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J. Anat. (1992), 180, pp.

233-237, with 2 figures Printed in Great Britain

233

Fast and slow intrafusal fibre type systems in chicken leg muscle spindles
ALFRED MAIER
Department of Cell Biology, University of Alabama at Birmingham, USA (Accepted 10 October 1991)

ABSTRACT

The results of this study indicate that in serial sections incubated with monoclonal antibodies against MHC and stained for mATPase, intrafusal fibres in chicken leg muscle spindles are separable into slow and fast types. As observed here, fast fibres are a homogeneous group; however, it had been shown earlier (Maier & Zak, 1990) that based on relative amounts of slow MHC, the slow fibres can be further divided into 2 subgroups. These 3 types of intrafusal fibre conform to 3 recently demonstrated patterns of motor innervation (Maier, 1991). It is suggested that the respective actions of slow and fast intrafusal fibres produce different components of the afferent discharge of chicken muscle spindles. with a monoclonal antibody against fast MHC to determine if avian intrafusal fibre populations could be subdivided according to their presumed contractile speed. Myosin heavy chain profiles were also compared with patterns of actomyosin ATPase (mATPase) staining to see if results from this histochemical reaction corroborated data obtained by immunohistochemistry.
MATERIALS AND METHODS

INTRODUCTION

Some intrafusal fibres in chicken leg muscle spindles react with a monoclonal antibody (CA83) (Maier and Zak, 1990; Maier, 1991) that also binds to atrial myosin heavy chain (MHC) and to the SM I and SM2 MHC isoforms of the slow-tonic chicken anterior latissimus dorsi muscle (Sweeney et al. 1987). From this reactivity profile it may be concluded that the antibody detects the presence of 'slow' MHC. It is currently not known what isoform is present in those intrafusal fibres which do not react with the CA83 antibody. In mammals there is diversity in contractile speed among infrafusal fibres. Nuclear chain fibres contract fastest and nuclear bag, fibres slowest, while nuclear bag2 fibres assume an intermediate position (Bessou & Pages, 1975; Boyd et al. 1977). Considering this tripartite system, and the relative proximity to each other on the evolutionary scale of birds and mammals, it would seem plausible that the intrafusal fibres in chicken leg muscle spindles which do not present slow MHC may contain fast MHC instead. Knowing this would be a major step towards a satisfactory classification of avian intrafusal fibres and towards deciphering the internal working of avian muscle spindles. Hence chicken intrafusal fibres in the current study were, besides the CA83 antibody, also incubated
USA.

Seventy-five muscle spindles from extensor digitorum longus were examined from 4 White Leghorn chickens age 8 wk. Animals were killed by injecting a lethal dose of sodium pentobarbital intraperitoneally. Muscles were dissected and cut into manageable blocks, which were then frozen in melting isopentane (-159 C) and cross-sectioned serially in a cryostat (-20 C). Sections were transferred to glass slides, allowed to dry at room temperature for 30 min and subsequently processed. Sections from 4 consecutive cross-sections* were, in order, incubated with a monoclonal antibody against 'slow' MHC (CA83) (Sweeney et al. 1987), incubated with a monoclonal antibody against fast MHC (MY32) (Sigma Chemical Company; Harris et al. 1989), incubated for mATPase, acid preincubation, or incubated for mATPase, alkaline preincubation (Guth & Samaha,

Correspondence to Dr Alfred Maier, Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294,

234

A. Maier

Table. Details of antibodies used


Primary antibody
Working dilution
Known reactivities

CA83*
MY32*

1:100
1:400

Chicken atria, SMI and SM2 MHC isoforms of chicken anterior latissimus dorsi muscle Adult fast and neonatal MHC in mammalian and avian skeletal muscle

* Secondary antibody: HRP-conjugated goat-antimouse IgG, diluted 1:75 (Organon Teknika, Durham, NC). 3,3'diaminobenzidine was used as chromogen.

CA83
U
U

.#Ati
=_ ZJ E ._>
_-!

Reference

Sweeney et al. 1987


Sigma Chemical Co., Harris et al. 1989

.,. ., . .A :E -. S C...

t. ,

..JEx.,._.

.. alli_
_. -

.. ..

a}.ff

...

