 428 312, 316, 317, 323, 325,
Cauna,N., Hinderer,K.H., Comrey,A.L. 237, 243 327, 331, 335, 336, 337,
Wentges,RT. 174, 178 Conner,J., see Winston,H. 339, 341, 346, 348
Cave,A.J.E. 20,23 65,73 - Rideal,E.K. 330,337,
Cavill, G. W.K., Robertson, Comad,H.E., see Werner,A. 348
P.L. 396,397,424 301,321 - Taylor,F.H. 327, 329,
Chaddock, B., see Taylor,H. Coombs, C. H. 238, 243 338,348
L. 456,464 Cooper,G.P. 457, 458, 459, - see Theimer,E.T. 288,
Chadwick,L.E., Dethier, V. 460,462 293, 312, 321, 337, 338,
G. 269,317 - Kimeldorf,D.J. 452, 339, 344, 350
Chalazonitis,N., see Arvani- 456,457,462 Davis,A.R., see Amoore,J.E.
taki,A. 69, 71, 133, 146, - - McCorley,G.C. 458, 247, 248, 251, 255, 309,
148 460,462 317
Chambers,RM., see Cop- Cope,F.W. 69,72 Davson,H., Danielli, T.F.
pock,H.W. 476,480 Cope, J., see Robbins, L. 450, 325,348
Champion,RA., Field, R K. 464 Dawson,W.W. 134,147,
479,480 Coppel,H.C., see Matsumura, 149,166,178
Cheesman,G.H. 305,308, F. 390,404,427 DeAmicis,E., Zorzoli,G.C.
317 - see Tai,A. 393,430 48,56
- Kirkby,H.M. 306,317 Dean,R.B., Fa-Si Li 337,
Coppock,H. W., Chambers, R.
- Mayne, S. 305, 317 M. 476,480 348
- Townsend,M.J. 224, - Hayes,K.E., Neville,R.
Corbiere,G. 424
242, 305, 317 G. 342,349
Corbin,K.B. 172,178
Chisholm,A. H. 445, 447 Deboldt,J. W., see Toba,H.
- Harrison,F. 172,178
Chung, S.-H., see Gesteland, H. 390,430
Corbit, T.E. 226,234,243
RC. 142,147,149 Deenen, L.L.M. van, see
Court-Brown, W.M., Mahler,
Clan, H., see Bean,RC. 417, De Gier,J. 336, 349
RF. 461,463
423 - see Veerkamp,J.H. 336,
- see Hunter,C.G. 461,
Clark, W.E., LeGros 28,31, 350
464
44, 56, 78, 79, 81, 83, 92, DeGier,J., van Deenen,L.L.
93,122,129,160,178,184, Cowan, W.M., see Powell, T. M. 336,349
202,209,215,264,319 P.S. 190, 191, 192, 193, Delbriick,M., see Adam,G.
- Mayer,M. 194, 202 194,203,204 354, 355, 360, 364, 384,
- Warwick,RT.T. 77,81, Cragg,B.G. 190, 194,202 410, 411, 423
93 Craigie,E.H. 435,447
DeLorenzo,A.J. 32, 56
Clausen,H.J., see Noble,G. Crocker, E. C., Henderson, L. Demerdache,A., Wright,R.
K. 162, 163, 180 F. 252,255
H. 331,349
Clay,J., see Harris,L.J. 471, Crosby,E.C., Humphrey, T. Demole, E. 299, 318
480 435,447 Dennis,B.J., Kerr,D.I.B.
Cloud,R, see Robbins,L. - see Huber,G.C. 435,447 190,195,202
450,464 Curie 449
Derscheid,J.M. 5,23
Cobb,S. 432,434,443,447 Desole, C., see Manni, E. 172,
- see Bang, B. G. 434, 435, Dahm,K.H., see Roller,H. 179
447 402,428
Dethier, V. G. 269, 318, 352,
Cogan,J., see Robbins,L. D'Angelo, D., Issidorides,M., 397,424
450,464 Shanklin, W.M. 71, 72 - Larsen,J.R,Adams,J.R.
Cole, K. S. 424 Danielli,J.F. 325, 348 360,424
Cole,RE., see Howard, W.E. - see Davson,H. 325,348 - Schoonhoven,L.M. 375,
480 D'Arcy,F.J., Porter,N.A. 388, 409, 424
Collander, see Overton 326 449,463 - Yost,M.T. 117,128,266,
Collier, C. W., see BierI, B. A. Darwin,C. 64, 72 267, 270, 318
390.391.393.423 Dastoli. F. R.. Lopiekes. D. V . - see Chadwick,L.E. 269,
Colosanti,G. 76,93 Doig,A.R. 249,255 317
Combs,C.M., see Bowman, - Price,S. 249,255 - see Schoonhoven, L.l\f.
J.P. 172,178 Davies 196 388,429
Author Index 487
Devos 470 Eager,RP., see Land,L.J. Engstrom,H., see Bloom,G.

Diamant,H., see Borg,G. 188,203 28,55
124, 125, 128 Eayrs,J.T. 79,93 Epstein,A.N., Teitelbaum,P.
Digiesi, V., Palehetti, R, Tor- - see Moulton,D.G. 234, 476,480
toli, V. 479, 480 244,267,320 - see McGinty,D. 476,481
Dine,H.I., Smith,J.C. 456, Ecker,A. 27, 56 - see Teitelbaum,P. 477,
463 Eckhard, C. 27, 56 482
Dingle,J. T., Luey,J.A. 67, Edinger, T., see Romer,A.S. Erickson,RP., see Miller,S.
72 11,25 D. 469,481
Disher,D.R. 467, 480 Edwards, M. 28 Erkel,G.A.van, see Kleere-
Dodson,C.H., Dressler,R.L., Ehrlich,P. 28,56 koper, H. 2, 3, 24, 28, 56,
Hills,H.G., Adams,RM., Eichner,J., see Bean,RC. 61,72
Williams,N.H. 391,399, 417,423 Ernst,K.D. 358,359,360,
424 Eichner,J.T., see Kay,E.R. 361, 362, 363, 425
Doving,K.B. 114, 115, 123, 380,426 Ervin,F.R., see Garcia,J.
128, 195, 198, 202, 242, Eisenbraun,E.J., see Vick,K. 461,463,471,480
243, 265, 282, 283, 284, W. 380,392,430 Evans,H.G.V., see Wright,
316, 318,341, 346, 349 Eisner, T., Meinwald,J. 353, RH. 70,74
- Gemme, G. 195, 196, 202 425 Exner,S. 76,81,91,93
- Hyvarinen,J. 198,202 - see Pliske, T.E. 353,400, Eyzaguirre, C., Kuffler, S. W.
- Lange,A.L. 202,242, 427 98,115,120,129,137,149
243,284,318 Eiter,K., Truscheit,E., Bo- - see Kuffler,S.W. 137,
- see Drake,B. 115, 124, ness,M 425 149
129, 271, 318 Ekman,G. 222,235,239,
Dogiel,A.S. 31,48,56 240, 243 Farbman,A.I. 263, 318
Doig,A.R, see Dastoli,F.R - Berglund, B., Berglund, Farquhar,M.G., Palade,G.E.
249, 255 U., Lindvall, T. 221,222, 33,56
Doll, W., Bournot,K. 263, 224,243 Fa-Si Li, see Dean, R. B. 337,
302,318 - Engen, T. 222, 240, 241, 348
Doolittle, R E., Beroza, M., 243 Fawcett,D. W., see Bloom, W.
Keisler, I., Schneider, E. L. - Froberg,J., Frankenhaeu- 55
344,349,390,425 ser,M. 222,223,243 Fechner, G. T. 218, 219, 243
- see Bittel, H. 423 Elsberg 478 Feder,B.H., see Buchwald,
Dostal,B. 387,425 EIsberg,C.A., Brewer,E.D., N.A. 458, 461, 462
Douek,E.E. 246,255 Levy,I. 168,179 - see Garcia,J. 452,453,
Drach,P. 21,24 - Levy,I., Brewer,E.D. 454, 455, 458, 460, 461,
Drake,B., Johansson,B., 168,179 463
Sydow,E.von, Doving,K. Emslie,A.G., see Hainer,R. - see Hull,C.D. 452, 453,
B. 115, 124, 129, 271, M. 264,319 455,460,463
318 Engen, T. 123,129,224,226, Feldman,M., see Ginsberg,R.
Dravnieks,A. 335, 345, 349 231, 234, 239, 240, 243, S. 468,480
Dressler,R.L., see Dodson,C. 284, 306, 318, 342, 349 Ferguson,J. 318
H. 391, 399, 424 - Bosack, T .N. 226, 243 Ferkovich,S.M., see Norris,
Drew, W.A., see Vick,K. W. - Cain,W.S., Rovee,C.K. D.M. 427
380, 392, 430 218, 233, 234, 243 Ferrer,N.G. 194,195,202
Dubose,W.P., Axtell,RC. - Lindstrom, C. O. 218, Field,R.K., see Champion,R
360,425 243 A. 479, 480
Dubrowsky,B., see Hull,C.D. - Lipsitt,L.P. 480 Fink,E. 440, 447
452,453,455,460,463 - - Kaye,H. 467, 480 Fisher,G.L., see Gesell,C.
Duhig,J. V. 62, 72 - McBurney,D.H. 218, 469,480
Duncan,C.J. 447 239,243 Flerova,G.J., see Byzov,A.
Duncan,RB., Briggs,M. 60, - Pfaffmann, C. 265, 318 L. 112, 128, 137, 149
62, 66, 67, 72, 246, 255 - see Cain, W. S. 219, 224, Flock,A., see Wersall,J. 58
- see Briggs,M.H. 62, 68, 226,242 Fliigge, C. 366, 425
72,264,317,333,334,348 - 8. Ekman,G. 222,240, Foster,D. 467, 480
Dyson,G.M. 311,318 241,243 Fraenkel,G. 352,425
488 Author Index
Frankenhaeuser,M., see Ek- Garcia,J., McGowan,B.K., Getchell, T. V. 136, 145, 149

