THE JOURNAL OF COMPARATIVE NEUROLOGY 364:6-15 (1996)

Commentary

Methods for Determining Numbers of

Cells and Synapses: A Case for More
Uniform Standards of Review
RICHARD E. COGGESHALL AND HELENA A. LEKAN
Marine Biomedical Institute, The University of Texas Medical Branch, Galveston,
Texas 77555-1069

ABSTRACT
Neuron and synapse numbers are important assays in neuroscience. These numbers are
estimated by one of four methods: 1) profile counts, 2) assumption-based methods, 3 ) serial
reconstructions, and 4)stereological methods. The criteria for these methods are diverse. This
creates a disparity in that some reviewers accept estimates from any of these methods, while
others accept only specific methods. An equally important issue is the diversity of sampling
strategies, since unbiased estimates of neuronal or synaptic numbers are contingent upon both
counting and sampling techniques. The purpose of this commentary is to institute a dialog that
will lead to a better understanding of the strengths and weaknesses of the above methods, and
to propose guidelines that should lead to more uniform and thus fairer judging of the studies
that provide estimates of neuron or synapse numbers. In addition, adoption of more uniform
standards for obtaining unbiased numerical estimates should result in the generation of an
unbiased database that will be of considerable use in future studies. D 1996 w i ~ e y - ~ i sInc.
s,