A--v.,.*
.v
4.1
.T.

S M
M

(a)

pH 4.6
U U
~~~~~~~~~~~~~~.
A.

M.~

*. ~~~~~~~~~ t..s.

(c)

(di)

X_

Fig. 1. Serial frozen cross sections at the encapsulated polar region of a chicken extensor digitorum longus muscle spindle incubated (a) with the anti-slow CA83 antibody, (b) with the anti-fast MY32 antibody, (c) for mATPase, acid preincubation, pH 4.6, and (d) for mATPase, alkaline preincubation, pH 9.6. The S and M fibres react with CA83 and stain dark after acid preincubation (a, c), while the U fibres react with MY32 and stain dark after alkaline preincubation (b, d). These profiles are characteristics of slow and fast muscle fibres, respectively. Note that S fibres are smallest, U fibres are largest, and M fibres are of intermediate size. Bar = 10 g.m.

1970). This sequence was repeated until a given block was completely cut. Incubation with the primary antibodies was for 2 h and with the secondary HRPconjugated antibodies I h (37 C). Stock solutions of antibodies were diluted with 0.5% Triton X-100 and 0.5 % BSA in PBS. Sources, dilutions and known reactivities of primary and secondary antibodies are listed in the Table. Acid preincubation (pH 4.6) lasted

7 min and alkaline preincubation (pH 9.6) 20 min (room temperature). Incubation with the substrate ATP was held to 45 min (room temperature). After all reactions were completed, sections were dehydrated and permanently mounted. Sections were examined on an Ortholux II Leitz microscope. Individual intrafusal fibres were traced through serial sections and photographed, or drawn

Chicken intrafusal fibres

235

with the aid of a camera lucida (100 x ). Specific fibre types were determined from their reactivities recorded on the drawings or on the photographic prints.
OBSERVATIONS

Previous work (Maier & Zak, 1990) established that at the encapsulated polar region intrafusal fibres in chicken leg muscle spindles can be reliably grouped into 3 types according to their reactivity with the antislow CA83 antibody. The degrees of reactivity of given intrafusal fibres with this antibody are either strong, moderate or unreactive. Thus, using the initial letters of the 3 grades of staining, types have been designated as S, M and U fibres, respectively. These terms will also be applied to intrafusal fibres in the current study. Fibres either reacted with the CA83 antibody that binds with slow MHC, or with the MY32 antibody that binds with neonatal and with fast MHC (Harris et al. 1989). There were no intrafusal fibres that reacted strongly with both. In some instances the M fibres reacted slightly after incubation with MY32. Antibody reactions and mATPase staining produced concordant patterns. Fibres reacting with the MY32 antibody (U fibres) stained dark after alkaline preincubation and were negative or stained only
E
CA83 (anti-slow MHC) M :

J
I
I

S. -I

UC
SC MY32 (anti-fast MHC) M C

M.l

UI
pH 4.6 mATPase

S
M

Ul
pH 9.6 mATPase

lightly after acid preincubation. On the other hand, S and M fibres reacting with the anti-slow CA83 antibody stained dark or moderate after acid preincubation and light to moderate after alkaline preincubation. The moderate mATPase staining after acid preincubation usually occurred in M fibres. The earlier reported (Maier & Zak, 1990) differences in size between S (small), M (medium) and U (large) fibres were again observed (Fig. 1). For the purpose of reconstructing MHC and mATPase profiles, intrafusal fibres were divided along their length into equator, juxtaequator and pole. The equator receives only sensory terminals, has no organised myofibrils, and is overlain by the collagenous perifibral sheath. The juxtaequator lies just distal to the equator. It receives both sensory and motor terminals and contains some myofibrils. In most cases the perifibral sheath is absent, typically ending near the equatorial-juxtaequatorial junction. The pole contains the greatest number of myofibrils and is only contacted by motor axons (Maier & Mayne, 1987; Maier, 1991). At the equator and juxtaequator M fibres did not react with CA83 or did so only very lightly. Similarly, the mATPase staining after acid preincubation became less towards the equator in both kinds of reactive (S and M) fibres. Myosin heavy chain content and mATPase staining from the pole to the equator were more uniform in U fibres than in S and M fibres (Fig. 2). The apportionment to muscle spindles of the 3 types of intrafusal fibre varied to some extent. Strongly reactive fibres were usually present in low numbers, occurring at about a rate of 1-3 per spindle. The M fibre had the most sporadic distribution. There might be as many as 3 or 4 per spindle (Fig. 1); however, the most frequent counts were 1 or none. Typically, there were more U fibres (2-4) than S fibres. The average counts in the 75 spindles that were surveyed were 1.8 S fibres, 0.9 M fibres and 3.2 U fibres. This means that fibres containing slow MHC were slightly outnumbered by fibres containing fast MHC.