man,G. 222, 223, 243 Ervin,F.R., Koelling,R. - Gesteland,R.C. 135,149
Franzen,O., Offenloch,K. A. 461,463 Gibbons,F.R., see Wald,P.
125,129 - see Buchwald,N.A. 458, 330, 350
- Osterhammel,P., Terkild- 461,462 Gilby,A.R., see Waterhouse,
sen,K., ZiIstorff,K. 108, - see Hull,C.D. 452, 453, D.F. 404,430
125,129 455, 460, 463 Gillies, M. T., Wilkes, T. J.
Friedman,L. 303,318 Gasser,H.S., 28, 56,109,129, 352, 366, 425
Friedman,M.P., see Furcht- 154,179,263,265,318 Ginsberg, R. S., Feldman,lL,
gott,E. 478,480 Gasteiger,E.L. 460 Necheles,H. 468,480
Frings,H. 367,425 - Helling,S.A. 459,463 Godfrey,E.W., Shenk,H.P.,
- Boyd,W.A. 445,447 - see Brust-Carmona, H. Silcox, L. E. 450, 463
Frisch,D. 44,47, 56 456,462 Goetzl,F.R.,Abel,M.S.,Aho-
Frisch,K. v. 367, 368, 378, - see Helling,S.A. 459, kas,A.J. 478, 480
379, 406, 407, 408, 409, 463 - Stone,F. 480
425 Gaston,L.K., see Payne, T.L. Goin,C.J., Goin,O.B. 12,24
373, 378, 427 Goin,O.B., see Goin,C.J.
Froberg,J., see Ekman,G.
- see Shorey,H.H. 352, 12,24
222,223,243
378,429 Golgi 186
Frohlich,F.W. 96, 97, 102, Gavaudan,P., Poussel,H.,
103, no, 129 Goll, W. 422, 425
Brebion,G., Schutzenber- Gomper,H.I. 48,56
Frommes,S.P., see Okano,M. ger,M.P. 267,270,318
39,57 Goodman,S.DeW., Huang,
Gegenbaur, C. 10, 24 H.S. 62,72
Friihwald, V. 76, 79, 81, 93 Gehuchten,A. van 28, 58
Fuchs, H. 10, 24 Goodwin,T.W. 62,72
- Martin,L. 190, 202 Gorbman,A., see Hara, T.J.
Fukumoto, Y., Nakajima,H., Geiger,M., see Huser,H.J.
Uetake,M., Matsuyama, 201,202
250,256
A., Yoshida, T. 249, 256 Gorman,J.E., see Rodin,J. O.
Gelvin,D.E., see Kay,E.R.
Fuortes,M.G.F. 98, 103, 428
380,426
129, 370, 425 Gottlieb,R., see Ikan,R.
Gemme,G., see DI'Jving,K.B.
Furchtgott, E., Friedman,lL 391,404,425
195, 196,202
P. 478,480 Graff,H., Stellar,E. 476,
Gerebtzoff,M.A., 263,318
480
- Philippot,E. 61, 63, 70,
Gall,J.G. 425 Granit, R., see Bernhard, C. G.
72
Garcia,J., Buchwald,N.A., 97,128
- Shapenko, G. 29, 56, 61,
Bach-y-Rita,G., Feder,B. 63,72 Grasse,P.P. 353,425
H., Koelling,R.A. 452, - see Heusghem, C. 63, 72 Grassi, V., Castronovo, A. 28,
453,463 - see Philippot, E. 63, 73 56
- - Feder,B.H., Koelling, Gesell,C., Fisher,G.L. 469, Gray,J.A.B., Sato,M. 102,
R.A. 454, 463 480 129
- - - - Tedrow,L.F. Gesteland,R.C. 120,129, Graziadei,P.P.C. 29,32,33,
454, 455, 458, 460, 461, 139, 146, 149, 196, 270, 34, 40, 50, 53, 56
463 271, 280, 318, 405 - Bannister,L.H. 32,56,
- - Hull,C.D., Koelling, - Lettvin,J.Y., Pitts,W.H. 434,447
R.A. 454, 455, 459, 463 120, 129, 137, 149, 214, - Metcalf,J.F. 29, 31, 38,
- Ervin,F.R. 461,463 215, 271, 279, 318, 336, 44, 54, 56
- - Koelling,R.A. 461, 341, 346, 347, 349 - Pierantoni,R.L. 56
463 - - - Chung,S.-H. 142, - Tucker,D. 50, 56, 160,
- - Yorke, C. H., Koelling, 147,149 179
R.A. 471, 480 - - - Rojas,A. 120, Green,D.M., Swets,J.A.
- Green,K.F., McGowan,B. 121, 129, 142, 149, 265, 227, 232, 243
K. 459,463 271, 318, 336, 346, 347, Green,J.D., see Baumgarten,
- Kimeldorf,D.J. 461,463 349 R. von 188, 191, 201
- - Hunt,E.L. 461, 463 - see Getchell,T.V. 135, - see Mancia,M. 186, 198,
- - Koelling,R.A. 461, 149 203
463 - see Lettvin,J.Y. 147, Green,K.F., see Garcia,J.
- Koelling,R.A. 461, 463 149, 303, 319 459,463
Author Index 489
Green,N., Jacobson,M., Kel- Harding, C., see Jacobson, M. Henton, W. W., Smith,J. C.,
ler,J.C. 399,425 390, 399, 426 Tucker,D. 166,179,443,
- see Beroza, M. 353, 404, Hargraeves,F.J., see Harris, 447, 456, 463
423 L.J. 471,480 Herberhold,C. 61,72
- see Jacobson,M. 390, Harman, H. 240, 243 Herrick,C.J. 22, 24, 175,
399, 426 Harper, R., Bate-Smith, B. C., 179,191,202,446,447
- see Toba,H.H. 390,430 Land,D.G. 237,243 Heusghem,C., Gerebtzoff,M.
Grewe,F.J. 18, 24 - Bate-Smith, E. C., Land, A. 63,72
Griffith,P.H., Siisskind,C. D.G. 153,179 Hewitt,P.H., see Read,J.S.
425 - - - Griffiths,N.M. 390,428
Griffiths,N.M., see Harper,R 252, 256 Higashino,S., Takagi,S.F.
252,256 109,129
Harris,L.J., Clay,J., Hargre-
Grigor'yev, Yu. G., Tsypin, A. - see Takagi,S.F. no, 131
aves,F.J., Ward,A. 471,
B. 452, 463 Hille,B. 416,425
480
- s. Tsypin,A.B. 452,464 Hills,H.G., see Dodson,C.H.
Harrison,F., see Corbin,K.B. 391, 399, 424
Guild,S.R., see Huber,C.G. 172,178
174,179 Hinderer,K.H., see Cauna,N.
Hartline,H.K. 97, 101, 129 174,178
Grossman, M. I., see Janowitz, - Wagner,H.G., Mac Nichol Hinds,J.W. 187,202
H.D. 478,480 E.F. 103,129
Grubb,W.J., see Read,J. Hodgkin, A. L., Katz, B. 323,
- - Ratliff,F. 189,202 349
302, 320
Hasler,A.D. 201,202 Hodgson,E.S., 141, 149,
Gueldner,R.C., see Tumlin- Haslewood,G.A.D., see 352, 425
son,J.H. 390, 391, 400, Brooksbank,B.W.L. - see Levandowsky,M.
404, 430 478,480 141,149
Guillot,M. 246, 251, 255, Hatfield,C.A., see Taylor,H. Hoebel,B.G. 477,480
256, 302, 305, 309, 318, L. 456,464 Hoffmann, C. 352, 425
319, 347,349
Hayes,K.E., see Dean,RB. Hoffmann,C.K. 76, 77, 81,
- Babin,R. 302, 319 93
342,349
- Thibaut,P. 302,319
Hecher,E., see Butenandt,A. Holl, A. 5, 7, 24
384,424 Hopf, H. C., Hufschmidt, H.
Haberly,L.B. 199, 202 J., Stroder,J. 170,179
Haensel, W., see Braun,J. von Hedin,P.A., see Tumlinson,
301,317 J. H. 390, 391, 400, 404, Hopkins,A.E. 28, 56, 263,
430 319
Hagbarth,K.E., see Kerr,D. Hoppe,G. 13,24
I. B. 196, 202 Heggie,R., see Milas,N.A.
60, 62, 67, 73 Horsley, C. H. 64, 72
Hagelin,L.-O., Johnels,A.G. Hosaya, Y., Yoshida,H. 89,
3,24 Heintz,A. 3, 24
Helling,H. 11,24 93, 99, 129, 135, 149
Hagins,W.A. 417,425 Howard,W.E., Marsh,R.E.,
Hainer,RM., Emslie,A.G., HelIing,S.A., Gasteiger,E.L.
459, 463 Cole,RE. 480
Jacobson,A. 264, 319 Howe,P.R., see Wolbach,S.
Haley,T.J., Snider,R.S. - see Gasteiger,E.L. 459,
B. 66,73
450,463 463 Huang, H. S., see Goodman,
Halpern,B.P., see Bernard, Helmholtz, H. v. 450 S.DeW. 62,72
RA. 66,72 Henderson,L.F., see Crocker, Huber,C.G., Guild,S.R
- see Tapper,D.N. 461, E. C. 252, 255
174,179
464 Hendrix,D.E., s. Hughes,J. Huber,G.C., Crosby,E.C.
Halpern,M., see Scalia,F. R. 285, 319, 332, 349 435,447
175,180 - see Wright, R H. 332, Hufschmidt,H.J., see Hopf,
Hamlin,H.E. 161, 179 333, 350 H.C. 170,179
Hammer,F.J. 478, 480 Henke,K., Ronsch,G. 358, Hug,C.D. 450,463
Hamrum, C. L. 443, 447 425 Hughes,J.R., Hendrix,D.E.
Hara, T.J.,Ueda,K., Gorb- Henkin, R. I. 247, 248, 256 285, 319, 332, 349
man,A. 201,202 - Kopin,I.J. 247,256 - - Wetzel,N. 285, 319,
Hardee,D.D., see Tumlinson, - PoweU,G.F. 245, 247, 332,349
J.H. 390, 391, 400, 404, 248,256 - see Wright, R H. 332,
430 Henning, H. 252, 256 333,350
490 Author Index
Hull,C.D., Garcia,J., Buch- Jacobson,M. 352, 399, 425 Kaissling, K. E., J acobson,l\L
wald,N.A., Dubrowsky, - Beroza,M., Jones, W.A. 399
B., Feder, B. H. 452,453, 304,319 - Priesner,E. 355, 357,
455, 460, 463 - Green,N., Warthen,D., 365, 366, 375, 384, 385,
- see Garcia, J. 454, 455, Harding,C., Toba,H.H. 386, 389, 410, 4ll, 412,
459, 463 390, 399, 426 418, 426
Humphrey,T. 176,179 - Schwarz,M., Waters,R. - Renner,M. 262,319,358,
- see Crosby,E.C. 435,447 M. 390, 399, 426 365, 372, 388, 403, 404,
Hunt,E.L., Carroll,H. W., - see Green,N. 399,425 408,426
Kimeldorf,D.J. 461,463 - see Jones, W.A. 399,426 - Thorson 415
- Kimeldorf,D.J. 452, - see Kaissling,K.-E. 399 - see Boeckh,J. 140, 141,
454" 461, 464 - see Toba, H. H. 390, 430 149, 272, 276, 307, 317,
- see Garcia,J. 461,463 Jagodowski,K.P. 28,31,56 326, 339, 348, 353, 354,
- see Kimeldorf,D.J. 450, Janowitz,H.D., Grossman, 373,392,395,403,424
452,461,464 M. I. 478, 480 - see Kasang,G. 419
Hunter,C.G., Munson,R.J., Jarvik,E. 8,9, 10, 21, 24 - see Ruttner,F. 372, 428
Court Brown, W.M., Ab- Jensen,K. 467, 480 - see Schneider,D. 276,
batt,J.D. 461, 464 Jessop,G., see Adam,N.K. 321, 353, 357, 365, 371,
Huser,H.J., Moor-Jan- 339,347 383, 388, 395, 405, 406,
kowski,J.K., Truog,G., Johansson, B., see Drake,B. 429
Geiger, M. 250, 256 ll5, 124, 129, 271, 318 - see Vareschi, E. 368,408,
Huyser,H.W., see Ruzicka, Johnels,A.G., see Hagelin,L.- 430
L. 298,320
O. 3,24 Kalmus,H. 366,426
Johnston,J.B. 173,179 Kandel,E.R. 194,202
Hyden, H. 71, 72
Johnston,J.W., Moulton,D. Kare,M.R. 433,447
Hyvarinen,J. see D0ving,K.
G., Turk,A. 422,426 Karli,P.,see Vergnes,M. 445,
B. 198,202
- see Amoore,J.E. 348 448
Jones,A. W.,Levi-Montalcini, Karlson,P. 352,426
lino,M., see Takagi,S.F. 92,
R. 435, 447 - Liischer,M. 352,426
94
Jones,F. 218, 243 Karpman,B., see Woodrow,
Ikan,R., Bergmann,E.D., Jones,F.N. 124, 129, 234, H.F. 223,244
Yinon, U., Shulov,A. 243 Kasang, G. 384, 420, 426
391, 404, 425 - Jones,M.H. 260, 263, - Kaissling,K.-E. 419
- Gottlieb, R., Bergmann, 319 - see Riemschneider,R. 428
E.D.,Ishay,J. 391,404, Jones,F.W. 17,18,24 - see Schneider,D. 321,
425 Jones,M.H., see Jones,F.N. 383, 429
Inman,D.R. 331,349 260, 263, 319 - see Steinbrecht, R. A.
Ishay,J., see Ikan,R. 391, Jones, W.-A., Jacobson,M. 355, 384, 419
404, 425, 426 399, 426 Kasprzak, H., see Brust-Car-
Ishiko,N., Loewenstein, W. - see Jacobson,~I. 304, mona, H. 456, 462
R. 349 319 Katz,B. 98, 120,129
Issidorides,M., see D'Angelo, - see Hodgkin, A. L. 323,
D. 71,72 Kafka, W.A. 356, 373, 377, 349
Ito,K., see Takagi,S.F. 106, 382, 383, 388, 400, 401, Katz, S. H., Talbert, E. J.
109, llO, 131, 137, 150 402, 403, 405, 418, 421, 167,179
Ivanoff 363 426 Kay, E. R., Eichner, J. T.,
Ivanov,L.L., see Pyatnova, Kahmann,H. 162,179, 187, Gelvin,D.E. 380,426
Y. 352,428 202 Kaye, H., see Engen, T. 467,
Ivanov, V.P., see Bronshtein, Kahn,M., Stellar,E. 468, 480
A.A. 44,55 480 Keeler,C.A. 65, 72
Iwama,K.K., s. Yamamoto, Kaiser, W., see Braun,J. von Keilbach,R. 18,24
C. 186, 188, 204 301,317 Keiser, I., see Doolittle, R. E.
Kaissling,K.-E. 355, 356, 344, 349, 390, 425
Jackson,R. T. 61, 63, 72 372, 373, 376, 377, 384, Keller,J.C., see Green,N.
- Lee,Ch.-Ch. 80, 93 392, 394, 403, 405, 4ll, 399,425
Jacobson,A., see Hainer,R. 412, 413, 414, 417, 418, Kellogg,F.E. 409,426
M. 264,319 419, 421, 426 - Wright, R. H. 366, 426
Author Index 491
Kellogg,R. 153,179 Kramer,E. 376 Larsen,J.R., see Dethier, V.

Kennedy,J.S., Moorhouse,J. Krause, B. 360, 363, 426 G. 360,424
E. 366, 426 Krause, W. 27, 56 Larue,C., LeMagnen,J. 476,
Kerkhoff,H. 175,179 Kroper,H., see Braun,J.von 481
Kerr,D.l.B., Hagbarth,K.E. 300,317 Lauder,B.A., see Patterson,
196,202 Kruskal,J.B. 238, 243, 244 P.M. 247, 249, 256
- see Dennis,B.J. 190, Kuffler, S. W., Eyzaguirre, C. Laverack,M.S. 142,149
195,202 137,149 Law,J.H., see Sheffield,L.P.
Keverne,E.B., see Michael, - see Eyzaguirre, C. 98, 164,180
R.P. 175,180 115, 120, 129, 137, 149 Lawrence,B., Schevill, W.E.
Khairy,M., Morgan, T.B., Kuhlenbeck, H., Malewitz, T. 18,24
Yudkin,J. 472, 476, 480 D., Beasley,A.B. 163, Lawrence,H.G., see Wolf,G.
Kimeldorf,D.J., Hunt,E.L. 179 65,73
450, 452, 461, 464 Kullenberg, B. 352, 426 Lederer,E., see Pain,J. 390,
- see Cooper,G.P. 452, - Bergstrom,G., Stallberg- 427
456,457,458,460,462 Stenhagen, S. 404, 426 Lee,Ch.-Ch., see Jackson,R.
- see Garcia,J. 461, 463 - see Andersson, C. O. 404, T. 80,93
- see Hunt, E. L. 452, 454, 423 Lees,A.D., see Slifer,E.H.
461, 463, 464 362, 363, 430
- see Bergstrom,G. 404,
Kimura,K. 114, 115, 120, 423 LeMagnen,J. 194, 199, 203,
129, 134, 146, 149 Kumpf,K.F., see Noble, G. K. 224, 244, 305, 319, 469,
King,A., see Kovach,J.K. 470, 472, 473, 474, 478,
163,180
468,480 481
Kirk, R. L., Stenhouse, N. S. Kurihara, K. 29, 56, 62, 63,
- Berger,P. 481
249,256 72
- Rapaport,A. 66, 67, 73
Kirkby,H.M., see Cheesman, Kurosawa, T., see Yama- - Tallon,S.474,475,476,481
G.H. 306,317 moto,T. 58
- see Berger,P. 467,479
Kiseleva,Z.N. 17,24 Kuwabara,M. 367,426 - see Larue, C. 476, 481
Kishaba,A.N., see Toba,H. Kyskina,A.S., see Pyatnova, - see Pierson,A. 468, 481
H. 390,430 Y. 352,428 Lenhossek,M.von 28,57
Kistiakowskyi, G. B. 334, Lettvin,J. Y., Gesteland,R.
349 Lachner, V. 272, 274, 275, C. 147,149, 303, 319
Kitamura,H., see Takagi,S. 319, 352, 360, 365, 367, - see Gesteland, R. C., 120,
F. 106, 109, UO, 131, 368, 369, 370, 374, 375, 121, 129, 137, 142, 147,
137,150 379,388,405,409,426 149, 214, 215, 265, 271,
Kleerekoper,H. 23,201,202 - see Schneider,D. 276, 279, 318, 336, 341, 346,
- van Erkel,G.A. 2, 3, 24, 321, 357, 365, 371, 388, 347, 349
28, 56, 61, 72 395, 405, 406, 429 Lcvandowsky,M., Hodgson,
Klimova-Cherkasova, V.I., Laffort,P. 189,203,313,319 E.S. 141,149
see But,V.I. 165,178 Laibach, E. 5, 24 Leveteau,J., MacLeod,P.
Klouwen,M.H. 290,319 Lal,A.B., see Singh,B.K. 122, 129, 197, 200, 203,
- Ruys,A.H. 298,312,319 302,321 284, 319, 467, 481
Knapp,H.,seeScalia,F. 175, Lammers,H.G., see Lohman, - see MacLeod,P. 106,
180 A.H.M. 190, 203 U5,129
Kneip,D.H., Morgan, W.L., Lamparter,H.E., Akert,K., Levi-Montalcini,R., see Jo-
Young,P.T. 467,480 Sandri, C. 422, 426 nes,A. W. 435, 447
Koelling, R. A., see Garcia,J. Land,D.G., see Harper,R. Levy,C.K. 450, 464
452, 453, 454, 455, 458, 153, 179, 237, 243, 252, Levy,I., see Elsberg,C.A.
459, 460, 461, 463, 471, 256 168,179
480 Land,L.J., Eager,R.P., Lewis, C. T. 427
Koster, E. P. 222, 244, 306, Shepherd,G.M. 188,203 Liebermann,K. 5,7,24
307,319 Lange,A.L., see D0ving,K. Lindstrom,C.O., see Engen,
Kolmer,W. 61, 72, 81, 87, B. 202, 242, 243, 284, T. 218,243
94,160,174,179, 318 Lindvall, T., see Ekman, G.
K0ster,E.P. 199,202 Langohr,J., see Robbins,L. 221, 222, 224, 243
Kovach,J.K., King,A. 468, 450,464 Lindzey,G., see Winston,H.
480 Larsell, O. 174, 179 65,73
492 Author Index
Linnaeus, C. 252, 256 Malhorta,S.K. 71, 73 McWhirter,K.G. 250

Lipetz,L.E. 449,464 Mancia,M., Baumgarten,R. Meinwald,J., Meinwald, Y. C.,
Lipsitt,L.P., see Engen, T. von, Green,J.D. 186, Mazzocchi,P.H. 391,
467,480 198,203 427
Livanov,M.N. 452,464 - see Baumgarten, R. von - see Eisner, T. 353,425
Locke,M. 364,410,427 188, 191, 201 Meinwald, Y.C., see Mein-
LOfqvist,J., see Bergstrom, Mann, G. 176, 179 wald,J. 391, 427
G. 404,423 Manni, E., Bortolami, R., Mendelson,M., see Loewen-
Loewenstein,W.R. 325, Desole, C. 172, 179 stein, W. R. 98, 115, 129
329, 331, 349 Marcarian,H.Q., see Smith, Mentink,G.M., see Lohman,
- Mendelson, M. 98, 115, R.D. 172,173,181 A.H.M. 186, 190, 191,
129 Marsh,R.E., see Howard,W. 203
- see Ishiko, N. 349 E. 480 Merrill, O. E., see Robbins, L.
Loftus, R. 422 Martin,A.J.P. 260,319 450,464
Loher, W. 353, 427 Martin,H. 366, 367, 368, MetcaH,J.F., see Graziadei,
Lohman,A.H.M. 191,203 378, 379, 427 P.P.C. 29,31,38,44,54,
- Lammers,H.G. 190,203 l\Iartin, L., see Gehuchten, A. 56
- Mentink,G.M. 186, 190, van 190,202 Metzler,O., see Prelog, V.
191,203 Maschwitz,U.W. 353,397, 297,320
Long,C.J., Tapp,J.T. 471, 427 Meyer, see Overton 326
481 Mathews,D.P., Tucker,D.
Meyer,M., see Clark,W.E.Le
Lopiekes, D. V., see Dastoli, F. 142, 143, 149
Gros 194,202
R. 249,255 Matsumura,F., Coppel,H.C.,
Lorenzo,A.J.D.de 186,203, l\fichael,R.P., Keverne,E.B.
Tai,A. 390, 404, 427
263,319 - see Tai, A. 393, 430 175,180
Lucretius, T. C. 322, 325, Matsuyama,A., see Fuku- :a.fichels,K.M., see Wright,R.
344,349 moto, Y. 249, 256 H. 252,256
Lucy,J.A., see Dingle,J. T. Matthes,E. 1,2,4,5,8, 11, Michelsen, W.J. 442, 447
67,72 13,17,18,21,24,164,179 Mihalkovics, V. von 161,180
LUscher,M., see Karlsson,P. Matthews,R., see Adrian,E. :a.filas,N.A., Postman, W.M.,
352,426 D. 124,128 Heggie,R. 60,62,67,73
Lundberg,P.O. 194,203 Mayer,M.S., 141,149 Wes,W.R., Beck,L.H. 101,
Lundquist,Per-G., see Wer- Mayne,S., see Cheesman,G. 129
sall,J. 58 H. 305,317 Miller,S.D., Erickson,R.P.
Lustig,A. 77, 81,94 Mazzocchi,P.H., see Mein- 469, 481
Lutmer,R., see Rubin,M. wald,J. 391, 427 Millot,J., Anthony,J. 9, 24
298,320 McAdam,D.W., Way,J.S. Wne-Edwards,P. 57
Lysen,M. 422 468,481 Milstein, T. 164, 180
McBurney,D.H., see Engen, Minyard, J. P., see Tumlinson,
MacLeod,P. 107, 129, 264, T. 218, 239, 243 J.H. 390, 391, 400, 404,
319 McCartney, W. 65, 73 430
- Perrin,C.M., Leveteau,J. McCorley,G.C., see Cooper, :a.fiscellaneous 252
106, 115, 129 G.P. 458,460, 462 :a.fisra, T.N., Rosenberg,B.,
- see Leveteau,J. 122, McCotter,R.E. 173,174, Switzer,R. 68, 73
129, 197, 200, 203, 284, 175,179 - see Rosenberg, B. 68, 69,
319, 467, 481 McDaniel,M.R., see Theimer, 70, 73, 334, 349
MacNichol,E.F., see Hart- E. T. 303, 321 Miyahita,D., see McGovern,
line, H. K. 103, 129 McGinty,D., Epstein,A.N., T.P. 390,391,427
!-Iahler,R.F., see Court- Teitelbaum,P. 476, 481 Moncrieff,R.W. 29, 57, 65,
Brown,W.M. 461,463 McGovern,T.P., Beroza,M., 73,153,160,166,180,212,
Maillet,M., see Ardouin,P. Ohinata,K., Miyahita,D., 214, 215, 224, 226, 244,
29, 55, 61, 71 Steiner,L.F. 390, 391, 306, 319, 335, 349, 402,
Mainland,R.C. 247,256 427 427
Malan,M.E. 13,24,164,179 McGowan, B. K., see Garcia, l\foor-Jankowski, J. K., see
Malcomson,K.G. 171, 179 J. 459,463 Huser,H.J. 250, 256
Malewitz, T.D., see Kuhlen- l\fcGurk,D.J., see Vick,K. W. Moore, B. P. 352, 427
beck,H. 163,179 380, 392, 430 Moore, T. 67,73
Author Index 493
Moorhouse,J.E., Yeadon,R., Nachman,M. 461, 464, 471, Ooyama,H., see Oomura, Y.