Indexing terms: counting methods, sampling, stereology, profiles

Neuron and synapse numbers are important. For ex-
ample, losses of neurons or synapses are assays of the
severity of a disease or the effects of an experimental
procedure. Because neurons and synapses are small, these
numbers must usually be determined by counts in histologi-
cal sections. Investigators who need such numbers often
have difficulty because of disagreements about the validity
of different methods (West, 1994). A particular difficulty,
and the reason for this commentary, is that reviews of
articles are disparate, with some reviewers accepting counts
by any method, and others rejecting any estimates except
by particular methods. The aim of this commentary is to
initiate a discussion that will lead to a better understanding
and more uniform judging of estimates of neuron and
synapse numbers.
I. CATEGORIES OF COUNTING METHODS
Counting methods for histologically sectioned material
fall into four categories: 1)profile counts (a profile is what is
seen of a cell or synapse in a histologic section); 2) serial
(section) reconstructions; 3 ) assumption-based methods,
which are profile counts plus geometric assumptions that
convert the profile counts into cell or synapse numbers; and
4)stereological methods.
11. PRESENTLY USED
METHODS-FREQUENCIES AND TYPES
A survey was done to get an idea of the proportion of
neuroscience studies that determine cell or synapse num-
bers and the frequency of the various types of methods
presently in use. From a recent survey of four major
neuroscience journals (Table 11, it can be seen that within
our sample 18%of all articles and 30%of the articles that
use histological sections report numerical data indicating
cell or synapse numbers (numbers of cells in a ganglion,
changes in numbers of synaptic profiles per mm2, etc.).
These percentages are low because we do not include those
Accepted October 4,1995.
Address reprint requests to Richard E. Coggeshall, M.D., Marine Biomedi-
cal Institute, The University of Texas Medical Branch, 301 University Blvd,
Galveston, TX 77555.1069.
COMMENTARY ON NUMBERS OF CELLS AND SYNAPSES
TABLE 1. Survey’
Articles
using
Total histological
Journal articles sections
Brain Kesearch (Vols 662-6671 276 93
.l. Comp. Neurol. (Vols. 345-3501 243 237
J. Neuropathol. Exp. Neurol. (Vol. 4 4 ) 66 66
.J Neurosr.1. ~ V i r l 14.
. July-Dec.! 243 93
‘rotdl 828 489
‘ A survey 0 1 nonreview (original data) articles published in four major neuroscience
journals in 1994. More specifically, these studies were published in the last part of 1994,
with the exception of Journal 01 Neuropathology and Experimental Neuroloa, which
spansail of 1994.
‘rABLE 2. Methods for Counting Cells and Synapses’
Profile counts
Limited aerial reconstructions
Assumption based methods
Stereological analyses
1A determination of the types and frequencies of counting methods used in the 148
articles that determine cell or synapse numbers. Note that profile counts make up the
large majority of these studies.
studies that simply say that “many” or “few” cells or
synapses were labelled, nor those that include counts of
cells in tissue culture or whole-mounts because these were
not done in histological sections. The sample is small, but
the conclusion that cell and synapse counts are a n impor-
tant part of many modern neuroscience studies is secure.
In the same survey, the counting methods each study
used were determined (Table 2 ) . It can be seen that the
large majority (76%) of studies presenting numerical data
count profiles of neurons or synapses. Then there are
smaller proportions of counts by limited serial reconstruc-
tions (11%),assumption-based methods (7%),and stereologi-
cal analyses (5%).
111. COUNTING AND SAMPLING
All counting methods, except complete serial reconstruc-
tions, sample. As a result, every study that reported neuron
or synaptic counts in histologically sectioned material
determined numbers of counted objects in a fraction of a
reference space (numbers of cell profiles per high power
field, numbers of synapses per volume of lamina, etc.).
What is specific to each method is how the units are
counted. These differences are considered in Counting
Methods (section IV). Equally important are Sampling
Strategies (section V).
IV. COUNTING METHODS
A. Serial (section) reconstructions
Complete serial reconstructions give a n accurate determi-
nation of neuron or synapse numbers. This is because there
is no sampling or estimating; one simply reconstructs all
the cells or synapses and then one knows how many there
are. To illustrate, Figure 1. depicts a reference space
(ganglion, nucleus, etc., Vrefin the stereological literature)
containing 10 particles (synapses, or cells, numbered 1-10)
that is cut into 8 (A-H) histological sections. In a serial
reconstruction, each profile would be numbered as it first
appears (cf. 1, 2, and 3 in section A), and then given the
same number in subsequent sections so it is not counted
again. New objects would then be labelled in subsequent
7
sections as they appear. As long as all the objects can be
Articles seen in at least one histological section, a requirement for
reporting any counting method, and can be followed from section to
cell or section, a n accurate count will result (SER column, Figs.
synapse
counts 1-3). This method is not in general use because it is too
labor intensive (inefficient), but serial reconstructions are
28
79
useful in restricted situations.
18 No study that we sampled did complete serial reconstruc-
23 tions. The limited serial reconstructions that we found were
148