DISCUSSION

S
M

U
Fig. 2. Diagram reconstructed from serial cross sections, showing MHC content and mATPase staining at the equator (E), juxtaequator (J) and encapsulated pole (P) in the 3 types of chicken intrafusal fibre. Solid dark indicates strong reaction, light no reaction and diagonal lines moderate (lines closer together) or weak (lines farther apart) reactions. Myosin heavy chain content and mATPase staining are complementary in each fibre type. (1) reacts sometimes slightly at the pole; (2) may react only moderately at the pole.
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It has been demonstrated in the current study that the U fibres which do not bind with the CA83 anti-slow MHC antibody react with the anti-neonatal/fast MY32 antibody. Mammalian intrafusal fibres react with an antibody against embryonic MHC (Maier et al. 1988); therefore, it cannot be ruled out that despite the age of the animals used, the staining observed after incubation with MY32 reflects the presence of
ANA 180

236

A. Maier

neonatal instead of adult MHC. Yet several lines of evidence favour the notion that staining with the MY32 antibody identifies in 8 wk postnatal chicken intrafusal fibres a fast isoform. For one, the ultrastructure of small intrafusal fibres (presumptive S fibres) and large intrafusal fibres (presumptive U fibres) resembles slow-tonic and fast-twitch extrafusal fibres, respectively (Ovalle, 1989). Secondly, motor endings on the larger U intrafusal fibres are reminiscent of the plate type found on twitch fibres, while endings on the smaller S intrafusal fibres resemble the en grappe configuration typical of slow-tonic fibres (Maier, 1991). Lastly, the mATPase staining of U fibres conforms to a profile characteristic of fast fibres (Burke et al. 1971; Staron & Pette, 1986). Thus it appears that there are 2 broad intrafusal fibre systems in chicken leg muscle spindles, slow and fast. The fast group as identified with antibody MY32 is a homogeneous population, while the slow group contains fibres that react either strongly (S fibres) or moderately (M fibres) with antibody CA83. The M fibres often display a reduction in slow MHC towards the equator as detected by a decrease in reactivity with CA83, and sometimes show a slight reactivity with the anti-fast MY32 antibody at the polar region. Concurrence of fast and slow MHC is characteristic of transitional IIC extrafusal fibres (Staron & Pette, 1986). The notion of separateness of M and S fibres is further emphasised by the recognition of occasionally only moderate mATPase staining at the pole in M fibres after acid preincubation, although as a group both S and M fibres exhibit an mATPase profile characteristic of slow fibres (Burke et al. 1971; Staron & Pette, 1986). Motor innervation of M fibres also differs somewhat from that of S fibres (Maier, 1991), and in addition the M fibre does not occur in each receptor, while S and U fibres do. Hence there are several attributes which qualify M fibres as an intermediate type between S (slow) and U (fast) fibres, ranking closer, however, to S than to U fibres. It is not yet known whether this diversion of M from S fibres is the result of separate inductive sequences or if it represents an arrested developmental stage. Events involving regulation of gene expression might account for the inconsistent frequency and distribution of M fibres. Unlike in mammals, bird intrafusal fibres are not separable at the equator into nuclear bag and nuclear chain types (Saglam, 1968; Maier & Eldred, 1971; James & Meek, 1973; Hikida, 1985). The lack of differentiation at that level, however, does not preclude their typing by other criteria. Differences in mATPase staining among populations of pigeon (Maier, 1977) and chicken (Ovalle, 1978; Toutant et