Beevor,P.S., Nesbitt,B. 481 476,477, 481
F. 427 Nagahara, Y. 76, 77, 83, 87, Osterhammel,P., Terkildsen,
- see Kennedy,J.S. 366, 93,94 K., Zilstorffik 107, 108,
426 Naka,F., see Oomura, Y. 125,130
Morell 211 476, 477, 481 - see Franzen,O. 108,125,
Morgan,T.B., see Khairy,M. Nakajima,H., see Fukumoto, 129
472,476, 480 Y. 249,256 Ostrom,J.H. 16,25
Morgan,W.L., see Kneip,D. Nakajima,M., see Wood,D.L. Ottenberg,P., see Stein,M.
H. 467,480 422, 431 467,482
Morin,F. 194, 203 Nakamura, Y. 76, 81, 94 Ottoson,D. 37, 57, 89, 94,
Morita 414 Nalwalk, T., see Teichner, W. 98, 99, 100, 101, 105, 106,
Morrill,A.D. 28, 31, 57 H. 170,181 107, 109, 112, 113, 114,
Morris,D.D. 456,464 Naves, Y.R. 303,320 115, 116, 117, 118, 121,
Moser,J.e., Brownlee,R.e., Navratil, D. von 161, 180 124, 130, 135, 146, 149,
Silverstein, R 379, 392, Necheles,H., see Ginsberg,R 150, 154, 180, 219, 244,
427 S. 468,480 260, 268, 269, 270, 305,
Moulton,D.G. 29,62,63,64, Negus, V. 18, 20, 25, 29, 57, 307,320
65, 73, 134, 149, 160, 180, 60, 61, 73, 153, 160, 162, - Shepherd,G.M. 98, 102,
199, 203, 207, 208, 215, 180 112, 114, 115, 130, 188,
263, 264, 281, 282, 319, Nemours, P. R 161, 180 203,264,320
320,334,349,479,481 Nesbitt,B.F., see Moorhouse, - Sydow,E.von 119, 130
- Beidler,L.M. 29, 57, J.E. 427 - s. Shepherd,G.M. 120,
114, 130, 264, 320 Neuhaus, W. 368, 378, 427 130
- Eayrs,J. T. 234, 244, 440, 447 Overton, Meyer, Collander
267,320 Neville,RG., see Dean,R.B. 326
- Tucker,D. 258,281,320, 342,349 Ozeki,M., Sato,M. 98,130
342,349 Newell,RR, Borley,W.E.
- see Johnston,J.W. 422, 450, 464
426 Nicola,Massa 27, 57 Pages, E. 199, 203
Mountcastle, V.B. 189, 203 Nicoll,RA. 186,203 Pain,J., Barbier,M., Bog-
Mozell,M.M. 100, 130, 134, Noble,G.K., elausen,H.J. danovsky, D., Lederer,E.
145, 149, 196, 208, 209, 162, 163, 180 390,427
210, 211, 212, 213, 214, - Kumpf,K.F. 163,180 Pain,J.F., Booth,D.A. 471,
215, 218, 240, 242, 244, 472,481
263, 278, 279, 320 O'Connell, RJ., Mozell,M.M., Palade,G.E., see Farquhar,
- Pfaifmann,e. 117,130, 142,143,149,214,215 M.G. 33,56
198, 203, 206, 207, 215, Oifenloch,K., see Franzen,O. Palchetti, R, see Digiesi, V.
281,320 125,129 479,480
- see O'Connell,RJ. 142, Ogle, W. 29,57, 65, 73,349 Palmer,L.S. 60,73
143, 149, 214, 215 Ohinata, K., see McGovern, T. Palmieri, G., see Amoore,J. E.
Miiller,A. 29, 57, 61, 73 P. 390, 391, 427 254, 255, 309, 317, 345,
Muller,B., see Steinbrecht,R Ohloif,G., Becker,J., Schulte- 348, 402, 403, 408, 423
A. 361,430 Elte, K. H. 293, 320 Panchen,A.L. 2, 5, 7, 8, 9,
Muller, W. 366,367,397,427 - see Rienacker, R. 303, 25
- see Altner,H. 38,50,55, 320 Pangborn,R.M., see Berg,H.
154,178 Okano,M., Takagi,S.F. 92, W. 478,479
Mulder,I., see Veerkamp,J. 94 Pareto 422
H. 336,350 - Weber,A.F., Frommes,S. Parker,G.H. 28,37,57,153,
Muller,J. 450 P. 39,57 166, 180, 247, 256
Mullins,L.J. 197, 203, 267, Olson,E.e. 22, 25 Parsons,T.S. 8, 10, 11, 13,
320 Omura,K., see Takagi,S.F. 21,22,23,25,57,153,154,
Munson,RJ., see Hunter,e. 142,150 180
G. 461,464 Onagawa,K. 65,73 - Williams, E. E. 10,22, 25
Murray,RW. 331,349 Oomura, Y., Ooyama,H., Ya- Pascucci,F., see Bassi,M.
Myers,J. 360,362,427 mamoto,T., Naka,F. 479,479
Myers,J.H., Walter,M. 427 476,477, 481 Passy,J. 189, 203, 267, 320
494 Author Index
Paton,N., see Butler,C.G. Powell, T.P.S., Cowan, W.1tI. Read,J.S., Warren,F.L., Hc-
424 190, 192, 203 witt,P.H. 390,428
Patterson, P.M., Lauder, B.A. - - RaisDlan,G. 190, Reese,T.S. 40, 44, 53, 57,
247,249,256 191,193,194,204 101, 105, 112, 113, 115,
Pauling, L. 263, 320 - see Phillips, C. G. 188, 130,263,320
Paulsen,C.A., see Sparkes,R. 203 - BrightDlan,M. W. 185
S. 247,256 Pratt, C. M.,McE. 13,25, - Stevens,S.S. 218,244
Payne, T.L., Shorey,H.H., 163, 164, 180 Regine,F.E., see Wilson,E.
Gaston, L. K. 373, 378, Prelog, V., Ruzicka,L. 296, P. 380,431
427 297,320 Regnier,F.K, Wilson,KO.
Pearson,A.A. 172, 173, 180 - - Metzler, O. 297, 320 380,428
Perrin,C.M., see MacLeod,P. - - Wieland,P. 297, 320 Reid, C. 479, 481
106, 115, 129 Prestage,J.J., see Slifer,E.H. - see Wright,R.H. ';0, 74
F1affDlann,C. 172,180,219, 360, 362, 429 Reinke, W. 5, 25
223, 226, 244 Price,L.M., see Teichner, W. ReDlold, H. 400, 428
- Bare,J.K. 479,481 H. 170,181 Renner,M., see Kaissling,K.-
- see Engen, T. 265, 318 Price, S., see Dastoli, F. R. E. 262, 319, 358, 365,
- see Mozell,M.M. 117, 249,255 372, 388, 403, 404, 408,
130, 198, 203, 206, 207, Priesner,E. 352,357,365, 426
215,281,320 371, 378, 394, 395, 422, Renvick,J.A.A., Vite,J.P.
Petrasovits, A., see Boch, R. 427,428 399,400,428
408,424 - see Boeckh,J. 98,107,
Retzius,G. 28,57,92,94,
F1eiffer, W. 5,8,25 115,128
Revusky,S.H. 461,464
Philippot,K, Gerebtzoff,M. - see Kaissling,K.-E. 355,
Rhodin,J.A.G. 44,57
A. 63,73 357, 365, 366, 375, 384,
Ricci,A., see Buu-Hoi,N.P.
- see Gerebtzoff,M.A. 61, 385, 386, 389, 410, 411,
292,317
63,70,72 412, 418, 426
Phillips,C.G., Powell, T.P.S., - see Schneider,D. 365, Richards,A.G. 363,428
Shepherd,G.M. 188,203 389,429 Richter,S. 428
Riddiford,L.M. 399, 421,
Pierantoni, R. L., see Grazia- Pryor,G., see Stone,H. 248,
dei,P.P.C. 56 256 428
Pierson,A., LeMagnen,J. Pyatnova,B., see Pyatnova, Rideal,KK., see Davies,J. T.
468,481 Y. 352,428 330,337,348
Pillai,R.V., see Anand,B.K. Pyatnova, Y., Pyatnova,B., RieDlschneider,R., Kasang,
476,479 Ivanov,L.L., Kyskina,A. G., BohDle,C. 428
PitDlan,G.B. 400,427 S. 352,428 Rienacker,R., Ohloff,G.
- see Vite, J.P. 390,391, 303,320
430 Quastler,H., Weingartner,H. RiDlbeck, T. 422
Pitts, W.H., see Gesteland,R. 81,94 Riss,W., see Scalia,F. 175,
C. 120, 121, 129, 137, Quint,E., see Scott,E.M. 180
142, 147, 149, 214, 215, 469, 471, 481, 482 Robbins,L., Aub,J., Cope,J.,
265, 271, 279, 318, 336, Cogan,D., Langohr,J.,
341,346, 347, 349 Rad'ko,N.K. 468, 481 Cloud,R., Merrill,O.E.
Planel,H. 161,180,187,203 RaiSDlan, G., see Powell, T. P. 450,464
Pliske,T.E., Eisner,T. 353, S. 190,191,193,194,204 Robertson,P.L., seeCavill,G.
400, 427 RaIl, W., Shepherd, G.M. W.K. 396, 397, 424
Porter,N.A., see D'Arcy,F. 123,130 Robinette,R.R., see Brown,
J. 449,463 Randebrock,R.E. 331, 349 K. S. 250, 255
PortDlann,A. 17,25,433, Ranvier 325 Robson,A., see Wright,R.H.
447 Rapaport,A., see Magnen,J. 311, 312, 321
Posvic,H. 301,320 Le 66,67,73 Rodin,J.O., Silverstein,R.
PostDlan, W.M., see Milas,N. Ratliff,F., see Hartline,H.K. M., Burkholder, W.K,
A. 60, 62, 67, 73 189,202 GorDlan,J.E. 428
Poussel,H., see Gavaudan,P. Read,E.A. 160,164,174, - see Bedard, W. D. 399,
267, 270, 318 180 423
Powell,G.F., see Henkin,R. Read,J., Grubb, W.J. 302, - seeWood,D.L. 400,404,
I. 245, 248, 256 320 431
Author Index 495
RodoHo-Masera, T. 160,161, Sandri,C., see Lamparter,H. Schneider, R. A., Schmidt, C.

180 E. 422,426 E. 171, 180, 200, 201,
Roller,H., Dahm,K.H., Sappey, Ph. C. 27,28,40,57 204
Sweely,C.C., Trost,B.M. Sarasin,F., see Sarasin,P. - WoH,S. 67,73
402,428 H,25 Schnitzlein,H.N. 175, 180
Roelofs,W.L., Arn,H. 390, Sarasin,P., Sarasin,F. H, Schoonhoven, L. M., Dethier,
397,428 25 V.G. 388,429
- Comeau,A. 397,398,428 Sass,H., see Boeckh,J. 372, - see Dethier, V.G., 375,
- Tette,J.P. 397,398,428 422,424 388, 409, 424
Ronsch,G., see Henke,K. Schremmer, F. 352, 368, 429
Sato,M., see Gray,J.A.B.
358,425 102,129 Schuch,K. H,26
Roentgen 449 Schiirmann,F. W., Wechsler,
Roessler,E.B., see Berg,H. - see Ozeki,M. 98,130
Scalia,F. 190, 191, 192, 204 W. 422,429
W. 478,479 Schulte-Elte,K.H., see
Roessler,H.P. 409,428 - Halpern,M., Knapp,H.,
Riss,W. 175,180 Ohloif,G. 293,320
Rojas,A., see Gesteland,R.C. Schultz,E.W. 85,93,94
120, 121, 129, 142, 149, Scarpa,A. 27,57
Schaller,A. 368,428 Schultze,M. 27, 28, 37, 57
265, 271, 318, 336, 346, Schutz,H.G. 237, 244, 252,
347,349 Schanz,M. 387,428
Schevill, W. E., see Lawrence, 256
Roll,D.L., see Smith,J.C. Schutzenberger,M.P., see Ga-
461,464 B. 18,24
vaudan,P. 267,270,318
Romer,A.S., Edinger,T. H, Schiff,M. 76, 94
Schmalhausen, I. I. 2, 9, 11, Schwarz,M., see Jacobson,M.
25 390, 399, 426
Ropartz,P. 199,204 12,25
Schwarz, R. 358, 367, 368,
Rosenberg, B., Misra, T.N., Schmalhausen, 0.1. 5, 8, 25
376, 378, 379, 429
Switzer, R. 68, 69, 70, Schmidt,C.E., see Schneider,
R.A. 171,180,200,201, Schwinck,I. 365,366,429
73,334,349
204 Soott,E.M., Quint,E. 469,
- see Misra, T.N. 68, 73
471,481,482
Roth,L. 353,428 Schmidt,R., see Tiemann,F.
- Verney,E.L. 471, 482
Roulet,N., see Stein,M. 467, 301,321
Seibt, U., see Schneider,D.
482 Schneider,D. 107,130, 135, 400,429
Rovee, C. K., see Engen, T. 141, 150, 272, 273, 274,
Seifert,K. 57
218, 233, 234, 243 304, 321, 353, 357, 369,
- Uie,G. 37,44,47, 57
Roys,C.C. 134,146,150 371, 372, 395, 422, 428,
Sekhon,S.S., see Slifer,E.H.
Rubin,M., Apotheker,D., 429
359, 360, 362, 363, 429,
Lutmer, R. 298, 320 - Block,B.C., Boeckh,J.,
430
- see Amoore,J.E. 348 Priesner, E. 365, 389,
Rudebeck,B. 175,180 429 Semb,G.B. 230,232,233,
Rudinsky,J.A. 399,428 - Kaissling, K.-E. 353,429 234,244
Ruttner,F., Kaissling,K.E. - Kasang, G., Kaissling,K.- Sen Gupta,P. 79,81,94
372,428 E. 321,383,429 Seydel, O. 10, 11, 13, 25
Ruys,A.H., see Klouwen,M. - Lacher, V., Kaissling,K.- 161,175,180
H. 298, 312, 319 E. 276,321,357,365, Shanklin,W.M., see D'An-
Ruzicka,L., Stoll,M., Huy- 371, 388, 395, 405, 406, gelo,D. 71,72
ser,H.W., Boekenoogen, 429 Shantaveerappa, T.R., Bour-
H.A. 298, 320 - Seibt, U. 400, 429 ne,G.H. 186,204
- see Prelog, V. 296, 297, - Steinbrecht,R.A. 360, Shapenko,G., see Gerebtzoif,
320 363, 364, 365, 429 M.A. 29,56
Rzoska,J. 471, 481 - see Boeckh,J. 98, 107, Shearer,D.A., see Boch,R.
115, 128, 135, 140, 141, 404,408,423,424
Saario,C.A., see Shorey,H. 149, 150, 272, 276, 307, Sheffield,L.P., Law,J.H.,
H. 378,429 317, 326, 339, 348, 353, Burghardt,G.M. 164,
Salzman,A., see Wenzel,B.M. 354, 373, 392, 395, 403, 180
445,448 424 Sheinin,J.J. 172,180
Samesreuther,G. 422 Schneider,E.L., see Doolittle, Sheldon,R.E. 166,181
Sandri,C., see Boeckh,J. R.E. 344,349, 390,425 Shenk,H.P., see Godfrey,E.
372, 422, 424 Schneider,R.A. 246,256 W. 450,463
496 Author Index
Shepard,R.N. 198,204, Simpson,J., see Butler,C.G. Stallberg-Stenhagen, S., see