of 2 types; 1) counts of spines along measured lengths of
dendrites in thick sections or 2 ) serial or interrupted serial
electron micrographs for synaptic counts.
B. Profile counts
112
Because of the labor required to follow cells and synapses
17 through serial sections, most investigators count profiles of
11 these structures (Table 21, although they say they are
8
counting cells or synapses. However, if an investigator
counts in single optical planes (cf. counting labelled struc-
tures in a histologic section), profiles of neurons or synapses
are being counted and not the neurons or synapses them-
selves. Profile counts are done in different ways, which we
will consider in turn.
1. Total profile numbers. In some studies, total profile
numbers in a reference space are estimated (Table 3 ) . This
is usually done by counting profiles at constant intervals
through the reference space and multiplying by the interval
(cf. counting profiles in every 10th section and multiplying
by 10). This provides a n unbiased estimate of profile
numbers. But the claim is that numbers of cells or synapses
are being estimated. If numbers of profiles are to accurately
estimate numbers of cells or synapses, each profile must
represent a cell or synapse uniquely. But profiles usually do
not indicate cells or synapses uniquely, as shown in Figure
1, where there are 22 profiles but only 10 objects. The
conclusion seems inescapable that there are more profiles
than cells or synapses because the latter are split in the
process of histologic sectioning.
Another way to illustrate the problem is to show that
only changing section thickness changes profile numbers.
Thus, in Figure 2 where section thickness is doubled, there
are 14 profiles of the same 10 objects. Changing sizes of the
objects will also change profile numbers, as in Figure 3 ,
where there are the same 10 objects, only a little smaller,
and 18 profiles. To get some idea of how closely profile
counts estimate cell numbers in histologic sections, we
counted profiles of L4 ganglion cells in embryonic rats and
found that there were 2.5 times as many profiles as cells
(Coggeshall et al., 1994). We conclude that profile counts
estimate profile numbers, not neuron or synapse numbers.
2. Densities and ratios. Rather than estimating profile
numbers, it is more common to determine densities (num-
bers of profiles per some unit of size, usually area; see
below), or density ratios. Without exception, the studies
determining densities or their ratios use these data to
indicate changes in cell or synapse numbers.
a. Areal densities and ratios. The commonest way to
determine densities (Table 3 ) is to count profiles per unit
area (numbers of profiles per mm2, per slide, etc.). We call
this the method of areal densities. The commonest way to
do density ratios is to determine areal densities in the
control and experimental situation and determine their
ratios (numbers of labelled, dying, etc., profiles compared to
numbers of all profiles per mm2, per slide, etc.). We call this
 I.TITI T lI r T
(Ganglion, Nucleus, etc.)
H
Figures 1-3. Profile counts are different from object counts and can
be significantly changed by changes in section thickness as well as
changes in the size or shape of the counted objects. By contrast, serial
reconstructions and disector analyses accurately determine object
numbers and are not influenced by changes in section thickness or
object size and shape.
Fig. 1. A sketch of a reference space (ganglion, lamina, etc.)
containing 10 objects. The space is divided into 8 histological sections
(A-H) and the objects (cells or synapses) are indicated by the numbers
1-10, Serial reconstructions: To serially reconstruct the 10 objects in
Figure 1, one would start with the first section (A) and number the
profiles (1-3). The profiles from objects 1-3 would then be followed in
serial sections and given the same number so they would not be counted
again. Then one would proceed to the next section, B, and number any
new profiles (none are seen), and then to section C where 4 and 5
appear, etc. When the reference space is completely analyzed this way,
an accurate count results (SER column). Profile counts: The 10 objects
0p-r- LOUfllS
SER PRF DIS
0 3 2
2 3 2
1 2 0
2 4 2
0 2 2
each profile is indicated by a “P.” Note that there are 22 profiles (PRF
column), which is quite different from the 10 objects. A particular type
of profile count is central profile counts (see text). If we take central
profiles as those that are not edges (do not extend all the way through
the section), there are 8 of them in this figure (object 1, the central two
profiles of 3, the central profile of 4, object 6, the two central profiles of
7, and object 9). Stereological counts-physical disector counts: Physi-
cal disectors consist of congruent pairs of sections. The procedure is to
identify profiles of the objects of interest in one section (the reference
section) and see if a profile from the same object appears in the other
section (the look-up section). If it does not this is a top or count (Q-)
indicated by down pointing arrows ( 1 ). To do a disector analysis on this
reference space, an investigator would take A as the first reference
section, and B as the first look-up section. Any profile from an object in
A but not in B would be a count. In section A, there is one ( .1 ). Then if B
is taken as the reference section and C the look-up section, there is
another count ( .1 1. If all sections in this figure are done this way, an
exact count of 10 results (DIS column). If one reverses the disectors by
COMMENTARY ON NUMBERS OF CELLS AND SYNAPSES 9