al. 1981; Toutant, 1982) intrafusal fibres have been recognised for some time, but because of the uniform appearance of their equatorial regions, these findings were difficult to reconcile with separate fibre systems along the line of mammalian nuclear bag and nuclear chain types (Boyd, 1962). In view of our recent findings (Maier and Zak, 1990; Maier, 1991) and the current study, there is now strong evidence of a fundamental subdivision within the chicken intrafusal fibre bundle. The distinction is between presumptive slow and presumptive fast fibres as deduced from their MHC content and mATPase staining, regardless of the appearance of their equatorial regions. Both fibre groups are also characterised by differences in motor innervation. Unreactive (U) fibres mostly have plate endings, while (despite some differences between S and M fibres) endings reminiscent of the en grappe type are more common on these 2 types than on U fibres (Maier, 1989, 1991). It is suggested that the morphological subdivision of the intrafusal fibre bundle in chicken muscle spindles is of primary importance in the function of this receptor. Physiological data on the avian spindle are virtually absent (Dorward, 1971); nevertheless, it seems plausible that contraction of fast and slow intrafusal fibres at different rates would affect the lengthening of the equatorial region and distortion of primary afferents differently. Consequently each fibre system should impart distinct discharge characteristics to the primary afferent fibres, as is generally accepted to occur in mammalian spindles (Boyd, 1981).
ACKNOWLEDGEMENTS

The CA83 antibody was a gift from Dr Radovan Zak, Department of Medicine, University of Chicago. This work was supported in part by a grant from the Muscular Dystrophy Association of America.
REFERENCES

BFssou P, PAGES B (1975) Cinematographic analysis of contractile events produced in intrafusal muscle fibres by stimulation of static and dynamic fusimotor axons. Journal of Physiology 252,

397-427.
BoYD IA (1962) The structure and innervation of the nuclear bag muscle fibre system and the nuclear chain muscle fibre system in mammalian muscle spindles. Philosophical Transactions of the Royal Society, London B 245, 81-136. BOYD IA (1981) The action of the three types of intrafusal fibre in isolated cat muscle spindles on the dynamic and length sensitivities of primary and secondary endings. In Muscle Receptors and Movement (ed. A. Taylor and A. Prochazka), pp. 17-32. London: Macmillan. BOYD IA, GLADDEN MH, MCWILLIAM PN, WARD J (1977) Control of dynamic and static nuclear bag fibres and nuclear chain fibres by y and fti axons in isolated cat muscle spindles. Journal of Physiology 256, 133-162.

Chicken intrafusal fibres


BURKE RE, LEVINE DN, ZAJAC FE, TSAIRIS P, ENGEL WK (1971) Mammalian motor units: physiological-histochemical correlation in three types in cat gastrocnemius. Science 174, 709-712. DORWOOD PK (1971) Response characteristics of muscle afferents in the domestic duck. Journal of Physiology 211, 1-17. GUTH L, SAMAHA FJ (1970) Procedure for the histochemical demonstration of actomyosin ATPase. Experimental Neurology 28, 365-367. HARRIs AJ, FITZSIMONs RB, McEWAN JC (1989) Neural control of sequence of expression of myosin heavy chain isoforms in foetal mammalian muscles. Development 107, 751-769. HIKIDA RS (1985) Spaced serial section analysis of the avian muscle spindle. Anatomical Record 212, 255-267. JAMES NT, MEEK GA (1973) An electron microscopical study of avian muscle spindles. Journal of Ultrastructure Research 43, 193-204. MAIER A (1977) Variations in intrafusal fiber size within histochemically identified types of intrafusal fibers in the pigeon. American Journal of Anatomy 150, 375-380. MAIER A (1989) Contours and distribution of sites that react with antiacetylcholinesterase in chicken intrafusal fibers. American Journal of Anatomy 185, 33-41. MAIER A (1991) Axon contacts and acetylcholinesterase activity on chicken intrafusal muscle fiber types identified by their myosin heavy chain composition. Anatomy and Embryology, in press. MAIER A, ELDRED E (1971) Comparisons in the structure of avian muscle spindles. Journal of Comparative Neurology 143, 25-40. MAIER A, MAYNE R (1987) Distribution of connective tissue proteins in chick muscle spindles as revealed by monoclonal antibodies: A unique distribution of brachionectin/tenascin. American Journal of Anatomy 180, 226-236.

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