238, 240, 244 424 Bergstrom,G. 404, 423
Shephard,W.C., see Bean,R. Simpson,R.W., see Sparkes, - see KuIlenberg,B. 404,
C. 417,423 R.S. 247,256 426
Sheperd,G.M. 123,130, Sinclair,J.G. 153,181 Stager, K. E. 432, 434, 444,
186,204 Singer,M. 80,94 448
- Ottoson, D. 120, 130 Singh,B.K., Lal,A.B. 302, Stamm,D., see Butenandt,A.
- see Land,L.J. 188,203 321 384, 424
- see Ottoson,D. 98, 102, Sinoir, Y. 429 Stebbins,R.C., 13, 15, 25
112, 114, 115, 130, 188, Skoglund,C.R., see Bern- Stein,M., Ottenberg,P., Rou-
203, 264, 320 hard,C.G. 97,128 let,N. 467, 482
- see Phillips,C.G. 188, Skramlik,E.von 167,170, Steinbrecher, W. 468, 482
203 171, 181, 200, 204, 267, Steinbrecht,R.A. 354, 358,
- see RaIl, W. 123, 130 321, 379 361, 362, 364, 365, 410,
Shibuya, S., see Shibuya, T. Slifer,E.H. 358, 360, 362, 430
142, 150 429 - Kasang,G. 355,384,419
Shibuya, T. 60,73,113,114, - Prestage,J.J., Beams,H. - Muller,B. 361,430
115, 130, 135, 150, 160 W. 360, 362, 429 - see Schneider,D. 360,
- Ai,N., Takagi,S.F. 142, - Sekhon,S.S. 359, 360, 363, 364, 365, 429
150 362,363,429,430
Steiner,G. 366,430
- - Lees,A.D. 362, 363,
- Shibuya,S. 142,150 Steiner,L.F., see McGovern,
430
- Tucker,D. 142,150,161, T.P. 390,391,427
Slosson, E. E. 227, 244
436, 447 Steiner, W. 402,430
Slotwinski,J. 48, 57
- see Takagi,S.F. 99, 109, Stellar,E., see Graff,H. 476,
Smallman,R.L., see Beidler,
110, 111, 115, 130, 131 480
L.M. 87,88,93
- see Tucker,D. 107, 131, Smith,C.G. 79, 80, 81, 85, - see Kahn, M. 468, 480
145, 146, 150, 264, 321, 93, 94, 176, 181 - see Teitelbaum,P. 477,
Shkapenko,G., see Gerebt- 482
Smith,F.E., see Baylor,E.R.
zoff,M.A. 61, 63, 72 450,462 Stenhagen,E., see Bergstrom,
Shorey, H. H., Gaston, L. K. Smith,G.C. 57 G. 404,423
352, 378, 429 Smith, 1. C. 3, 25 Stenhouse,N.S., see Kirk,R.
- - Saario,C.A. 378,429 Smith,J.C., Roll,D.L. 461, L. 249,256
- see BarteIl,R.J. 378,423 464 Stensio,E. 3, 21, 25
- see Payne, T. L. 373,378, - Tucker,D. 456, 464 Stephan,H. 154,181
427 - see Dinc, H. 1. 456, 463 Stevens,C.F. 192,204
Shulov,A., see Ikan,R. 391, - see Henton, W. W. 166, - see Biedenbach, M. A.
404,425 179, 443, 447, 456, 463 193, 199, 201
Shumake,S.A., Smith,J.C., - see Shumake,S.A. 443, Stevens,J.C., Stevens,S.S.
Tucker,D. 443, 447 447 219,244
Sieck,M.H. 438,447 - see Taylor,H.L. 456, Stevens,S.S. 217, 244, 342,
- Wenzel,B.M. 437, 438, 464 349, 416, 430
439,448 Smith,R.D.,Marcarian,H.Q. - see Reese,T.S. 218,244
- see Wenzel, B. M. 448 172, 173, 181 - see Stevens,J.C. 219,
Silcox,L.E., see Godfrey,E. Smolic,N. 479,481 244
W. 450,463 Snider,R.S., see Haley, T.J. Stoll,M., see Ruzicka,L.
Silverstein, R. M., Brownlee, 450 298, 320
R.G., Bellas,T.E., Wood, Snow,D.W. 444,448 Stone, B. C., see Boch, R.
D.L., Browne,L.E. 390, Sokolov, W. 60,73 424
391, 429 Soudek, S. 432, 448 Stone, F., see Goetzl,F.R. 480
- see Bedard, W.D. 399, Sparkes,R.S., Simpson,R. Stone,H. 218,219,221,234,
423 W., Paulsen,C.A. 247, 244
- see Moser,J.C. 379,392, 256 - Bosley,J.J. 235, 236,
427 Spencer,M.M., see Barnett, 244
- seeRodin,J.O. 428 S.A. 468, 479 - Carregal,E.J.A., Willi-
- seeWood,D.L. 400,404, Stallberg-Stenhagen, S., see ams, B. 170, 181
422,431 Andersson, C. O. 404,423 - Pryor, G. 248, 256
Author Index 497
Stone, H., Williams, B., Takagi, S. F., see Higashino, S. Thompson,A.C., see Tumlin-
Carregal,E.J.A. 170, 109,129 son,J.H. 390, 391, 400,
181 - see Okano,M. 92,94 404, 430
Story,R.H. 175,181 - see Shibuya, T. 142, 150 Thompson,H.W. 70,73
Strahan, R. 3, 26 Takata,N. 57, 77, 81, 94 Thomson,K.S. 8,9,26
Stricht, 0., vander 58 Takeda, K. 368, 430 Thornhill,R.A. 42,57
Stroder,J., see Hopf,H.C. Takeuchi, H., see Arvanitaki, Thorpe,W.H. 445,448
170,179 A. 133, 146,148 Thorson, see Kaissling,K.-E.
Strom,L., see Borg,G. 124, Talbert,E.J., see Katz,S.H. 415
125,128 167,179 Thorson, J. 422
Strong, R. M. 432, 448 Tallon 470 - see Beroza,M. 416, 422,
Stuerckow,B. 375,430 Tallon,S., see LeMagnen,J. 423
Stuiver,M. 189, 204, 223, 474, 475, 476, 481 Thurm, U. 358, 369, 370,
244, 266, 302, 305, 321 Tapp,J.T., see Long,C.J. 371, 414, 415, 430
Siisskind, C., see Griffith, P. 471,481 Tiemann,F., Schmidt,R.
H.425 Tapper,D.N., Halpern,B.P. 301,321
Sugano, H., see Ueki, S. 445, 461,464 Tilden,P.E., see Bedard,W.
448 D. 399,423
Taylor,F.H., see Davies,J. T.
Svericenko,P.A. 468,482 - seeWood,D.L. 400,404,
327,329,338,348
Sweely,C.C., see Roller,H. 431
Taylor,H.L., Smith,J.C.,
402,428 Timmermans,J. 345,350
Hatfield,C.A., 456,464
Swets,J.A., see Green,D.M. Titova,L.K., see Vinnikov,J.
- - Wall,A.H., Chaddock, A. 32,58
227,232,243
B. 456, 464 Titow,A., see Vinnikov,J.A.
Switzer,R., see Misra, T.N.
68,73 Technau,G. 17,18,26 61,73
Tedrow,L.F., see Garcia,J. Toba,H.H., see Jacobson,M.
- Bee Rosenberg, B. 68, 69,
454, 455, 458, 460, 461, 390, 399, 426
70, 73, 334, 349
463
Sydow, E. von 124, 125,131, - Green,N., Kishaba,A.N.,
219,244 Teichmann, H. 5, 26, 189, Jacobson,M., Deboldt,J.
204 W. 390,430
- see Drake, B. 115, 124,
129, 271, 318 Teichner, W.H., Price,L.M., Todd,J., see Atema,J. 445,
- Bee Ottoson,D. 119, 130 Nalwalk, T. 170,181 446
Sziitz,A. v. 83, 94 Teitelbaum,P. 476, 482 Tonosaki, A., see Yamamoto,
- Epstein,A.N. 477,482 T. 58
Tai,A., Matsumara,F., Cop- - Stellar,E. 477,482 Torgerson,W.S. 237,244
pel, H. C., 393, 430 - see Epstein,A.N. 476, Tortoli, V., see Digiesi, V.
- see Matsumura,F. 390, 480 479,480
404,427 - Bee McGinty,D. 476,481 Townsend,M.J., see Chees-
Takagi,S.F. 29, 57, 89, 94, Terkildsen, K., see Franzen, man,G.H. 224,242,305,
109, 110, 114, 115, 130 O. 108, 125, 129 317
- Aoki,K.,lino,M., Yajima, - see Osterhammel,P. 107, Toyama, T. 454, 464
T. 92,94 108, 125, 130 Traynham,J.G., Bee Blum,
- Omura,K. 142, 150 Tester,A.L. 4,26 M. S. 392, 402, 403, 423
- Shibuya, T. 99, 109, 110, Tette,J.P., see Roelofs, W.L. Traynier,R.M.M. 366,430
111, 115, 130 397,398,428 Traynlam,J.G., see Bittel,H.
- - Higashino, S., Arai, T. Teuifert, W., see Braun,J. von 423
110,131 301,317 Tribble, M. T., see Bittel, H.
- Wyse,G.A., Kitamura, Theimer,E.T. 289,321 423
H., lto,K. 106,109,110, - Davies,J. T. 288, 293, Troland, L. T. 263, 321
131, 137, 150 312, 321, 337, 338, 339, Trost,B.M., Bee Roller,H.
- - Yajima,T. 109, 110, 344,350 402, 428
131, 137, 150, 324, 349 - McDaniel, M. R. 303, 321 Trujillo-Cenoz, O. 57
- Yajima, T. 29,57,77,89, - see Beets, M. G.J. 296, Truog,G., see Huser,H.J.
91, 92, 94, 110, 111, 131, 309, 310, 317 250,256
264,321 Thibaut,P., Bee Guillot,M. Truscheit,E., see Eiter,K.
- see Aoki, K. 133, 148 302,319 425
HZ fib. BeDlKll"Y Fhy.lology, Vol. IV/1
498 Author Index
Tsypin,A.B., Grigor'yev, Yu. Veerkamp,J.H., Mulder,I., Waters,R.M., see Jacobson,

G. 452,464 vanDeenen,L.L.M. 336, M. 390, 399, 426
- see Grigor'yev, Yu.G. 350 Watson,D.M.S. 16,26
452,463 Veldstra, H. 303, 321 Way,J.S., see McAdam,D.
Tucker,D. 28,38,40,55,57, Venstrom,D., Amoore,J.E. W. 468,481
134, 145, 146, 150, 154, 248, 256 Webb,A.D., see Berg,H.W.
155, 160, 164, 165, 169, - see Amoore,J.E. 242, 478, 479
170, 171, 181, 187, 204, 242, 247, 248, 251, 255, Weber,A.F., see Okano,M.
220, 244, 259, 278, 321, 287, 309, 317, 340, 345, 39,57
435, 436, 448, 479, 482 346, 348 Wechsler, W., see Schurmann,
- Beidler,L.M. 171,181 Vergnes,M., Karli,P. 445, F. W. 422, 429
- Shibuya, T. 107, 131, 448 Weingartner,H., see Quast
145, 146, 150, 264, 321 Verney,E.L., see Scott,E.M. ler,H. 81, 94
- see Beidler,L.M. 134, 471,482 Welker,W.I. 170,181
148, 165, 178, 258, 278, Vick,K.W., Drew,W.A., Ei Wentges,R. T., see Cauna,N.
317,336,346,347,348 senbraun,E.J., McGurk, 174,178
- see Graziadei,P.P.C. 50, D.J. 380, 392, 430 Wenzel,B.M. 438,440,441,
56,160,179 Vinnikov,J.A., Titova,L.K. 444, 448
- see Henton, W. W. 166, 32,58 - Albritton,P.F., Salzman,
179, 443, 447, 456, 463 - Titow,A. 61,73 A., Oberjat, T. E. 445,
- see Mathews,D.P. 142, Vite,J.P., Pitman,G.B. 448
143,149 390, 391, 430 - Salzman, A. 445, 448
- see Moulton,D.G. 258, - see Renvick,J.A.A. 399, - Sieck,M.H. 448
281, 320, 342, 349 400, 428 - see Sieck,M.H. 437,438,
- see Shibuya, T. 142,150, Vowles, S. M. 368, 430 439,448
161, 436, 447 Werner,A., Conrad,H.E.
- see Shumake, S. A. 443, Wagner,H.G., see Hartline, 301,321
447 H.K. 103,129, 189,202 Wersall,J., Flock, A., Lund
- see Smith,J.C. 456, 464 Wald,G., Brown,P.K., Gib quist,PerG. 58
Tumlinson,J.H., Hardee,D. bons,I.R. 330,350 Wesolowski,H. 58
D., Gueldner,R.C., Waldow, V. 352,360,374, Westerman,R.A., see Wilson,
Thompson,A.C., Hedin, 375, 409, 430 J.A.F. 32, 58
P.A., Minyard,J.P. 390, Wall,A.H., see Taylor,H.L. Westmoreland,N., see Calvin,
391, 400, 404, 430 456,464 A.D. 443, 447
Turk,A., see Johnston,J.W. Walsh,R.R. 198,204 Wetzel,N., see Hughes,J.R.
422,426 Walter,M., see Myers,J.H. 285, 319, 332, 349
427 Wharton,D.R.A., see
Ueda,K.,seeHara,T.J. 201, Walter,W.G. 432,440,442, Boeckh,J. 372, 422, 424
202 443,448 Wheeler, W.M. 353,430
Veki,S., Sugano,H. 445, Wanke,E., see Amoore,J.E. Whitaker,L.M. 445,448
448 254, 255, 309, 317, 345, White,L.E. 190,204
Vetake,M., see Fukumoto, Y. 348, 402, 403, 408, 423 White,T.H. 445,448
249, 256 Ward,A., see Harris,L.J. Whitten, W.K. 194, 199,
Vie,G., see Seifert,K. 37, 471,480 204
44,47,57 Warren,F.L., see Read,J.S. Whitteridge,D. 172,173,
390, 428 181
Valega,T.M., Beroza,M. Warter,S.L., see Blum,M.S. Wiedemann,E. 163,181
353,430 392, 402, 403, 423 Wiedersheim,R. 7, 11,26
Valverde,F. 190, 191, 204 Warthen,D., see Jacobson,M. Wieland,P., see Prelog,V.
VanCleave,C.D. 450,464 390, 399, 426 297, 320
Varner,J.E., see Bonner,J. Warwick, R. T., see Allison, A. Wigglesworth, V.B. 402,
70,72 C. 187,197,201,264,316 431
Vareschi,E. 388, 392, 408, - see Clark,W. E. LeGros Wilde, W. S. 163, 181, 187,
409,430 77,81,93 204
- Kaissling,K..E. 368, Waterhouse,D.F., Gilby,A. Wilkes, T.J., see Gillies,M. T.
408,430 R. 404,430 352, 366, 425
Author Index 499
Williams, B., see Stone, H. Wood, D. L., see Silverstein, Yamamoto, T., see
170,181 R.M. 390, 391, 429 Yamamoto,C_ 186, 188,
Williams,C.M., see Calvin,A. Wood, T.F. 288, 289, 294, 204
D. 443,447 295, 296, 321 Yasutake,S. 58
Williams, E. E., see Parsons, Woodrow,H.F., Karpman,B. Yeadon,R., see Moorhouse,J.
T.S. 10,22,25 223, 244 E. 427
Williams,N.H., see Dodson, Woskow,H.M. 237, 238, Yinon, U., see Ikan,R. 391,
C.H. 391,399,424 242,244 404, 425
Wilson,E.O. 352,431 Wright, R. H. 62, 70, 74, Yorke,C.H., see Garcia,J.
- see Bossert, W.H. 366, 311, 312, 321, 331, 332, 47l,480
424 333, 342, 343, 344, 345, Yoshida,H. 89,94
- see Regnier,F.E. 380, 350, 352, 366, 390, 431 - see Hosaya, Y. 89, 93,
428 - Hughes,J.R., Hendrix,D. 99, 129, 135, 149
E. 332, 333, 350 Yoshida, T., see Fukumoto,
Wilson,E.P., Bossert,W.H.,
- Michels, K. M. 252, 256 Y. 249,256
Reginer,F.E. 380,431
- Reid,C., Evans,H.G.V. Yost,M. T., see Dethier, V.G.
Wilson,J.A.F., Westerman,
70,74 117, 128, 266, 267, 270,
R.A. 32,58
- Robson,A. 311,312,321 318
Windle,W.F. 172,181 - see Demerdache,A. 331, Young,M.W. 191,204
Winston,H., Lindzey,G., 349 Young,P. T., see Kneip,D.H.
Conner,J. 65, 73 - see Kellogg, F. E. 366, 467, 480
Witkam,W.G.M. 176,181 426 Yudkin,J., see Khairy,M.
Wolbach,S.B. 66,73 Wyse,G.A., see Takagi,S.F. 472,476,480
- Bessey,O.A. 66,67, 73 106, 109, 110, 131, 137,
- Howe,P.R. 66,73 150, 324, 349 Zilstorff,K., see Franzen,O.
Wolbarsht,M.L. 370,431 108, 125, 129
Wolf,G., Lawrence,H.G. 65, Yajima,T., see Takagi,S.F. - see Osterhammel,P. 107,
73 29, 57, 77, 89, 91, 92, 94, 108, 125, 130
Wolf,S., see Schneider,R.A. 109, 110, Ill, 131, 137, Zilstorff-Pedersen,K. 478,
67,73 150,264,321,324,349 482
Wolff,D. 64, 74 Yamada,M. 141, 150, 422, Zorzoli, G. C., see De Amicis,
Wood,D.L., Brownlee,L.E., 431 E. 48,56
Bedard, W.D., Tilden,P. Yamamoto, C., Yamamoto, Zotterman, Y., see Borg,G.
E., Silverstein,R.M., Ro- T.,Iwama,K.K. 186, 124, 125, 128
din,J. O. 400, 404, 431 188,204 Zuckerkandl,E. 161,174,
- Silverstein,R.M., Naka- Yamamoto,T., Tonosaki,A., 181
jima,M. 422,431 Kurosawa, T. 58 Zwaardemaker, H. 189, 204,
- see Bedard,W.D. 399, - seeOomura,Y. 476,477, 219, 252, 256, 305, 321,
423 481 379
Subject Index
Ablation (see oHactory bulb) Alcohols