Reference Space counts

(Ganglion. Nucleus, etc.)
OPT

n SER
(#)
PRF
(P)
DIS
(1)

3 3 3

3.P

2 3 2

3 4 3

D
i/n
nu 10,P,t
9.P,?

2 4 2

TOTALS 10 14 10

Fig. 2. The same reference space and objects as in Figure 1 except
that the sections (A-D) are thicker. Serial reconstructions: The
procedure is as for Figure 1. Note that an accurate count results when
this space is reconstructed (SERcolumn). Thus as expected, section
thickness has no bearing on the counts from serial reconstructions.
Profile counts: As in Figure 1,the objects are divided into profiles by the
process of histological sectioning. Note that there are 14 profiles (PRF
column) which is different from both the numbers of objects and the
numbers of profiles in Figure 1. Thus, section thickness has a bearing
on profile numbers. Stereological counts-physical disector counts: As
with Figure 1, one would take the first section (A) and count any
profiles that are found in A but not in B. There are 2 in this picture
(unmarked]. If one does the whole tissue this way, an accurate count of
10 results. Thus, section thickness has no bearing on physical disector
counts. Ontical disector counts-Optical disectors are demarcated by 2
lines on the top and bottom of the reference space. To do a complete
optical disector analysis, an investigator would start with optical
section A and determine how many structures are in focus in the first
focal plane (indicated by the line above section A). There are none in
this plane. Then one would focus through the section to the second focal
plane (the line between A and B) and count all particles that come into
focus. There are 3 such particles in section A ( T ) . Thus the count for
this disector would be 3 (OPT DIS column). Then one repeats the
procedure for section B. In this case, the first focal plane is the line
between sections A and B. Objects 1 and 2 would not be seen in this
plane and object 3 would already be present (in focus), so none of these
would be counted-but particles 4 and 5 would come into focus ( t J for
a count of 2.When the entire reference space is analyzed this way, an
accurate count of 10 results. This is the same number as derived from
the serial analysis. The same numbers of objects are also found if
10 R.E. COGGESHALL AND H.A. LEKAN

Reference S p a c e Counts
(Ganglion, Nucleus, etc.)
! 1 S E R PRF DIS

0 2 1

2 3 1

D 0 2 2

E 2 2 1

2 2 1

2 3 2

0 1 1
H

I I
TOTALS 10 18 10

Fig. 3. The same reference space and sections (A-H) as in Fig. 1but
the objects are slightly smaller and different in shape. Serial reconstruc-
tions: Note that an accurate count of 10 results when this tissue is
reconstructed. Thus size or shape of the reconstructed objects has no
bearing on serial reconstruction counts. Profile counts: Note that there
are 18 profiles (PRF column), a number that differs from the profile
counts in Figures 1 and 3 and the true object numbers. Thus size and
shape have a bearing on profile counts. Stereological counts-physical
disector counts: Note that accurate counts ( 1 result when the
complete tissue is analyzed (DIS column). Thus size and shape of the
counted objects do not bear on physical disector counts. Optical disector
counts-Note that an accurate count of 10 (unmarked) results when
the tissue is completely analyzed as in Figure 2. Thus size and shape of
the counted objects have no bearing on optical disector counts.