Absorption of chemicals by the nasal epi- 236,238,247,249,252,259,268,269,271,
thelium 212 273-279, 281-285, 288, 289, 296, 299,
Acantlwmyops (see ant) 301-303, 306, 307, 312, 313, 327, 329,
Acceptor (insect) 393, 394, 399, 401, 402, 331-334, 341, 345, 355, 366, 371, 375,
403,405,410,412,417,418 379, 380, 382, 384-386, 389, 392, 393,
Accessory nasal sacs 6, 7 395, 400, 403--406, 408, 411, 413, 417,
- oHactory bulb 10, 22, 153, 154, 161, 164, 421, 437, 468 (see also specific alcohols)
174-176, 187 aldehydes 66-68, 117, 124, 166, 169, 211,
- - nerve (see vomeronasal nerve) 238, 248, 252, 254, 268-271, 274-278,
- - organs 3, 10 284,288,290,298,303,307,309,312,332,
- sinuses 20 375,377,382,396,399,402,406,408,418
Acetal 63 (see also specific aldehydes)
Acetaldehyde 268 Alkanals 269
Acetates 69,89,91,117, 146, 155, 158, 159, Alkanes 313
165-167, 169-171, 206, 207, 211, 240, -, C-I0, -12, -13 380
253, 275, 278, 281-283, 304, 306, 340, Alkanols 267, 269, 270, 278, 281, 306, 313
344,390,391,396,398,399,400,402--404, 2-Alkanones 283
406--408, 435, 437, 438, 441, 442, 443, Alkenoic acids 392
470, 471, see also individual acetates. Allicin 252
Acetic acid 157,236,254,276,367,476,477 Allyl alcohol 167, 268
- anhydride 166 - isothiocyanate 252
Acetone 268,281 ex-Allyl phenyl acetic acid 303
Acetophenone 283, 284, 338 Almond odor 290
1O-Acetoxy-hexadecen-7-cis-l-01 304 Amber 287, 296, 312, 313
Acrylic acid 377 Ambergris 309,347
Actinopterygii 21 Ambrette 311
Acyl indanes 296 AmbysWma 12
- tetralines 296 Amines 140, 141, 272, 405
Adamantane 252
Amino acids 141,409
Adaptation, central 69
6-Amino caproic acid 277,400
-, cross-adaptation 224-226, 304-306
-, effect of duration 104, 220--222 Ammonia 69,156,165,167,272,326,404
-, - of intensity 219 Ammonium hydroxide 248
-, insect 377-378, 417 n-Amphetaroine 473
-,odor 219,286,304-308 Amphibia, Jacobson's organ 153
-, selective 224 -, Labyrinthodonta
Adipic acid 400 -, -, nasal cavity 10
Adsorption isotherm 377,413 -, Lepospondyli
Aggregating pheromones 391 -, -, nasal cavity 10, 11
Agnatha, nasal anatomy 2 -, nasal anatomy 10--13
-, nasal evolution 21 -, - cavities 11, 12
Air stream velocity 356 -, - structure evolution 21, 22
Alarm pheromone 392,396,397,402,408 -, nervus terminalis 73
Albatross 433, 438, 442 -, olfactory pigments, carotenoids 60
Albinism (see olfactory pigment) -, - system 435
Alcohols 62, 63, 65, 71, 100, 106, 117, 118, -, secretions supporting cells of oHactory
122, 124, 140, 141, 146, 165-167, 169, mucosa 44
170, 207, 211, 220, 223-226, 233, 234, -, tracking behavior 163
Subject Index 501
AmphiOXUB, nasal structure evolution 21 Aplysia, photoconduction 69

AmphiBbaeniidae, nasal cavities 15 Apomorphine 479
Amylacetate 89,91,117,146,155,158,159, Approach flight 365
165, 166, 169-171, 206, 207, 211, 240, Argon 458
278,282,306, 435--438, 441--443, 471 Asafetida 160, 165, 166, 168, 169
-, iso-Amyl acetata 167, 396, 406-408 Autonomic efferents 171, 177
- alcohol 271
-, tert-Amyl alcohol 283
Basal bodies (see cilia)
iso-Amyl salicylate 406
- cells 28, 44, 55
Androstadienone 253
Androstan-3-o1 341 Bat (Chiroptera)
Androstane derivatives 288 -, Jacobson's organ, absence 176
5cx-Androstene-16-o1-3cx 312 Bees, alarm pheromones 396
Androstenolxylol 252 -, antennae adsorption 382, 384
,118-Androstenone-3 310 -, cas and humidity receptors 409
Anemotaxis 365, 366 -, EAG 139, 140, 275
Anise 168 -, female pheromones 372
Anisole 238,252,281-284,332 -, hair modification 360
Anosmia, inheritability 245,249-251,347 -, masking of odors 399
-, odor class 255, 286, 296, 308, 310, 347 -, odor discrimination 405
-, origin 248, 249, 309 -, - generalist cells 272
-, sweaty odor 251-254 -, pore cavities and tubule 364
-, test methods 247, 248 -, sensillum surface 370
-, types 246--248,252,308,309,346,347 -, slow potential 373
Ant 404 -, bumble bee (Bombus)
-, Acanthomyops claviger -, -, complex odor mixtures 404
-, - -, alarm pheromones 380 -, -, compounds, behavior response 391
-, - -, alarm response 397,404 -, honey bee (A pis melli/era)
-, Atta texana -, -, absolute odor 367
-, - -, alarm pheromones 379, 380 -, -, adaptation and recovery 378
-, - -, alarm response 392 -, -, adsorption quotient 384
-, - -, complex odor mixtures 404 -, -, antennae 354
-, Dolichoderinae -, -, behavior threshold 378, 379
-, -, alarm pheromones 396 -, -, CO. receptor 375, 379
-, Formicinae -,-,deutocerebrum 422
-, -, alarm pheromones 396 -,-,EAG 275
-,Iridomyrmex pruinosus -, -, filter coefficient 356
-, -, alarm releasing 402-404 -, -, generalist cells 388
-, Lasius -, -, humidity receptor response 374
-, -, masking substances 400, 402 -, -, Nassanoif gland 404
-, M yrmicinae 396 -, -, odor discrimination 358
-, Pogonomyrmex -, -, - distinction 408, 409
-, - alarm pheromones 380 -, -, oHactory and sensory threshold 368
Antenna, electrical recording 369, 373 (see -, -, - conditioning 367, 368
EAG) -, -, - sensilla morphology 365
-, filter coefficient 355-358,360,381-387 -, -, - threshold 379, 382
-, odor filter 354-358, 381 -, -, pore plate 363, 388
-, structure 353-358 -, -, receptor potential 376, 379
Anterior commiBBure 190, 191 -, -, response of single receptor cells 392
-, -, sensilla and sensitivity 387
Anterior oHactory nucleus 191,435
-, -, sensitivity 352, 379
Antheraea pernyi (see moth) -, -, specialized-generalist cells 388
Anura, nasal anatomy 10--13 -, -, - receptor cells 262
Aphids, plate organs 363 -, -, specificity 406--409
Aphrodisiacs 353 -, -, threshold of queen substance 384
Aplysia (Sea hase) -, orchid bees (Euglossinae)
-, effects of odors on other tiBBUes 146 -, -, odor masking 399
502 Subject Index
Beetles, complex odor mixture 404 Birds, odor reflexes 440, 442
-,EAG 139 -, olfactory bulb 434, 435
-, hair modification 360 -, - perception 439
-, pore cavity and tubule 364 -, - pigments 60, 61
-, receptor response 140 - , - receptors 434
-, bark beetle (Dendroctonu8) -, secretions from supporting cell 432
-, -, masking substances 399,400 -, Albatross
-, -, sex attractants 390, 391 -, -, importance of olfaction 435
-, carrion beetle (Necrophorus) -, -, nasal structures 18
- , - , antennae 354 -, -, olfactory bulb recording 437,438
-, -, basiconic sensilla recording 369 -, -, - pigments 61
-, -, carrion receptors 404 -, -, reaction to olfactory stimulus 442
-, -, EAG, response frequency 273 -, Canary (Serinu8 canaria)
-, -, hyperpolarization of receptor poten- -, -, olfactory bulb size 434
tial 405 -, chicken (Gallus gallu8)
-, -, inhibition of carrion excitation 374 -, -, olfactory bulb recording 437
-, -, neurotubules 358 -, -, - nerve responses 436
-, -, olfactory sensilla morphology 365 -, -, reaction to olfactory stimulus 442
-, -, receptor potential 369 -, coot
-, -, sensillum basiconicum 361, 370 -, -, anatomy of olfactory bulb 434
-, -, time course of carrion stimulation 373 -, -, olfactory bulb recording 437
-, kapra beetle (Trogoderma granarium) -, crow
-, -, odor mixtures 404 -, -, anatomy of olfactory bulb 434
-, potato bettle (Leptinotar8a) -,duck
-, -, sensilla and sensitivity 387 -, -, Mallard (Ana8 platyrhyncho8)
-, water beetle (Dyti8CUS) -, -, -, olfactory bulb recording 438
-, -, generator potentials 97,98 -, -, -, reactions to stimulus 442
Benzaldehyde 66, 124, 169, 211, 238, 27l, -, -, muscovy (Cairuna m08chata)
290, 298, 307, 309, 312, 332, 406 -, -, -, action potential 435
Benzene 168, 252, 281, 331, 337 -, -, -, olfactory nerve response 436
iso-Butyl benzoate 406, 407 -, goose
Benzol 168 -, -, grey lag (An8er anser)
Benzonitrile 27l, 279, 290, 305 -, -, -, action potential 435
Benzophenone 283 -, -, -, odor in food locating 444, 446
-, -, -, olfaction pattern of respiration
Benzothiazole 281,282
440
Benzyl acetate 301, 399, 406, 470
-, -, gruiformes
- alcohol 252
-, -, -, olfactory bulb size 435
- amine 154, 155, 165, 166, 169, 170, 404
-, -, laysan
Benzylmercaptan 272, 404 -, -, -, olfactory epithelium 433
Benzyl salicylate 252, 309 -, hawk (sparrow)
Bicyclic and monocyclic keto musks 296 -, -, olfactory nerve responses 436
Bilateral stimulation 184, 200, 201 -, indicator (Indicator indicator) (I. minor)
Birds (Aves), anatomy 433, 435 -, -, odor in food locating 442
-, anatomy of nasal structure 17,18 -, kiwi (Apteryx aU8trali8)
-, bulb recording 437,439 -, -, external nares 433
-, buccal cavity 434 -, -, importance of olfaction 435
-, electrophysiology 435, 436 -, -, nasal structures 18
-,EOG 436 -, -, olfactory bulb size 434
-, existence and function of olfactory system
-, -, olfacto-habenular tract 435
432
-, feeding 443, 444 -, -, reaction to olfactory stimulus 442
-, learned responses 442, 443 -, loon
-, nasal cavity 433 -, -, olfactory bulb anatomy 434
-, - structure evolution 22 -, oil bird (Steatorni8 caripensi8)
-, odor in food finding 443, 444 -, -, odors in food locating 444
-, - perception 435, 438 -, -, olfactory epithelium 433
Subject Index 503
Birds, oil, olfactory pigments, carotenoids 6 Blast-injection 168, 478

-, parakeet Blowfly (Oalliphora)
-, -, anatomy of olfactory bulb 434 -, carrion receptor 404
-, parrot -, receptor responses 140
-, -, reaction to olfactory stimulus 442 -, stimulating effectiveness of alcohols 269
-, passiformes Bollweevil (A nthonomus grandis)
-, -, olfactory bulb size 435 -, complex odor mixture 404
-, pelicaniformes -, sex attractant 390, 391
-, -, olfactory bulb size 435 Bombykol (hexadecadiene-lO-trans-cis-12-1-
-, piciformes 01) 141,273-275,355,366,375,382,384,
-, -, olfactory bulb size 435 385, 386, 389, 393, 395, 410, 411, 413,
-, pigeon (Oolumba livia) 417--421
-, -, learned response to olfactory stimulus - receptor 385,411,417,418
442,443,446 Bombyx (silk worm moth)
-, -, normal adaptive behavior 445 -, adsorption isotherm 412--414
-, -, olfactory bulb ablation 445 -, - quotient 384
-, -, - bulb recording 437, 438 -, antenna size, flying speed 357
-, -, respiration relation to odor 440,441 -, behavioral responses 365, 386, 389
-, -, threshold behavior 166 -, EAG 141, 272, 275, 372, 375, 377, 385,
-, podicipediformes 388, 389, 395, 421
-, -, olfactory bulb size 434 -, filter coefficient 356, 360
-, procellarieformes -, fluttering responses 366
-, -, olfactory bulb size 435 -, fractionated elution of H3 bombykol
-, psittaciformes 419, 420
-, -, olfactory bulb size 435 -, molecular adsorption by antennae 355
-, quail (bobwhite) (Oolinus virginianus) -, olfactory sensilla morphology 363
-, -, learned response to odor 443 -, - threshold 366, 382, 383
-, raven (Oorvus corax) -, pheromone receptors 395, 418
-, -, reaction to olfactory stimulus 442 -, pore system 363
-, robin (Turdus migratorus) -, receptor potential 369, 416
-, -, nasal cavity 17 -, sensory transduction 410, 411, 420, 421
-, shearwater (Putfinus putfinus opisthome- -, specialized sensitivity 357
las) -, threshold of single receptor cells 383
-, -, nature of reaction to stimulus 442 iso-Borneol 302
-, -, olfactory bulb recording 437, 438 Bowman's glands
-, snow petrel (Pogodroma nivea) -, anatomy 31,44,47,48, 174, 175
-, -, anatomy of olfactory bulb 434 -, location 10, 27, 28, 30, 76
-, sparrow (Passer domesticus) -, pigment 29, 61, 64, 66, 70, 263
-, -, action potential 435 -, secretion 47,48, 177
-, -, anatomy of olfactory bulb 434 -, vomeronasal organ 160,161
-, vulture Brevicomin 390-392, 399
-, -, nasal structures 18 2-Bromooctane 403
-, -, olfactory mucosa 46 Bromstyrol (w-phenyl ethyl bromide) 368,
-, -, olfactory pigments, carotenoids 60, 408,409
61 n-Butanol 63, 100, 106, 117, 122, 124,211,
-, -, black (Ooragyps atratus) 232-234, 238, 254, 268, 269, 271, 285,
-, -, -, action potential 435 329,420
-, -, olfactory nerve responses 436 -, iso-Butanol 268, 269
-, - -turkey (Oathartes (Rhinogryphus) -, sec-Butanol 268, 269
aura) -, tert-Butanol 284
-, -, - , action potential 435 Butanone 282
-, -, importance of olfaction 435 Butterfly, Florida Queen (Danaus gilippus
-, -, odor in prey detection 432, 444, 446 berenice)
-, -, olfactory epithelium 433 -, -, EAG to male aphrodisiac 372, 373
-, -, reaction to odors stimuli 442 -, -, masking odors 400
Bitter almond 287,297,298,305 -, -, sensillum basiconicum 362
Blastema cell 86-88, 93 - (Vanessa) antennae 354
504 Subject Index
n-Butyl acetate 211, 283, 402, 403 Chiroptera (see Bat)

-, sec-Butyl acetate 442 Chloroform 92, 109, 115, 372, 419
test-Butyl alcohol 207,268,269,281,282 Choana (see Nasal cavity)
iso-Butyl benzoate 406 Cholesterol 340
tert-Butyl benzene 298 - acetate 340
- carbinol 252 - formate 340
- cyclohexane 290 ChoriBtoneura rosaceana (see Moth)
n-Butyl ether 207,281,282 iso-Chromane derivative 292
- mercaptan 249 Chromoproteins (see Olfactory pigment)
Butyl-aldehyde 254 Cilia, basal bodies 32, 40, 41
Butylamine 404 -, lack on vomeronasal receptors 38-59,
Butylmethyl ether 282 160
iso-Butyraldehyde 252 -, mobility 28, 40, 53
n-Butyric acid 157, 166, 168, 169,238,254, -, necessity for olfaction 38, 39, 264
271,272,367,377,379,404 -, number per receptor 40
iso-Butyric acid 251,253, 254, 347 -, transduction 112, 113, 281
-, transport 164
Calliphora (see blowfly) -, ultrastructure 27,37,39,263
Camphor 238,271,283,284,285,287, 305, Cineole 252, 281, 282, 391, 399
345 Cinnamaldehyde 252,271,275,406
Caproic acid 175-177,253,254,356, 376, Cinnamyl alcohol 406
377,379,382,384,392,400,403,404,408 Citral 210, 238, 279, 282-284, 332, 379,
Caprylic acid 254 380,396,404,407,469,470,472,474
Carbon dioxide 69, 141,352,366,368,375, Citronellol 170, 282, 301, 380, 396, 399
388,409 p-Citronellol 107, 303, 380, 404
- disulfide 271 Civetone 297
- tetrachloride-trichloro ethane 283 Cloves 168
Carboxylic acid 251 f'A)caine 97-99, 114
,13-Carene 400 Cockroach, 139
fJ-Carotene 66-69, 71, 334 -, effects of odors on other tissue 146
Carotenoids 59, 60-63, 66-70, 333, 334 -, on-off responses 141
Carrion 370, 373, 374, 404 -, recording from neurons of deutocerebrum
Carvone 211, 303 422
Cat (Feli8), albinism linked to anosmia 64 -, whole nerve or filament recording 134
-, blastema cell 88 Coffee 165, 166, 169
-, ethmoidal nerve recording 165, 176 Colchicine 80, 92
-, ethmoturbinal region 171 Common chemical sense 153,166,247
-, Jacobson's organ 154 Concha 10, 15-17,22,433
-, kittens, innate feeding 468 Conductance 410, 414-416
-, mesencephalic nucleus 172 Coreoid bugs, complex odor mixtures 404
-, muscle spindles 172 Cortical potentials 125
-, nasal cavity 20 Coumarin 271,282,283,443
-, - innervation 164 Crabs, arousal by X-rays 450
-, nervus terminalis 173,174 meta-Cresyl methyl ether 406
-, olfactory bulb recording 457 para-Cresyl methyl ether 406
-, - epithelium 86 Crocodilia, nasal cavities 16
-, - system 183 -, - structure evolution 22
-, pigments in olfactory epithelium 63 Crossopterygii, nasal anatomy 8-10
Caterpillars, CO 2 and humidity receptors 409 Crustacea, nasal cavities 3
CatostomU8 catostomus (longnose sucker) (see - , - structure 4
Fish) -, positive phototropism 449
Cattle, Jacobson's organ 162 -, stretch receptor 98
-, olfactory pigments, carotenoids, vitamin A Cue-lure 390
62,63,71,333 Cumin 298
Cavum nasi proprium 10-13, 15-18, 20 Cyanopsin 59
Cedarwood oil 281 Cycloheptanone 275
Cedryl acetate 253 Cyclohexanol 271
Subject Index 505
Cyclopentadecanone 282 3-cis, 6-cis, 8-trans-Dodeca-triene-1-o1 390,

Cyclopentanol 306 393, 404
Cyclopentanone 282, 306 Dodecenyl esters 390, 391
Cyclophosphoamide 472 -, cis-5-Dodecenyl acetate 390, 391
Cyclostomata, nasal cavities 3 -, cis-6-Dodecenyl acetate 390, 391
-, - structure 2 -, cis-7-Dodecenyl acetate 390,391
-, - - evolution 21 -, cis-9-Dodecenyl acetate 390, 391
-, olfactory pigments, carotenoids 60 -, trans-7 -Dodecenyl acetate 390
Cyclotene 252 -, cis-7-Dodecenyl butyrate 390
Cystic fibrosis 245 -, - propionate 390
Dodecyl acetate 398, 400, 404
Dog, blastema cell 88
cis-Decalin 290
Decanoic acid 157 -, degeneration of olfactory nerves 76
-, interruption of nasal trigeminal innerva-
Deet (N,N,-Diethyl-m-toluamide) 399, 409
tion 168
Defense agents 353, 397
-, Jacobson's organ 162
3-Dehydro-retinol 62
-, mesencephalic nucleus cell types 172
Dendrite model 416, 417
-, nasal innervation 164
Dendritic potentials 99
-, nervus terminalis 173
Dendroctonus brevicomis (see bark beetle)
-, olfactory and trigeminal conditioned
- frontalis (see bark beetle)
reflexes 168
- pseudotsugae (see bark beetle)
-, - bulb recording 457
Detection of odors 226-236
-, - mucosa cilia, variations in 39, 40
-, detection theory 227-232
-, - organ 265
-, individual differences 234, 235
-, - pigments 333
-, odor concentration 232-234
-, - thresholds 117
-, odor thresholds 189, 227
-, pigments in olfactory epithelium 62,63,
Deutocerebrum 421,422
Diastereomers 301 71
-, sensitivity compared with moth 352
p-Dichlorobenzene 252
-, stimulation of olfactory nerves 326
Dichloroethane 282
-, testing "odorless" compounds 344
Diethylamino ethanol 279
-, threshold concentration 379
Diethyl ether 342
Dolichoderinae (see Ant)
- phthalate 232
Drosophila (fruit fly), adsorption of antennae
- sulfide 313
356
N,N-Diethyl-m-toluamide 399,409
-, positive anemotaxis 366
Diffusion 41, 354, 410, 412, 419
Dihydrobombykol 417,418,420,421
2,3-Dihydrofarnesol 404 Effective fraction of adsorption 355, 356
2,3-Dihydro-7 -methyl pyrrolizinone 391, Efficiency of the antenna 356
392 Electroantennagram (EAG) 135, 141, 272,
Diisopropyl ether 420 274, 275, 371-373, 375, 377, 388, 389,
394, 395, 417, 421
trans-3, 7-Dimethyldeca-2,6-dien-l,10-diol
Electrodes permanently implanted in bulb
400
281
Dimethyl disulfide 396
Electromyography 169
Dimethyl disulfide acetic acid 253 Electro-olfactogram (EOG) 95-128, 135 to
6,6-Dimethyl-3-heptanone 402, 403 139, 282, 283
2,6-Dimethyl-5-hepten-l-01 380 -, and bulb activity 121-123
2,4-Dimethyl-3-hexanone 392,393 -, and neural activity 120, 121, 282
- -trans-hexahydrophthalates 301 -, chemical correlate 268-272, 283-285
- octanols 301 -, cilia 105, 112
- phenyl carbinol 289 -, concentration dependence 100, 101, 103
- trisulfide 396 -, discovery of 98, 99
Dinosaurs, nasal anatomy 16 -, in birds 436
Dipeptides 141 -, in receptor degeneration 89, 90, 92
Diphenols 409 -, latent period 101, 102
Dipnoi (lungfish) 8, 21 -, negative 92
506 Subject Index
Electro-olfactogram, odor type 116-119, Fenchone 332

146 Fish, coding of odors 147
-, olfactory transduction 112 -, degeneration of olfactory cell 81
-, "on" and "off" responses 109-111, -,EOG 107
137-139 -, nervus terminalis 173
-, origin of 111-116, 137 -, olfactory arousal 201
-, oscillations 108, 109 -, - bulb 195
-, peak amplitudes 271 -, - - ablation 445
-, plasic response 102, 103 -, - microvilli 32
-, positive 92 -, - pigments 60
-, psychophysical 124-126 -, secretions from supporting cells 44
--, relation to EAG 141 -, vomeronasal nerve 177
-, species dependence 106-108 -, Actinopterygii, nasal structure evolution
-, use in chemotography 119 21
-, vomeronasal organ 154 -, -, - anatomy 5
Eledone (mollusc), generator potentials -, (Astroscopus), nasal anatomy 2,7
96-98,110 -, bIennie (Zoarces), nasal anatomy 7
-, on-off response 110 -, burbot, olfactory bulb 196
Enantiomers 286, 301, 302 -, carp, olfactory pigments, carotenoids 60
Endoturbinalia 19, 20 -, cartilaginous, nasal anatomy 4
Enzymes 105,260,334,420 -, (Channa argus), olfactory pigments,
Epithelium (see olfactory epithelium) carotenoids 60
Esters 124,211,232,252,309,313,340,379, -, Chondrichthyes (Raja), nasal anatomy 4
380,406,407,444,459 -, -, nasal structure evolution 21
Ethane 326 -, dogfish, nasal anatomy 4
- thiol 313 -, -, subjective and reflex responses 166
Ethanol 303,327,329,334,468 -, drum, freshwater (A plodinotus grunniens)
Ether 165, 326, 336, 372 nasal anatomy 6
-, sec-n-octyl 403 -, ectostereorchachis, nasal cavity 9
Ethmoid 6,7 -, Esox, nasal anatomy 7
Ethmoidal nerve 153, 164, 165, 169, 172 -, EU8lhenopteron, nasal anatomy 9
Ethmoturbinals 16, 18-20 -, Gnathostomata, nasal structure evolution
2-Ethoxyethyl acetate 402, 403 21
Ethyl esters 313 -, hagfish, nasal cavity 2, 3
-, acetate 281 -, -, nasal structure 2, 4
-, butyrate 459 -, longnose sucker, nasal anatomy 7
-, caprate 379 -, lungfish, African (Protopteru8)
-, caproate 379 accessory olfactory bulb 175
-, caprylate 379 -, minnow, olfactory receptors, cilia 38
-, mercaptan 444 -, mudfish, olfactory pigments, carotenoids
--, pelargonate 379 60
-, undecylate 379 -, osteostracens, nasal structure 4
Eucalyptol 332, 469, 470 -, -, - cavities 3
Eugenol 281, 332, 379, 400, 406 -, paddlefish (Polyodon) , accessory olfactory
Exaltolid (see pentadecanolide) bulb 175
Exo-brevicomin 391, 392, 399 -, Polypterus, nasal anatomy 7,9
External nares 5, 6, 8, 10-13, 16-18, 21 -, -, - structure evolution 21
- olfactory peduncle 190 -, salmon, carotenoids 63
-, -, olfactory arousal 201
Factor analysis 237 -, -, - selectivity and sensitivity 183
False alarm 230 -, Sarcopterygii, nasal anatomy 8
Familial dysautonomia 247 -, shark, behavior approach to prey 201
Far infrared spectra 311, 312 -, -, nervus terminalis 173
Farnesol 252, 309 -, stickleback (Gasteristeus)
Fatty acids 63, 140, 157, 213, 267, 272, nasal anatomy 6, 7
274--276, 278, 279, 309, 404, 408, 409 -, teleost, nasal anatomy 5-8
- aldehyde 275 Flea (water), phototropism 450
Subject Index 507
Flowrate and response 220 Gallocyanin 91

Fly 139,380 Garlic 65
-, hyperpolarization 405 Gasserian ganglion 177
-, olfactory pits 359 Gasterosteus (see stickleback)
-, threshold concentration 381 Gene frequencies 250
-, fruit fly (see Drosophila) -, mapping 251
-, mediterranean fruit fly (Oertatitis capi- Generator potential (see Receptor potential
tata) and also EOG)
sex attractants 390 Genetic polymorphism 250
-, melon fly (Dacus) Genetics 249-251
sex attractants 390 Geranial 210, 252, 271, 279, 282, 283
Formaldehyde 166, 248, 288 Geraniol 284,307,371,404,405,408
Formic acid 157,253,254,276,353,367,396 Geranoic acid 404
Formicillae (see Ant) Geranyl-acetate 404
Freesia flowers 250 Glomerular layer 185
Frequency of smelling 233 Glomerular potential 197
Frog, basal bodies related to cilia 40 Glomerular response 122
-, Bowman's glands 48 Glomeruli 197,264,265,286,422
-, degeneration of olfactory cells 81 Glucose 472,476
-, - of olfactory nerves 76, 77, III Glutaraldehyde 88
-, effects of odors on other tissues 147 Grasshopper (Locusta), EAG 139
-,embryo 81 -, green odor receptor 400, 401, 403, 404
-, EOG 89, 91, 92, 106-108, lll, ll5, -, humidity receptor 374
ll8, 120, 121, 124, 136, 138 -, olfactory sensilla, morphology 365
-, extracellular action potentials 145 -, - sensilla, type 359
-, - recording 134 -, - threshold 382, 383
-, latent period 101, 102 -, stimulus-response of olfactory receptors
-, membrane recording 270 377
-, nasal anatomy 10-13 Gruiformes (see Birds)
-, - cavities 12, 13 Guaiacol 238
-, odor properties and response 145 Guinea pig, interruption of nasal trigeminal
-, olfactory bulb 175 innervation 168
-, - cilia 40, 49, ll3 -, Jacobson's organ 162
-, - mucosa dendrites 37, 38 -, main and accessory olfactory bulb 176
-, - nerve specificity 336 -, olfactory system 183
-, septal olfactory organ 161
-, - neurons 54, 55
-, vomeronasal nerve 174
-, - pigments 60 Gyptol 390
-, - receptor, ultrastructure 34
-, - vesicle, basal bodies 32, 33 Habenula 435
-, on-off response 109 Hamster, main and accessory olfactory bulb
176
-, oscillatory potentials 108
Heart rate 440,442,443
-, polyphasic response 109 Hedgehog, olfactory brain 183
-, receptor membrane 271 -, - bulb (stimulation) 198
-, recording from nerve fibers 278 -, recording from olfactory bulb 280
-, - - olfactory bulb 282 Hedonic dimension 239
-, relative activity of two mucosal regions Heliotropine odor 297
209 Heptadecanoic acid 417
-, single axon response 144 Heptaldehyde 2ll
-, slow olfactory potential 99 Heptane 206, 380
-, stimulation of olfactory bulb 198 Heptanoic acid 157, 272, 276
n-Heptanol 403
-, supporting cells 47 2-Heptanol 403
-, whole nerve or filament recording 134 Heptanone 392, 403
-, zinc sulphate on olfactory epithelium 85 2-Heptanone 236, 253, 392, 396, 402-404
Frontalin 391, 392, 399 3-Heptanone 2ll,403
Furfural 2ll 4-Heptanone 403
508 Subject Index
6-methyl-5-heptenone 403 Human, hunger and satiety effect on oHactory

n-Heptyl alcohol 236 threshold 479
Heterostraci, nasal cavities 3 -, individual differences in detection 234
-, nasal structure 4 -, infant electromyogram 170
-, - - evolution 21 -, inhaled vapor effects 167, 168
lO-trans, 12-cis-Hexadecadien-l-01 273, 384 -, interference of oHaction in reproductive
-, lO-cis-12cis 273 mechanisms 199
-, lO-cis-12trans 273 -, irradiation illness 461
-, lO-trans-12trans 273 -, mechanisms to assess side of unilateral
n-Hexadecalactone 391 stimulation 200
Hexadecanol 331, 404, 417 -, menstruation and olfactory threshold 478
-, multidimensional analysis of odors 241,
Hexadecanolide 293
242
Hexadecenol 404
-, nasal anatomy 21
lO-trans-Hexadecen-l-01 417
-, odors and blood glucose 479
cis-7-Hexadecen-l-01 acetate 399 -, olfactory abnormalities 245, 247
2,4-Hexadienal -, - bulb recordings 280
Hexadienoic acid 277 -, - cilia 263
Hexa-fluro derivatives 289 -, - thresholds 329
Hexalure 399 -, - -trigeminal respiratory reflex 168,
n-Hexane 337 169
Hexanoic acid 157, 277, 278, 400 -, perceived intensity 232
-, and its derivatives 401 -, pigments 71
n-Hexanol 65 -, primary odors 255
Hexanone 392 -, qualitative similarity of odors 237
Hexenal 140, 276, 277, 383, 384, 403-405 -, radio sensitivity of eye 449
-, 2- 276, 278 -, sensations in nose 279
-, trans 277 -, sensitivity compared with bees 352, 379
-, trans-2- 377, 382, 396, 399, 402, 418 -, sensitivity of sensory nerves 322, 323
-,2-trans- 277 -, sickening odors 468
Hexen-2-an-ol-l 277 -, specific receptor site types 262
3-Hexen-2,5-diol 277, 400 -, subjective and reflex response 166
-, subject's expectation causing bias 223,
Hexene-l 276
2-Hexenoic acid 276 227,231
-, swallowing movements 467
trans-2-Hexenoic acid 377, 402, 403
-, taste thresholds for heptanol and octanol
Hexenol 140, 238, 276-278, 283, 400, 403, 269
404 -, trigeminal system 151,153
Hexenyl acetate 400 -, unmyelinated nerve terminals 174
Hexyl amine 253 Humidity receptor 374, 409
Hologram 265, 279, 314 Hydrocarbons 280, 290, 329
Hormone action 402 Hydrochloric acid 248
Horse, Jacobson's organ 162 Hydrogen cyanide 249, 250, 252, 309, 372
-, pigments in oHactory epithelium 63 - sulfide 221, 222, 272, 281, 313, 326, 404
Human, absolute and differential threshold 4-Hydroxy-benzaldehyde 268,269
235 9-Hydroxy-decenoic acid 140, 392
-, adverse odor of ozone 459 15-Hydroxypentadecanoic acid lactone 346
-, chemical senses and food intake 466 4-P-Hydroxy
-, cross-adaptation 224, 225 4-Hydroxyphenyl-2-butanone acetate 344,
-, decline in sensitivity with constant stimu- 390
lation 221 Hymenoptera, alarm pheromones 396
-, description of odors (see Primaries)
-, EOG 107, 108, 117, 125, 126 Impedance changes 146,370
-, factors for statistical control 248 Incisive foramen 154
-, feeding behavior 467 Incurrent aperture 4, 5
-, frequency components in oHactory bulb Indane musks 338,341
332 Index of detectability 234
Subject Index 509
Indole 253, 305, 332 Kephalin 336