the method of (simple) areal density ratios. As an example conclusion would be that the treatment caused a 10-fold
of the use of areal densities, if an investigator found 10 increase in the numbers of dying cells. As an example of the
dying cell profiles per high power field in an untreated use of density ratios, if an investigator found 10% of
tumor and 100 dying profiles per field after treatment, the synaptic profiles labelled for glutamate in the normal and
COMMENTARY ON NUMBERS OF CELLS AND SYNAPSES
TABLE 3. Types of Profile Counts ( 112 Total S t u d i e s ) '
Total nuinhers
Areal derinitws
a. Simple densities
h. Ratios oi areal densities
Volume densities
Section separation
Central prolilr counts
Small objects In thick sectionb
' A survey of the profile-counting methods in the 112 studies that do such counts. The
total is larger than the 112 articles from which this survey was done because many of the
studies used inore than one of the methods lcf. estimated densities of central prnfiles!.
TABLE 4. A s s u m p t i o n Based M e t h o d s (11 S t u d i e s ) '
Abercromhie I 19461
Beaudet and Sotelri (1981!
Konlgsmark I 19701
Na D
Focus o n top dsection
'Numbers of assumptinn-based methods used in our survey. Note that the method of
Abercromhie iAhercrnmhie. 19461 is the most common nf these, and corroborates the
recent finding (if a considerable and steadily Increasing use of this method IWilliams and
Ksluc. 19XR!. The methods nf Konigsmark 11970) and Reaudet and Sotelo 119R1! are also
used. N e I) 15 L commonly used older method where the areal density is divided by the
mean "height" o f t h e objects (Weihel, 19791. The focus on top of section method means
that tht. mvrstigator counted nii cells that were in focus of the top (IS the section hut
counted all other cells that came into focus as the thickness o f t h e section was examined.
30% labelled after nerve injury, the conclusion would be that
nerve injury caused a 3-fold increase in labelled terminals.
Let us examine the above reasoning. The first problem is
that there is no simple relation between profile counts and
neuron or synapse numbers. But investigators doing densi-
ties or their ratios would say that they are interested in
proportional changes and not in the numbers themselves.
The necessary assumption, therefore, is that changes in
profile densities or ratios indicate changes in cell and
synapse numbers, even if the profile numbers are not the
same as cell or synapse numbers. But cells or synapses
usually change in size and shape, etc., after a perturbation,
which modifies profile densities and ratios unrelated to
changes in cell or synapse numbers. Also if a population of
cells or synapses is lost, it is usually a specific population
which changes profile densities and ratios disproportion-
ately to alterations in cell or synapse numbers (cf. if large
cells are lost, there is a disproportionate drop in profile
numbers because large cells are sectioned into more profiles
than small cells). Also the reference space may change,
which changes densities and ratios even if cell or synapse
numbers are constant (cf. if the volume of cerebral cortex
doubled because of edema, cell densities would fall by 50% if
no cells were lost). This is the "reference trap" (Braeden-
gaard and Gundersen, 1986; Pakkenberg et al., 1991). To
illustrate the effect of size changes, compare Figures 1 and
3, which contain the same numbers of objects but the
objects are smaller in Figure 3. There are 22 profiles in
Figure 1 and 18 in Figure 3 . Thus there are 18% more
profiles in Figure 1, but this does not indicate 18% more
cells or synapses. Admittedly biases may balance each
other, but there is no way another investigator can tell if
this is the case unless the counts are calibrated (to calibrate,
one estimates known populations with the chosen method)
(Coggeshall et al., 1990). Thus, uncalibrated densities or
ratios do not allow unambiguous conclusions about changes
in synapse or neuron numbers (Braedengaard and Gun-
dersen, 1986).
6. Volume densities and ratios. Occasionally profile
volume densities (numbers of profiles/unit volume) are
determined by counting profiles in measured areas and
11
multiplying by section thickness and separation to provide
27 a volume (Table 3). The same difficulties as for areal
93 densities hold for these analyses-namely, that changes in
47
46 volume densities or ratios do not indicate proportional
2 changes in numbers of cells or synapses unambiguously.
2
23 3. Section separation. Some investigators count profiles
2 but stipulate that each profile must come from a different
cell. This is done by separating the sections from which the
counts are done by a distance greater than the diameter of
the largest cell. We refer to this as the method of section
separation. But this is still counting profiles. To illustrate,
none of the objects in Figure 1 extend across more than 4
sections, so if we counted the profiles in sections A and E, all
would come from different objects. We could use this as the
basis of a n estimate, in which case we would multiply the 6
profiles in A and E by 4 (since we counted in 2 of 8 sections).
The estimate would be 24 profiles, which is close to the total
number of profiles and not the total number of objects. If we
analyze the whole tissue this way, we would find 6 profiles
in A and E, 4 profiles in B and F, 7 profiles in C and G, and 5
profiles in D and H for a total of 22, which is the true
number of profiles, and not the true number of cells or
synapses. Thus profile counts, even if the profiles come
from different cells or synapses, estimate profile numbers
and not cell or synapse numbers.
4. Central profile counts. Investigators sometimes only
count central profiles, which are profiles of cells that
contain a prominent nuclear profile and often the nuclear
profile must also contain a nucleolar profile. We refer to this
as the method of central profile counts, implying that edges
are excluded. The assumption is that, if edges are not
counted, profile counts will accurately estimate cell num-
bers. But if we exclude edges (profiles that do not extend all
the way through the section), there are 8 such profiles in
Figure 1, and 6 such profiles in Figures 2 and 3, yet in each
case the number of objects is 10. Admittedly, the numbers
of central profiles occasionally may be the same as the
numbers of objects that gave rise to the profiles, but there is
no way another investigator can tell whether this is true
unless the counts are calibrated. So estimates from central
profile counts, although they may deviate less from true
numbers than estimates from total profile counts, will
usually not estimate cell or synapse numbers accurately. A
greater problem is that another investigator cannot tell
whether the estimates are biased or not (a biased estimate
approaches a number different from the true number as
sample size increases).
5. Small objects in thick sections. Two studies noted
that no correction factors were needed because the objects