Induced waves 120, 121 9Ketodecenoic acid (see 90xodecenoic
Inferior cavities 9, 12, 13 acid)
Information pattern 279, 308, 314 Ketones 117, 169, 207, 211, 236, 252, 253,
Informational capacity of the receptor mem 268, 270, 279-285, 296, 297, 300, 303,
brane 265 305, 306, 309-311, 323, 326, 327, 329,
Inhibition 123, 198,272,276,278,282, 397, 338, 347, 368, 379, 380, 391-393, 396,
495 397, 400, 402----404, 406, 408, 409
Inhibitors 398, 399 Kinetic model 421
Insects (see also specific insects) Kiwi (see Birds)
-, behavioral responses 117, 266, 267, Kolliker's pit 21
273-278, 351, 365-368, 391, 422
-, central pathways 421, 422 Labyrinthodonta (see Amphibians)
-, comparison of antennae 365 Lacerta vivipara (see Lizard)
-, contact chemoreception 134 Lachrymal duct 9,11-13,15,17,162,163
-, EAG (see Electroantennagram) Lactic acid 268, 269, 367
-, latency 375 Lampreys, nasal cavities 3
-, neural activation 450 -, - structure 2, 4
-, non contact chemoreceptors 133 -, - structure evolution 21
-, odor generalists 405-409 -, olfactory pigments 60, 61
-, - specialists 388, 404 -, supporting cells, cilia 42, 43
-, odors as communication 352, 353 Latent period 41, 95, 101, 211, 213, 412
-, - - food guides 352 Lateral nasal gland 162
-, - - repellants 353 - - sinus 11
-, olfactory cells 358 - olfactory tract 190, 435
-, - receptors on antennae 272 Latimeria, nasal anatomy 8, 9
-, perception of "odorless" compounds 352 -, - structure evolution 21
-, positive phototropism 449 Lavender 168
-, recording from brains 333 Laysan (see Birds)
-, receptor potential 369-372, 373, 374, LBjMB ratio 209
376 Lecithins 63, 336
-, reflexlike response to odors 365 Length. constant 415----417
-, selectivity of odors 351 Lepidoptera (see also Moths, Butterflies)
-, sensory fibers 422 -, generalist cells 388
-, single type receptor sites 262 -, olfactory receptor cells 375
-, social interaction of pheromones 397 -, pore cavities and tubuli 364
-, thermodynamic activity 118 Lepo8pondyli (see Amphibia)
-, transduction 410----421 Leucine 409
-, unit recordings 273-278, 375-378 Limonene 207, 209-211, 271, 279, 281,
-, use of antennae in scent perception 353, 282, 332, 396
354 Limulu8, generator potentials 97-99, 101,
I nsectivora, olfactory and accessory olfactory 103
bulb 154 Linalool 249, 334, 404
Insulin 479 Linalyl isobutyrate 334
Internal naris 10 Lipids 63
Interneurons 422 Lithium chloride 461, 466
Intracellular recording 133 Lizards, EOG 154
Iodoform 253 -, Jacobson's organ 154, 162, 163
Iodopsin 59 -, nasal anatomy 15, 16
Ion flux 410 -, nasal trigeminal innveration 164
-,pump 371,415 -, neural recording 164, 176
Ionone 207, 252, 281, 282 -, stimulus transport 176
IXIonone 285, 303, 309, 379 -, vomeronasal nerve 176
p.Ionone 285, 323, 326, 327, 329, 347 Locust, adsorption quotient 356, 383, 384
dlaionone, 303 -, CO 2 and humidity receptors 409
Isosensitivity function 230 -,EAG 276
Juvenile hormone 402 -, green odor receptor 418
510 Subject Index
Locust, hyperpolarization of receptor poten- Methylcyclopentanol 345

tials 405 Methylcyclo-propylketone 252
-, positive anemotaxis 366 Methyl decanoate 380
-, specialized generalist cells 388 4-Methyl-3-heptanol 392
-, stimulus desorption 421 3-Methyl-2-heptanones and its isomers 392
Loligo (see Squid) 2-Methyl-4-heptanone 396,409
4-Methyl-3-heptanone 380, 391-393, 396,
Macrocyclic diones 293 397,404
6-Methyl-5-heptenone
- lactones 298, 341, 404
6-Methyl-5-hepten-2-one 392
- musk civetone 286, 297
Methylheptenone 368
- musks 286
-2-Methyl-2-Hepten-6-one 396
- oxa-Iactones 293
-Methyl-5-hepten-2-one 408
Magnitude estimation 217, 225
Mammals (see also Human) -6-Methyl-5-hepten-2-one 392, 396, 402,
-, accessory olfactory bulb 187 406,408
-, dolphin 153 14-Methyl-cis-8-hexadecen-1-o1 404
-, electrical activity 446 -4-Methyl-2-hexanone 396
-, embryonic 173 -4-Methyl-3-hexanone 392
-, -, nervus terminalis 173, 174 Methyl ionone 347
-, -, vomeronasal nerve 174 - methacrylate 437
-, lipids 336 - sec-n-octyl ether 403
-, nasal anatomy 18-21 - salicylate 238,332,391,399,437,438
-, neural activity 450 Mice, Bowman's glands 47
-, olfactory bulb 195 -, olfactory bulb 161, 188
-, - pigments 60, 61 -, olfactory influence on sexual behavior
-, - system 435 199
-, radiation 450 Microvilli 105, 263, 371, 434
-, rhinitis 81 Middle concha 17, 18, 22
-, secretions from supporting cells of olfac- Mitosis 76, 79, 80, 83, 86, 92, 93
tory mucosa 44 NUtralcells 188,190,198,264,265,281,286
-, septal olfactory organ 160 -, recording from 123, 281-285
Masking 397, 399 -, single units 198,284
Mass action law 412 Modomyristin 340
Masticatory reflexes 172 Molecular migration 213
Maxillary nerve 153, 166-168 - profiles 262, 316
Maxilloturbinal 18-20,22 - structure 257, 316
Mechanoreceptors 104, 330 - -, optical antipods 300-304
Medial forebrain bundle 194 Moles 39,40
- nasal gland 160, 161
Mollusca (see Eledone)
- recesses 9, 12, 13
Monkey, degeneration and regeneration 85,
Melanin 63
92,93
Menthol 252, 282-284, 329, 332
d-Menthol 302 -, irradiation illness 461
-, muscle spindles 172
I-Menthol 238,249,279,281,283,302
-, olfactory nerve endings 186
Menthone 252, 279, 283
Mercaptans 140,253,278,281 -, vomeronasal nerve 176
-, x-ray detection 456
Mercapto-acetic acid 271
Mesencephalic nucleus V 172, 173, 177 Mono alkyl derivatives 312
Metabolism 421 Monocyclic musks 300
Methanol 279, 288, 302, 334, 419 Mono osmatic odorant 262, 266, 287, 308,
Methional 253 310
Methyl acetate 69 Mosquito (Aede8 aegypti)
- anthranilate 379, 406, 407 -, attractiveness of repellants 366, 367
- benzoate 124, 211 -, complex behavior reaction 366, 367
3-Methyl-2-butanone 284 -, CO 2 and humidity receptors 409
2-Methyl-2-( 4-tert-butylcyclohexyl-4- -, odor spectra 409
pentanone) 310 -, specialized generalists cells 388
Subject Index 511
Moth (see also Lepidoptera) Musk, indane 338, 341

-, antennae, scent perception 353 -, isochroman 338
-, complex odor mixtures 404 musk ketone 169, 283, 305
-, EAG 139 -, macrocyclic 252, 309, 311, 335, 338
-, masking substances 400 -, nitro 286, 294, 309, 311, 312
-, odor generalist cells 272 -, steroid 309
-, sensitivity 352 -, tetralin 338, 341
-, sex attractant 390, 399 -, xylene 326
-, stimulation of olfactory nerve 326 - xylol 271
- , Aglia, EAG effects of bombykol 395 Myrcene 399
- , Argyroploce, sex attractants 390 Myristic acid 340
-, Argyrotaenia, inhibition of pheromone
Myrtenal 303
receptors 397, 398
- diethyl acetal 303
-, -, sex attractants 390, 397, 398
-, Asiatic silk (Antheraea pernyi)
-, -, EAG 276, 421 Naphthalene 252, 279
-, -, generalist cells 388 ,B-Napthol methyl ether 406
-, -, hyperpolarization of receptor poten- Nasal cavity 123
tial 405 -, amphibia 10-13
-, -, nerve impulses 371 -, birds 17,18,433,434
-, -, olfactory sensilla morphology 365 -, cartilaginous fishes 4, 5
-, -, sensilla basiconica, reaction spectra -, cribriform plate 19
406 -, cyclostomes 2, 3
-, Cabbage looper (Trichoplusia ni) -, evolution 2, 21, 23
sex attractants 389, 390 -, lachrymal duct 9, 11, 12, 16, 18, 19, 162
-, -, temperature dependent potential -, mammals 18-21
changes 373 -, media nares 2, 3
-, Choristoneura rosaceana, sex attractants -, naris 3-10, 12, 14, 16-18, 22
387,398 -, nasal sac 2, 3, 6, 7
-, Deilephila, EAG effects to bombykol 395 -, nasopharyngeal duct 2, 3, 10-14, 16,
-, Endromis EAG effects to bombykol 395 18,21
-, gypsy moth (Porthetriadispar),EAG 375 -, oral-nasal connection 2
-, -, lure substances 304, 380 -,reptiles 13-17
-, Pink bollworm (Pectinophora gaUypiella) , -, sacropterygian fish 8-16
masking 399 -, septum 3,14,17
-, saturniid moths 353, 354, 394, 395, 421 -, teleosts 5-7
-, silk worm moth (see Bombyx) -, turbinate 18-20
--, telea, adsorption efficiency of antennae Nasal flow rate 155,168-170
356 - irradiation 167
-, -, antennae 372 - reflexes 167
-, -, - size 357 - trigeminal innervation 164, 166, 168,
-, -, masking substances 399 169
-, -, olfactory sensilla type 359 Nasociliary nerve 16,166-168,172
-, -, proteins on antennae 421 Nasopalatine canal 154, 162
-, -, sex attractants 394, 395, 397 - duct 153
-, -, specialized sensitivity 357 - nerve 162,164, 177
Mouse, sectioned olfactory nerve 83 Nasopharyngeal duct 10-16, 18,20
- (see Mice) - pouch 2, 3, 21
Mucic acid 400 Nasopharynx 20
Muconic acid 277 Nasoturbinal 18-20,22
Multidimensional scaling 237 Nassanoff gland 388,404,408,409
Muscle spindle 98
Neo-IX irones 303
Musk 252,267,280,283,284,288-300,303,
305, 311, 312, 323, 330, 336-339, 343, Nerol 368, 379
345-347 Nervus terminalis system 173, 177
- ambrette 290, 295 Neural coding 132, 148
-, aromatic carbonyl 338 Nitroacetophenone musk 341
512 Subject Index
Nitrobenzene 271, 279, 290, 298, 312, 406, Olfactory bulb, latency 209
407 - -, mitral cells 186
Nitrogen 69, 458 - -,-,anatomy 186
Nitro groups 294 - -, -, inhibition 188, 189
Nitrophenols 266 - -, -, number 188
- -, -, recording from 186, 197, 198,
5-Nitro-ortho-toluidino methylene camphor
200,285
302
- -, response to X-rays 456--458
3-Nitro-ortho-toluidino methylene camphor - -, tufted cells 186, 188
302 Olfactory cortex 192
Nonane 211 -, recording from 193, 199
Nonanone 157, 392, 396, 403 Olfactory discrimination 206, 265, 315
Nonanol 238 - eminence 13
Nonylphenol 340 - epithelium 3, 5, 10, 13, 15, 19-22, 66,
Number of olfactory cells 79, 91 75, 76, 78, 86, 156, 161, 263, 434
Nutrition, food monitoring 466-468 - -, electrophysiology 211
-, innate responses 468, 469 - -, odor flow rate 212
-, ora-pharyngeal control 475--479 - -, of the frog 214
-, palatability 469--471 - -, single unit recording 214
-, self regulation 471--474 - -, spatio-temporal patterns 211
Olfactory fatigue 69
- hallucination 227
Octane 207,209-211,279,281,282,442 - lamellae 3-10, 12, 17, 21
iso-Octane 442 - mucosa 27,152,153,160,165,209
Octanic acid 157 - -,size 29
Octanol 238, 269, 283, 437 Olfactory nerves 27, 45--47, 114, 120, 134,
-2-0ctanol 302, 437 153,163,165,167,169,209,435,436
Octanone 392, 403 - -, antidromic stimulation 114
- -, recordings 145,278,279,436
-, 2-0ctanone 236, 403
- -, sectioning 76-79, 83, 91
Ocular irritation 166, 167 - -, size 28
Odor blindness (see Anosmia) - -, ultrastructure 35-37
- classification (see Primaries) Olfactory nerve fibers 95
- discrimination 405 - -, sectioning of 76, 89
- generalists 272, 406 - organ 153, 162-164, 177
- mixtures 95, 119 - pigment 59-71
- similarity 287,306 - -,age 61
- space 238 - -, albinism 64-66
Oleic acid 272 - -, bird 61
Olfacto-habenular tract 435 - -, Bowman's glands 29, 61
Olfactometer 154, 306 - -, chromoproteins 63, 70
Olfactory and Trigeminal affluents 169 - -, carotenoids 59,61,62,66-69,334
- axons 28, 45 - -, cattle 62, 333
- bulb, ablation 76, 78, 79, 83, 164, 167 - -,fish 60,70
- -, anatomy 10, 22, 29, 185 - -, olfactory sensitivity 64-66, 264, 333
- -, centrifugal fibers 167, 168, 190, 195, - -, - transduction 68-70,331-334
196 - -, vitamin A 62, 66, 67
- -, comparitive 434, 435 - -, vomeronasal organ 160
- -, efferents 190 Olfactory placode 29, 48
- -, electrophysiology 99, 121-123, 206 Olfactory projections 193
to 209, 212, 280, 281-285, 341-347, - -, amygdaloid 194
438, 439 - -, hypothalamus 194, 199
- -, glomerulus 185 - -, thalamus 193,199
- -, granular layer, anatomy 187 Olfactory receptors 27, 28, 31, 154
- -, induced waves 121-123 - -, cell body 34
- -, interneurons 186 - -, cilia 32, 37--40, 264
- -, isomorphism 209 - -, insect antennae 272
Subject Index 513
Olfactory receptors, microvilli 32 Peme carbinol 252

- -, mobility 32 Pentadecanolide (exaltolid) 267, 281-283,
- -, olfactory rod 34 305,478
- -, olfactory vesicle 31 Pentane 281, 380, 419
- -, recording techniques 133 Pentanol 220, 223-226, 269, 282
- -, regeneration 29, 31, 54, 55, 76--93 iso-Pentanol 284
- -, ultrastructure 31 tert-Pentanol 284
- -, unit recording 133, 271, 279, 280 3-Pentanone 392
Olfactory receptor recording (see also Insects) 2-Pentanone 392
- -, coding 143 Pentyl acetate 283
- -, inhibition 143 Pepperminty 285
- -, insect 139 Periamygdaloid area 192
- -, marine invertebrates 141 Petromyzon 2, 60
- -, single units 134,135,142,271,279,280 Phagocytes 44, 79, 91
- -, temporal patterns 143 Phenol 326, 327
Olfactory reflex 164 Phenyl acetaldehyde 406
- rod 34 - acetic acid 253, 332
Olfactory supporting cells 42-44, 264 - ether 279
- -, degeneration 76, 91, 104, 115 - ethyl acetate 275,406
- -, microvilli 42 - - alcohol 146,165-167,169,170,238,
- -, phagacytosis 105 252, 279, 406
- -, secretion 43, 54 Phenyl ethylbromide 406, 407, 408
- -, ultrastructure 44 Phenylethylmethylethyl carbinol 247
Olfactory transduction 112, 116, 324, 331 Phenyl isocyanide 253
- -, insect 410-421 - isothiocyanate 252
Olfactory threshold (see Threshold) - propyl alcohol 379
- tubercle 190, 192 Pheromones 352-405
- vesicle 31, 32 -, definition 352, 353
On and off responses 95, 100 -, specificity 389, 400
Opossum (Didelphys), nasal mucosa 165 Phytochrome 70
-, nerve fiber recordings 278 Pici/orrnes 435
-, olfactory brain 183 Pig (Sus scrota), albinism linked to anosmia
-, - nerve specificity 336 64
-, septal olfactory organ 161 -, extraocular muscle spindles 172
Optical antipods 300 -, lack of carotenoids in olfactory epithelia
Orchids 391, 399 334
Organ of Corti 64 -, nasal cavities 18, 19
- of Jacobson (see vomeronasal epithelium) -,nasal septum 152
- of Masera 161 -, pigments in olfactory epithelium 62, 63
Oro-nasal connection 2 Pigment (see Olfactory pigment)
Oscillations 95, 108 Pimelic acid 277
Osteotraci, nasal cavities 3 Pinane 238, 271
Ostracoderms, nasal cavities 3 Pinene 303, 400
Oxal-octahydro-anthracene musks 341 Pinoacetaldehyde 303
Oxygen 69,334,458 I-trans-Pinocarveol 303
9-0xo-Decenoic acid 392, 403 d-trans-Pinocarveol 303
-9-0xo-trans-2-Decenoic acid 353, 382,390,
Placodeum 365
392
-9-0xo-cis-2-Decenoic acid 390 Placoid sensilla of the honey bee 272
Ozone 459, 460 Planaria, neural activation 450
Pogonomyrmex (see Ant)
-, ~lu.rm pheromones 79, 86
Pacinian corpuscle 98
Poliomyelitis 79, 86
Parosmia 254, 308
Passeri/ormes 435 Polypterus 8, 9, 21
Passerinae 434 Ponerinae (Ant) 396
Pelargonic acid 254 Population genetics 251, 255
Pelecaniformes, olfactory bulb 435 Pores 410
33 Hb. Sensory Physiology, Vol. IV!l
514 Subject Index
Pore plates and tubules 360-365, 370, 382, Pungent 287