they were counting were small in relation to section thick-
ness. It is true that profile and object counts are the same
thing if no objects are split during sectioning. On the other
hand, some of the objects will almost always be split during
histological sectioning, and the numbers that are split
depend on many variables. So if one is going to use profile
counts to estimate numbers because the objects are small in
relation to section thickness, we still think it necessary to
calibrate to show that bias due to overcounting because of
object splitting does not jeopardize the conclusions.
Summary. Profile counts usually do not provide accu-
rate estimates of total numbers or changes in numbers of
neurons or synapses because numbers of profiles depend on
many variables besides cell and/or synapse numbers. The
bottom line is that profile counts estimate profile numbers
and not cell or synapse numbers.
12
C. Assumption-basedmethods
Assumption-based methods (Table 4) were devised to
control for the difficulties with profile counts. Although
these methods appear disparate, they all determine profile
numbers and then make assumptions that convert the
profile counts to cell or synapse numbers. Usually the
assumptions require that something else be measured, for
example, nuclear diameters (Abercrombie, 1946). These
procedures are efficient, especially if they are computerized
(Hendry, 1976; Devor et al., 1985; Rose and Rohrlich,
1987). Many such methods are in existence, of which we
only list some of the commonly used ones (Agduhr, 1944;
Floderus, 1944; Abercrombie, 1946; Konigsmark, 1970;
Anker and Cragg, 1974; Offord et al., 1974; Hendry, 1976;
Beaudet and Sotelo, 1981; Smolen et al., 1983; Coggeshall
e t al., 1984; Devor et al., 1985; Rose and Rohrlich, 1987).
We will consider two of these methods, not because they are
intrinsically superior to the others, but because this is
enough to make our points.
1. The method o f Abercrombie. Probably the most fre-
quently used assumption-based method is that of Abercrom-
bie (1946), and the number of studies that use this method
is increasing (Williams and Rakic, 1988). In this method,
nuclear profile counts (n) are multiplied by mean nuclear
3iameter (D) divided by mean nuclear diameter plus section
thickness (T) to yield neuron numbers (N); N = (n x D)/
[D + T). The reasoning is sound if the assumptions are
met. Some of the assumptions are that nuclei are spherical,
that one can recognize any fragment of a nucleus sectioned
by the microtome knife (no lost caps or invisible fragments),
and that the sections are perfectly smooth. But nuclei are
not true spheres, thin slices of nuclei do fade into back-
g-ound, and sections are not perfectly smooth. And to the
zxtent that the assumptions are not met, the method will
not estimate cell or synapse numbers accurately. We sup-
2ort this note of caution, for we determined numbers of a
sample population of cells in adult rat dorsal root ganglia by
serial reconstructions and then estimated this population
Nith the method of Abercrombie, and the estimates were
naccurate (Coggeshall et al., 1990). Our unpublished data
;how the same for counts of embryonic dorsal root ganglion
:ells.
2. The method ofKonigsmark (Konigsmark, 1970). This
nodification of the methods of Abercrombie (1946) and
Floderus (1944) is particularly concerned with invisible
i.agments. Konigsmark’s formula is N = (n x T)/(T +
2Jlr2_(k/2)2]), where N = total neuronal numbers, n =
,otal neuronal nucleolar numbers (Konigsmark counted
iucleoli rather than nuclei because the former are rounder,
;maller and more intensely stained), T = section thickness,
= mean nucleolar diameter, and k = the height of the
nvisible nucleolar fragment. The assumptions are the same
is for the Abercrombie method and, in addition, one must
issume that the height of the invisible fragment can be
letermined, which is problematical because of limitations
if the resolution of the light microscope. If these assump-
ions are met, the method would provide unbiased esti-
nates of neuron numbers; but when we calibrated, the
:stirnates were inaccurate (biased) (Coggeshallet al., 19901,
ndicating that the assumptions were not met.
3. Other assumption-based methods. We could have a
;imilar discussion for the other assumption-based methods,
)ut the real point is that it is not possible for another
nvestigator to tell whether the assumptions are met unless
R.E. COGGESHALL AND H.A. LEKAN
ods, inaccurate estimates of known populations resulted
(Coggeshall et al., 1990; Pakkenberget al., 1991).
Summary. Assumption-based methods make geometric
assumptions that allow profile counts to be converted to
neuron or synapse numbers. Such assumptions require
measuring other variables (e.g., nuclear diameters). The
assumptions are reasonable, and if they are met, unbiased
estimates would result. The difficulty is that the assump-
tions are usually not met, and hence, bias will be present.
And unless calibration is done, there is no way to know
whether bias is present or not.
D. Stereological methods
The difficulties with both profile counts and assumption-
based methods arise because neurons or synapses are
counted in single optical planes. This necessitates equating
profile counts with cell or synapse numbers (profile count-
ing methods), or making assumptions that allow profile
counts to be converted to cell or synapse numbers (assump-
tion-based methods). The difficulties with these procedures
have been considered. Prior to 1984, the only ways to be
certain of unbiased estimates of cell or synapse numbers
were to calibrate or reconstruct in serial sections. But
calibration is rarely done, and serial reconstructions are so
labor intensive that such counts usually cannot be com-
pleted in a reasonable amount of time. Then in 1984, the
disector principle was described (Sterio, 1984). The remark-
able insight was that one need not find all the profiles of
each neuron or synapse, as in serial reconstructions; one
need only to find the “last” profile of the object or of a
unique point associated with the object. The “last” profile
is a profile seen in one section but not the next (called a top
or count, symbolized by Q- in the stereological literature).
Unique associated points are defined by the investigator
and are such things as cell bodies, nuclei, nucleoli (provid-
ing cells have only one nucleolus), or postsynaptic densities.
The disector principle led to a type of serial analysis well
suited for sampling, and so provided unbiased estimates of
cell and synapse numbers efficiently. This was first done by
using pairs of sections, the physical disector, so named
because each pair of sections is a di-sector (Sterio, 19841,
and more recently two optical planes in one section, the
optical disector (Gundersen, 1986; Gundersen et al., 1988a,b;
West, 1993, 1994) or three-dimensional counting (Williams
and Rakic, 1988, 1989). We will consider these methods in
turn with the aim of showing that these are types of serial
reconstructions that are well suited for sampling.
1. The physical disector. The basic procedure of the
physical disector is to locate profiles of the objects under
study in one section (the reference section) but count only
those that are not found in a serial section (the look-up
section). To show that this is a serial reconstruction
method, one could take A in Figure 1 as the first reference
section, and B as the first look-up section. Any profile in A
but not B would be a count. Then one would take B as the
reference section and C as the look-up section, and continue
this procedure until the reference space is analyzed. When
this is done an exact count of 10 results (Fig. 1, DIS
column). If one uses thicker sections (Fig. 2) or smaller
objects (Fig. 31, accurate counts still result (Figs. 2 and 3,
DIS column). Thus the physical disector can be considered a
type of serial analysis, and gives the same numbers as a
classic serial reconstruction. The only difference is that the
section in which each particle is actually counted is usually
COMMENTARY ON NUMBERS OF CELLS AND SYNAPSES
A complete physical disector reconstruction, where all
sections are examined as parts of disector pairs, would be no
more efficient than a classic serial reconstruction, where all
sections are examined individually. Physical disectors are
suited for sampling, however (Gundersen et al., 1988a,b),
and so an investigator only need examine widely spaced
section pairs (or more commonly only small parts of widely
spaced section pairs) to get a n unbiased estimate efficiently
(Pakkenberg and Gundersen, 1989; Coggeshall, 1992). This
is essentially impossible for classic serial reconstructions.
a. Uses of the physical disector. The physical disector is
usually used where resolution (the ability to see profiles of
the particles of interest clearly) is important. This would
include almost all electron microscopic studies.
b. Disadvantages of the physical disector. i. Inefficiency.
Although the physical disector is much more efficient than
serial reconstructions, two sections must still be compared.
One can reduce the time of comparison by projecting images
of the sections onto each other, or photographs or drawings
of profiles can be matched, but these procedures consume
time.
ii. Matching. Any time numerous objects are present,
there may be difficulty in matching (deciding which profiles
come from the same object and which from different objects
in the two sections). This is particularly difficult when the
elements to be counted are small and closely packed (cf.
granule cells of the cerebellum). One solution is to use the
optical disector (see below), but if this is not feasible, then
one can only reduce section thickness and/or separation
with resultant increase in clarity but loss of efficiency.
2. The optical disector. The optical disector is similar to
the physical disector except that instead of two sections, one
uses two optical planes in one thick section, and instead of
counting profiles in one section not found in another, one
counts cells that come into focus as one passes from the first
to the second optical plane. To show that this is a type of
serial analysis, we can start with section A in Figure 2 and
determine how many structures come into focus as one
focuses down through the section (in this case there are 3).
If one does all the sections this way, a n accurate count
results (Fig. 2, OPT DIS column). If we reverse the
sections, a similar accurate count emerges. If we use
thinner sections (Fig. 1) and smaller objects (Fig. 3),
accurate counts still result. Note that classic serial recon-
structions and the disector analyses give the same num-
bers. And as with physical disectors, it would be no more
efficient than classic serial analyses to analyze every section
in a whole tissue with the optical disector (West, 1993); but
the optical disector gives estimates more efficiently than
either the physical disector or classic serial analyses be-
cause one deals only with widely spaced single sections
instead of pairs of sections (the physical disector) or all
sections (classic serial reconstructions) (West et al., 1991).
a. Uses of the optical disector. The major use of the
optical disector is for counting structures in the light
microscope, and one can proceed with the counts almost as
if one was counting profiles. This method also solves the
profile matching problem encountered with the physical
disector because there is no matching with the optical
disector, one simply counts the objects that come into focus
between two focal planes in a single section.
6. Disadvantages of the optical disector. The main
disadvantage of the optical disector is that the section must
be relatively thick. This prohibits most electron microscopic
studies. In addition, it may become difficult to see or stain
13
used, there is no question that the optical disector is a major
time saver when trying to get unbiased estimates of cell
numbers efficiently.
Summary. Stereologic methods using the disector prin-
ciple provide unbiased estimates because they are a type of
serial section analysis and are relatively efficient because
they are suited for sampling.
Overall summary of counting methods
There are four ways to determine numbers of neurons
and synapses in histological sections. The first is serial
reconstructions. This method gives completely accurate
counts but is inefficient. The second is profile counts. This
is the most common method, but what is being estimated is
numbers of profiles rather than numbers of cells and
synapses. The third is assumption-based methods. These
methods make assumptions that allow profile counts to be
converted to cell or synapse numbers, but the assumptions
are abstractions and so are rarely met. The real difficulty is
that a reviewer or another investigator cannot tell whether
the assumptions are met or not without calibrations.
Finally, there are stereological methods that provide unbi-
ased estimates of cell and synapse numbers relatively
efficiently.
V. SAMPLING STRATEGIES
No matter what counting method was used, every study
we investigated that counted in histologic sections was
estimating numbers or densities of cells or synapses. There
are two major issues in evaluating such estimates. The first
is what was counted. This is considered in the section on
counting methods. The second, of equal importance, is how
the material was sampled. Sampling strategies have been
discussed in considerable detail (Weibel, 1979; Gundersen
and Jensen, 1987; Gundersen et al., 1988a,b; West, 1993,
1994), but the guiding principle is simple, namely, that
every neuron or synapse in the chosen reference space must
have an equal chance of being sampled. We will consider
this principle and two practical issues relating to it: system-
atic random sampling and the fractionator.
The equal opportunity rule
The basic requirement for unbiased sampling is that
every cell, synapse, profile, or indeed anything whose
numbers are being estimated must have a n equal chance of
being selected for the sample that leads to that estimate. We
call this the equal opportunity rule. The necessity of equal