410 Pure olfactory stimuli 166-168, 177
Porolepi8 9 Putrescine 253
-, nasal anatomy 9 Putrid 287
Porphyropsin 59 Pyridine 238,248,271,437,438,442
Potassium cyanide 249, 250 Pyridine silver 174
Power function, defined 217
-, odor intensity 124, 217, 218, 416 Queen substance 356, 388, 392, 403, 404,
Preconcha 16, 22 409
Prepyriform area 192, 435, 439 Quinine sulfate 66, 163, 164
Primaries 340
-, anosmia 255,310
-, classification technique 236 Rabbit, decrease in number of olfactory cells
-, content model 239 79
-, distance models 237 -, degeneration of olfactory cells 77
-, factor analysis 237, 238 -, - of septal mucosa 78, 79
-, historical 252, 253 -, enzymes in nasal mucosa 334
-, molecular basis 246, 251, 262, 266, 271, -, EOG 106, 154, 284, 285
281,284,285,287,334,341,344--347 -, ethmoidal nerve 169, 171
-, multidimensional scaling 237,242 -, - - recording 165, 176
Primary aliphatic alcohols 117 -, glomerular potential recording 200
- information pattern 262, 265, 267, 276, -, Jacobson's organ 153, 154, 164
280, 285, 287, 313-315 -, lack of carotenoids in olfactory epithelia
Primates, absence of accessory olfactory bulb 334
187 -, learning of olfactory detection 199
-, (higher) absence of Jacobson's organ 177 -, nasal mucosa 165
-, olfactory bulb 154 -, olfactory bulb 187
Proboscis reflex 408 -, - - removal 81
Proeellarii/orme8 434 -, - epithelium recovery 79
-, adaptation 305 -, - potential 98, 99
Propanediol 1, 2, 268, 269 -, - trigeminal reflex 168
iso-Propanol 306, 334 -, pigments in olfactory epithelium 63
n-Propanol 207, 224-226, 234, 238, 268, -, projection of the mucosa upon the bulb
269, 281, 282, 285 209
-, pure olfactory stimulation 167
Propenylsulfenic acid 252
-, receptor potential resistence 114-116
Propionic acid 151, 236, 254, 268, 269, 272,
-, recording from olfactory bulb 280, 281,
367, 368, 374, 379 457
Propyl acetate 283 -, regional analysis of odorants by olfactory
10-propyl-trans-5-9-tridecadienyl acetate mucosa 206
399 -, septal olfactory organ 161
Propylure 309 -, sustained stimulation 104
Prosimians, accessory bulb 154 -, trigeminal responses 169, 170
Protein 63, 64, 70, 249, 334, 421 -, vomeronasal epithelia 160, 176
Proteus 12 -, X-rays and EEG 452, 454
-, nasal cavities 12 Radiation theories (see Theories)
Psithyrus sp. 404 Raman frequencies 311
Psittaei/ormes 435 Rana catesbeiana (bullfrog) (see also Frog)
Psychophysical measurements 95, 108, 124, -, vomeronasal system 174
216, 222, 284 Rank correlation coefficients 284
Psychophysics 216, 242 Ranodon, nasal cavities 12
-, correlation with electrophysiology 284 Rat, behavior after hypothalamic lesions
-, detection 226, 236 476
-, odor classification 236-242, 252, 253, -, blastema cell 88
255, 286, 287 -, chemical effect on olfactory epithelium
-, power law 416 79-81
-,scaling 216-226 -, - mediation of X-ray stimulus 459
Subject Index 515
Rat, cilia, filaments 40 Reptilia, Lacerta (see also Lizard)

-, destruction by X-rays 454--456 -, -, vomeronasal epithelium 50, 51
-, discrimination of pulse X-rays 459, 460 -, Lizard (see also Lizard)
-, effect of gases on neuron sensitivity 455 -, -, nasal anatomy 15, 16
-, - of removal of olfactory bulb 200 -, Phytosauru8, nasal anatomy 16
-, food preferences based on odor preferences -, Snakes (see Snake)
474 -, Squamata (see also Squamata)
-, intracranial self-stimulation and food -, -, nasal anatomy 15, 16
deprivation 477 -, -, - structure evolution 22
-, intragastrical feeding 476 Respiratory mucosa 27, 152
-, irradiation illness 461 - rate 440, 442
-, main and accessory olfactory bulbs 176 Resting activity in the olfactory cells 315
-, nasal septum 152 - cell 83, 88, 93
-, - trigeminal interruption 168 Retinal 62
-, neural coding of taste preferences 461 -, retinene 67
-, odor and food intake 469---472 Retinol 62, 71
-, - discrimination depending on color 65 Retinoic acid 62
- , olfactory bulb removal 81 Retrograde degeneration 111
-, - cortex stimulation 199 Rhinitis 83
-, - identification of sexual partner 199 Rhipidistia, nasal anatomy 8-10,21
-, - impairment due to albinism 65 Rhodopsin 59, 330, 333
-, - nerve responses 436 Rhynchocephalia (Spherwdon) 141
-, - neurons 54, 55 Ribonucleic acid 71
-, - pigments 61 Rodents, nasopalatine canal 162
-, optic nerve section 79
-, pigments in olfactory epithelium 62, 66, Safrol 238
71,334 Safrole 305
-, preference of rapid solutions 468 -, iso-Safrole 371, 406
-, responses to X-rays 453 Salamander (axolotls), nasal cavities 12
-, sectioned olfactory nerves 77-79 -, Ambystoma, nasal anatomy 10-13
-, sniffing sounds 170 -, -, pigments in olfactory epithelium 63
-, stimulation with irradiated air 460 -, -, tracking behavior 163
-, suppression of licking 459 Salicylic aldehyde 284
-, vitamin A deficiency 66 Salmon (see Fish)
-, vomeronasal nerve 173 Sandalwood 290, 299
-, vomeronasal receptor 163, 164 cx-Santalol 299
-, X-ray exposure, heart rate 452 fJ-Santalol 299
-, - exposure threshold 458 Saturniid moths (see Moths)
-, zinc sulphate on olfactory epithelium 85 Sclareol 309
Reaction times (see Latent period) Sea hare (see Aplysia)
Receptor potential 95,96,98,99,114-116, Secondary palate 10, 16, 20
249,251,254,369,370,371,373,410,412, Sectioning of olfactory nerve 76, 83, 89, 92
416 Semiconductive properties 69
- site 112, 147, 261, 264, 266, 271 Semilunar 177
Reciprocal inhibition 189, 193 Sensilla basiconica 276, 406
Regeneration (see olfactory receptors) - - of antheraea pernyi 276
Repellent 399 - coeloconica 409
Reptilia, electrical activity 446 - trichodea 354, 355
-, (embryonic) nerve terminalis 173 - - of antheraea 276
-, Jacobson's organ 153 Sensillae 353
-, nasal anatomy 13-16 -, filter properties 360
-, neural activation 450 -, morphology 358-365
-, olfactory pigments, carotenoids 60 -, pore plate 363-365, 382, 410, 411
-, - system 435 -, tubules 360-362, 365
-, para olfactory functional unit 187 Sensillum 365
-, secretions from supporting cells of ol- - coeloconicum 405
factory mucosa 44 - liquor 371
516 Subject Index
Sensillum trichodeum 385 Taste receptors 412, 414

SerillUS canarias 442 Termite (Reticulotermes), complex odor
Sex-linked 249,250 mixtures 404
Shark, behavior approach to prey 201 -, pheromone specificity 390, 393
-, nervus terminalis 173 Terpenes 281, 399, 400, 409
-, dogfish shark (Squalus acanthias), nasal Terpenoids 281
cavity 4
Terpineal 406
Sheep, albinism linked to anosmia 64
Terpineol 305, 371, 406
-, extraocular muscle 172
-, lack of carotenoids 334 Terrapene carolina (box turtle), vomeronasal
-, pigments in olfactory epithelium 63 receptors 160
Similarity judgements 123, 198, 284 Tetradecenyl acetate 397, 398
Siren (Trachystomata), nasal cavities 12 -, -cis-7-Tetradecenyl acetate 390
Skate (Raja), nasal anatomy 4 -, -cis-9-Tetradecenyl acetate 391
Skatole 207, 238, 253, 440 -, -cis-11-Tetradecenyl acetate 390, 391,
Slow bulb potential 95 393,397
- negative potential 268 -, -l1-cis-Tetradecenyl acetate 398
Snails, phototropism 450 -, -transoU-Tetra decenyl acetate 390
Snake, Jacobson's organ 153, 154, 162, 163 -, -11-trans-Tetradecenyl acetate 399
-, nasal anatomy 15, 16 Tetraodon (teleost), nasal anatomy 7
-, nasal trigeminal innervation 164 Tetraethyl tin 279
-, neural recording 164 Tetralin 305
-, stimulus transport 176 - musk 341
-, vomeronasal organ 163 Tetrapods, nasal structure evolution 21, 22
-, European grass, Jacobson's organ 163 -, - terminology, definitions 10
-, European viper, Jacobson's organ 162 -, Eryops, nasal cavity 11
-, garter, Jacobson's organ 65, 163 Thalamic taste nucleus 163
-, rattle (Crotalus), nasal cavity 15, 163 Thalamus 193
Spatio-temporal analysis 211 Thanatophilus, carrion receptors 404
- code 279 -, receptor potential of carrion 374
- patterns 205-214
Theories, enzyme 334, 335
Specialists 388
-, molecular profiles 251-254, 276, 287,
Specific anosmia (see Anosmia)
345-347, 402
Specificity 254, 387, 388, 397, 402, 409 -, penetration and puncturing 322-331,
Sphenodon (see also Rhynchocephalia)
335
-, nasal cavity 14, 15
-, profile and functional group 261-316
-, - structure evolution 22
-, radiation 260, 311-313, 331-334,
Sphenoidal sinus 20 342-344
Sphingomyelin 336 -, spatio-temporal 212-214
Squalus acanthias (see Dogfish shark)
Thermodynamic activity U8, 267, 269
Squamata 14, 22, 154, 163
Squid (Loligo), excitation of giant axons 323 Thiamine 471
Stearic acid 337, 342 Thiophane 253
Steers, olfactory pigments, carotenoids 60 Threshold 219,245,266,267,305,385,388
Stereochemical theory (see Theories) -, absolute 235, 236
Steroid 297, 409 -, anosmia 248
- ketones 296,310,311 -, before and after adaptation 306
- - and alcohols 296 -, differential 235, 236
Straight chain fatty aldehydes 275 -, errors 266
Struniiformes, nasal anatomy 8-10 -, insect 365,367,368,378,379-389
Sulfur dioxide 326 -, man and bee 379
Supporting cells (see Olfactory supporting -, molecular basis 266,267,274,305-307,
cells) 328
Sustained stimulation 95, 103 Thrirwxodon, nasal bones 16
Sweat 409 Thymol 252
Sympathetic nasal reflex 171 Toad (Xenopus), olfactory pigments, caro-
Synergism 397,398,399 tenoids 60
Subject Index 517
Toluene 238, 281 Turtles, green, nasal cavity 14

Toluylene-bis-amino-methylene camphor 302 -, -, vomeronasal epithelium 161
Tongue flicking 162, 164
Tortoise, electrically recorded responses 176 gamma-Undecalactone 252
-, EOG 107, 115 Undecane 380
-, gopher, EOG 154, 155, 159 -2-Undecanone 207, 300
-, -, nasal flow rate, dependence of fre- Unit techniques 133
sponses 165 Urine 296, 310
-, -, olfactory mucosa histological section -, nasal anatomy 10-13
30 Urodela, nasal structure evolution 22
-, -, respiratory epithelium and vomero-
-, pigment 70
nasal epithelium 50 -, vomeronasal nerve 174
-, -, respiratory mucosa cilia 49
-, -, vomeronasal receptors 160
Trachystomata (Siren), nasal cavity 12 Valeric acid 279, 400
Trail substance 393, 397 -, iso-Valeric acid 157,251, 253, 254, 274,
Trans-siglure 391 347, 379
-, -n-valeric acid 157, 207, 254, 271
Triatoma (blood-sucking bug)
Vanillin 238, 252, 290, 297, 305
-, responses of specialist cells 141, 283
Ventral ganglion 146
Trichlorethylene 252
- nerve cord 134
Tricyclic isochromanes 296
Verbal expression of odor sensation 285,286
2-Tridecanone 380, 400
trans-Verbenol 391, 399
Triethyl amine 166
Verbenone 399
Trigeminal mesencephalic nucleus 172, 173
Versalide 252
Trigeminal nerve 28, 78, 134, 159, 164,
Vestibulum 10, 12-18
167-169, 177, 441
-, anatomy 164, 171 Vitamin A 61, 62, 66, 67, 69, 71, 246, 334
-, fibers in olfactory mucosa 47,48 (see also Olfactory pigments)
-, recording from 55, 165, 166 Vomeronasal epithelium 8, 10-12, 15, 16,
-,reflexes 164,166-169 20,48, 151-177, 187
-, responses to odors 155,165-171 -, Amphibia 11, 12, 154
Trigeminal sensory ganglion 170, 172 -, embryology 23, 48, 153
-, evolution 23
Trigeminus 153, 167, 169, 170, 177
-, lack of Bowman's glands 51
trans-Trimedlure 390, 391 -, lack of receptor cilia 38, 39, 51-53,
Trimethylamine 281, 404 160
Trimethylpentane 437,438,441,442 -, lizards and snakes 15, 16, 154,160-164
Trinitro-tert-butyl-xylene 323 -, mammals 20, 21, 154, 160-164
Tritium 275 -, neural activity 154-159
Triton, degeneration and regeneration 83, -, other reptiles 14, 154, 160-164
92,93 -, response to odors 154-159
Tufted cell 186, 188, 190 -, stimulus transport 161-164
Turbinals 433 -, ultrastructure 50, 51
Turtles, basal bodies, related to cilia 40
-, medial nasal gland 154, 176 Weber's law 235
-, nasal cavities 13-15 Whale, absence of olfactory organ 135
-, - structure evolution 22 Wintergreen 168
-, neural recording 176
Worm, earth, mucus 164
-, olfactory vesicle, basal bodies 32, 33
-, slow (Anguis), olfactory pigments, caro-
-, vomeronasal system 174, 176
tenoids 60
-, box, olfactory mucosa 36, 41-43, 45
-, -, vomeronasal epithelium 50, 51, 160
-, -, olfactory receptor, electronmicrograph
33
-, -, vomeronasal epithelium 52 Xenopus toad, olfactory pigments, ca-
-, -, - receptor 43, 54, 160 rotenoids 60
-, green (Chelonia nydas) X-rays, arousal 452-454
518 Subject Index
X-rays, aversion to 460-462 Xylocaine 170

-, odor detection 454-456 Xyol 168-170 (see also Xylene)
-, properties 449, 452
-, response of olfactory bulb 456-459 Zinc sulfate 79, 80, 85, 86, 92
Xylene 168, 169, 281 Zoarces (Blenniidae), nasal anatomy 7
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(Handbook of Sensory Physiology 4 - 1 - Chemical Senses.) J. E. Amoore, M. G. J. Beets, J. T. Davies, T. Engen, J. Garcia, R. C. Gesteland, P. P. C. Graziadei, K.-E. Kaissling, R. A. Koelling, J. LeMa

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Handbook

H. Autrum R. Jung . W. R. Loewenstein

J. E. Amoore . M. G. J. Beets' J. T. Davies' T. Engen

With 212 Fignres

Springer-Verlag Berlin Heidelberg New York 1971

Olfaction is involved intimately in two of the most basic functions of animals:

Tallahassee, July 1971

Chapter 1 Anatomy of Nasal Structures from a Comparative

AMOORE, John E., Composition Investigations, Vegetable Laboratory, United

Anatomy of Nasal Structures

The primary function of the nasal cavities is olfaction. Although in tetrapods

Anurans vary considerably in their nasal anatomy, and, although it applies to

Sphenodon, the only living rhynchocephalian, has a small vestibulum leading

often referred to as ventral (inferior), middle, and dorsal (superior) meatuses

may be 0 to 7 endoturbinalia in each nasal cavity, but most macrosmatic mammals

The nasal anatomy of various mammals is much modified. There may be a

Evolution of Nasal Structure

logous could represent parallel developments in separate lines. Only speculation is

The Olfactory Mucosa of Vertebrates

that the olfactory mucosa in vertebrates is composed of three cellular elements:

The Nasal Cavities

Histology of the Olfactory Mucosa

The olfactory epithelium as seen in histological sections is thicker than the

Ultrastructure of the Olfactory Mucosa

The Olfactory Receptor

The Olfactory Vesicle

also a constant organelle in the olfactory vesicle. A constricted zone normally

and supporting cell surface membranes is observed. This is characterized by the

The Dendritic Process

Receptor Cell Bodies

Fig. 5. In a number of physiological and experimental conditions the secretory activity of

hundreds, running in reciprocal close contact in single pockets of the Schwann's

preparative procedures. The most prominent organelle observed in supporting cells is

nerves. Trigeminal fibers, however, have been described by early histologists to

ER and the number of mitochondria are expressions of the particular secretory

The Vomeronasal Epithelium

anatomical and functional similarities the VN epithelium has some characteristic

tortuous than the dendrite of olfactory neurons. Neurotubules are observed in

in the olfactory mechanism. Morphologically the secretory granules appear dif-

operated by a continuous duplication and maturation of stem cells recognized as

The Olfactory Pigment

Distribution and Location

medin, causes expansion. The olfactory melanophores do differ in one respect,

a) Carotenoids and Vitamin A

Fig. 1. Structure of members of the vitamin A series and p-carotene

Pigment and Olfactory Sensitivity

(b) Albinism is associated with the lack or reduction in intensity of olfactory

b) Vitamin A, Carotenoids and Sensitivity to Odors

the knowledge that vitamin A is needed to prevent drying of mucous membranes

Theories Implicating Pigment

60 100 I~O lBO 220 250

While ROSENBERG'S theory may, in essence, be applicable to non-carotenoid

epithelium would not be surprising. Beginning at the age of 7 -8, a proportion of

LE MAGNEN, J., RAPAPORT, A.: Essai de determination du role de la vitamine A dans Ie

Degeneration and Regeneration

The olfactory epithelium is generally made up of olfactory, sustentacular and

A. Experiments which Showed no Degeneration

B. Degeneration not Accompanied by Regeneration

2. Closing of the Nasal Opening

were found in most regions of the olfactory epithelium. Intraperitoneal injection

C. Regeneration of the Olfactory Epithelium after Degeneration

2. Sectioning of the Olfactory Nerve

Fig. 4. Degeneration and regeneration of the olfactory epithelium. a Normal olfactory

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