opportunity is intuitively apparent. If a profile, cell, or
synapse does not have equal opportunity, this means that
some profiles, cells, or synapses are preferentially selected;
but then one is estimating only the selected population. For
example, if one were trying to estimate numbers of syn-
apses in a nucleus, labelled cell profiles in a ganglion, or
dying cells in a tumor, one would not sample only large
synapses, heavily labelled profiles, or cells in the center of
the tumor because this would mean that only the numbers
of large synapses, heavily labelled profiles and central cells
are being estimated. This seems simplistic, but 46% of the
studies we sampled either violated the equal opportunity
rule (cf. counted profiles in only part of a region, usually the
center, or only where labelling was intense), or did not
provide enough information to allow another investigator
to tell whether equal opportunity was provided (cf. counted
labelled profiles in 3 otherwise unspecified sections of a
14
A. Systematic random sampling
How can one can be assured that all neurons or synapses
in the structures of interest have an equal opportunity for
being sampled? There are several ways (Weibel, 19791, but
systematic random sampling has come to the fore because it
is well suited for histologically sectioned material (Gun-
dersen and Jensen, 1987; Gundersen et al., 1988a,b). To do
systematic random sampling of sectioned material, one
chooses an appropriate section separation (k in the stereo-
logic literature), and starts counting from a section chosen
randomly between the first and the kth section of the
reference space (the R-th section in the stereologic litera-
ture) and then counts from sections R, R + k, R + 2k, etc.,
until the space is analyzed (Pakkenberg and Gundersen,
1989; Coggeshall, 1992).We emphasize that this is no more
work than is already being done. For example, it would still
be possible to count labelled cells in very few sections of a
nucleus, it is just necessary to choose the sections appropri-
ately. An advantage of systematic random sampling is that
one need count only about 100 cells or synapses to get
sampling variances to be negligible in comparison with
interanimal variances. Paradigmatic analyses on large tis-
sues are available (Pakkenberg and Gundersen, 1988,1989;
West et al., 1991; West, 1993, 1994).
B. The fractionator
Ideally, densities should be converted to total numbers. A
common way to do this is to multiply a density (numbers
per unit size) by the total size of the reference space (cf.
N, x Vref). Sometimes this must be done. Other times,
however, one can do fractionator sampling. A simple ex-
ample of fractionator sampling is to count objects in every
kth section and multiply by k. The fractionation can be
reduced to much smaller samples, however, in that one only
need count small regions of only a few sections taken from
only a few parts of even a large reference space (Gundersen
et al., 1988a; West et al., 1991). Fractionator sampling is
very appealing because one need not determine the volume
of the reference space (Vref).As long as all parts of the
reference space have an equal chance of being sampled and
one knows what fraction of the space is being sampled, one
simply multiplies the counts from the sampled regions by
the reciprocal of that fraction to get an unbiased estimate of
total numbers.
VI. GENERAL COMMENTS
In our experience, most investigators accept that serial or
stereological methods are the most accurate presently
available, but these investigators also see these methods as
labor intensive and unnecessarily complicated. Criticisms
of profile or assumption-based counts are not accepted,
particularly when 1) no obvious changes are seen in the
size, shape, or orientation of the counted objects; 2) the
investigators are simply determining densities or density
ratios; and 3) the conclusions are only that there are
significantly more or fewer neurons or synapses in the
experimental case. So we can turn the question around and
ask if profile and/or assumption-based counts always lead
to biased data and inaccurate conclusions. The answer is
certainly not. For profile counts, it is perfectly possible that
with certain material, counts excluding edges would lead to
estimates statistically indistinguishable from the true num-
bers. Similarlv. who is to sav that the assumDtions in anv
R.E. COGGESHALL AND H.A. LEKAN
particular assumption-based study are not met? But inves-
tigators must also realize that just as the critiquer cannot
prove that profile or assumption based counts are inad-
equate, the investigator cannot prove that they are ad-
equate unless they calibrate (estimate known populations
using the chosen method). It must be remembered, how-
ever, that when calibrations were done, most of the assump-
tion based methods that were tried gave estimates that
differed from true numbers by 15-60% and gave very high
standard deviations (Coggeshall et al., 1990; Pakkenberg et
al., 1991). In addition, a single calibration is not enough
because each time conditions change (age, sex, disease,
experimental conditions, fixative, etc.), cell or synapse sizes,
shapes, etc., may also change. Therefore, one needs to
recalibrate every time a condition changes. This is ineffi-
cient. Admittedly, however, if the calibrations show that
the biases are under control, then profile or assumption
based counts or ratios are certainly acceptable.
VII. RECOMMENDATIONS
1. Profile counts
A. Neither profile counts nor profile ratios have any
direct relation to neuron and synapse numbers. So, as a
short term goal, we recommend that reviewers insist that, if
profiles are counted, investigators say this clearly and
discuss their reasons for using profile counts as indicators
of cell or synapse numbers.
B. As a long-term goal, we recommend that reviewers not
accept profile counts as indicating either absolute numbers
or changes in numbers of cells or synapses unless calibra-
tions have been done.
2. Assumption based counts
A. As a short term goal, we recommend that investigators
who use assumption-based methods be required to discuss
the major assumptions that they make to convert the
profile counts to cell numbers.
B. As a long term goal, we recommend that numerical
estimates by assumption-based methods not be accepted
unless the counts are calibrated.
3. Strategies
No matter what counting method is used, we recommend
that such estimates be accepted only if all parts of the
anatomical region under study have an equal chance of
being sampled. As an aside, we emphasize that it is as easy
to estimate total numbers as it is to estimate densities; it is
just that attention must be paid to appropriate sampling
strategies.
We feel that, if accepted, these recommendations will lead
to more uniform and thus fairer evaluations of estimates of
cell and/or synapse numbers. This would also result in a
compilation of a database consisting of unbiased estimates
that would be a useful baseline for future studies.
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