muscle fibres and efferent nerves in a crustacean receptor … · 2006. 5. 26. · muscle fibres...

551

Muscle fibres and efferent nerves in a crustaceanreceptor muscle

By MELVIN J. COHEN(From the Department of Biology, University of Oregon, Eugene, Oregon, U.S.A.)

With 4 plates (figs. 2 to 5)

SummaryThe accessory flexor muscle of the crab myochordotonal organ consists of two kindsof muscle-fibres. In one type the fibrillar material has a uniform punctate appearancein cross-section. The Z bands are often broken across the fibre width and are spaced2 to 3/n apart. These resemble the 'Fibrillenstruktur' muscle of vertebrates. In thesecond fibre type the fibrillar material appears as large clumps in cross-section and itis often difficult to distinguish the outlines of individual fibres. The sarcomere lengthis 10 to i2/x. These resemble the 'Felderstruktur' muscle of vertebrates.

Two types of expanded structures are seen in conjunction with the two efferentneurones innervating this muscle. With fresh material under phase microscopy onetype appears as a 20 by 40 fj. rectangular plaque containing vacuoles as well as darkclumps and granules. The other kind of enlargement consists of a group of spheresgiving the appearance of a grape cluster and measuring 40 /x in its greatest dimension.It is suggested that these expanded structures may be associated with efferent nerveterminations in this muscle.

IntroductionT H E accessory flexor muscle in the walking legs of decapod crustaceans wasdescribed by Barth (1934) as forming part of a sense organ called the myo-chordotonal organ. During a physiological investigation of this sensory system(Cohen, 1960, 1963) the accessory flexor muscle was seen to have severalunique morphological characteristics. Two distinct structural types ofmuscle-fibres were observed. The efferent neurones innervating this muscleseem to have large specialized expansions unlike the fine tapered terminalscommonly described for crustacean motor terminations (Mangold, 1905; vanHarreveld, 1939; Hoyle, 1957; Wiersma, 1961).

The accessory flexor muscle plays a specialized role in a sensory system verymuch analogous to the intrafusal fibres of the vertebrate muscle-spindle(Cohen, 1963). The muscle seems to have been modified both functionallyand structurally for this special role. The present study describes the peculiarmorphology of this muscle and its efferent innervation and suggests somefunctional implications of this unique structure.

Methods and materialsThe local market crab Cancer magister Dana was used throughout this study.

Males ranging in weight between 900 and 1700 g were used. The animalswere obtained and kept in the laboratory as described by Cohen (1963).

[Quart. J. micr. Sci., Vol. 104, pt. 4, pp. 551-9, 1963.]

552 Cohen—Crustacean muscle and nerve

Walking legs 2, 3, and 4 were cut off either at the joint between the bodyand the coxipodite or between the coxipodite and ischiopodite. The bodystump was packed with soft wax to reduce bleeding. The anterior cuticle ofthe meropodite was removed and the accessory flexor muscle and associatedsensory structures exposed. All dissection was carried out with the limbimmersed in sea water kept between io° and 12° C.

The methylene-blue preparations were made after the method of Alexan-drowicz (1951). For phase contrast examination of muscle and efferent nervejunctions the fresh material was teased out on a slide and then gently flattenedwith a cover slip.

Two types of silver stains were used on paraffin sections cut at 10 and 20/x.The protargol silver technique of Stotler (1951) proved excellent for showingthe various types of muscle-fibres, while the modified Bielschowsky techniqueof Weiss (1934) proved most suitable for examination of efferent neuronesand their endings.

A modification of Masson's trichrome technique was also used for examina-tion of muscle-fibres. The technique was essentially that described by Masson(1929) except that the light green stain was made up as a 0-5% solution in90% ethyl alcohol rather than in acetic acid. The slides were transferred fromthe green stain directly into 95% ethyl alcohol, dehydrated through xyleneand mounted in xylene damar. The tissue was fixed in Bouin's fluid.

For the cholinesterase staining, fresh material was teased out on a slide andexcess water blotted from around the preparation. It was then fixed in a solu-tion of 10% formalin in sea water for 14 to 24 h and stained for cholinesteraseby the technique of Koelle (1951, 1955).

ResultsGross anatomy

The gross anatomy of the accessory flexor muscle and the entire myo-chordotonal organ has been described in detail by Cohen (1963) and will onlybe reviewed briefly here for purposes of orientation. As seen in fig. 1, theaccessory flexor muscle consists of a spindle-shaped proximal head and abroad flat distal head. The proximal head originates at the ventral proximaledge of the meropodite (figs. 1; 2, A) and sends a fine tendon the full lengthof the meropodite to insert on the tendon of the main flexor muscle. Thedistal head originates from the anterior cuticle of the meropodite and insertson the accessory flexor tendon just before the latter joins the tendon of themain flexor muscle.

A thick and a thin efferent nerve-fibre innervate the proximal and distalaccessory flexor heads (figs. 1, 4). As far as can be determined from examina-tion of well over 100 methylene-blue-stained preparations, these two neuronesdo not innervate any other musculature of the leg and provide the sole efferentinnervation for both heads of the accessory flexor.

The following results are obtained primarily from the proximal head of theaccessory flexor, but the situation in the distal head seems essentially similar.

Cohen—Crustacean muscle and nerve 553

Two types of muscle-fibres

If one looks at the proximal head of the accessory flexor (fig. 2), the muscle-fibres along the dorsal edge in the region of the attached elastic strand are seento be in loose bundles and less densely packed than in the remainder of themuscle. A transverse section of the muscle taken through the distal edge of the

FIG. 1. Anterior view of a right walking leg with the anterior cuticle removed to expose themyochordotonal organ sensory system and the main limb musculature. Dorsal is above andmedial is to the right. View of the ischiopodite (i), meropodite (m), and carpopodite (c) to showthe relationship of the accessory flexor system to limb musculature and joints. The accessoryflexor tendon (taf) runs the full length of the meropodite to attach to the main flexor tendon((/) near the m-c joint. Note the relationship between the proximal head of the accessoryflexor muscle (afp) elastic strand (es) and sense cell bodies (sc) in the region of the ischio-meropodite (i-m) joint. Two efferent nerve-fibres (en) leave the leg nerve (In) to innervateboth the proximal and distal (afd) accessory flexor heads. The main extensor tendon (te) is

also seen (modified from Cohen, 1963).

elastic strand (fig. 2, A) and stained with Masson's trichrome (fig. 2, B) showstwo distinct types of muscle-fibres. Along the dorsal edge adjacent to theelastic strand, there are a group of 18 to 30 loosely packed muscle-fibres rang-ing from 10 to 25 ju in diameter. The fibrillar material gives a punctateappearance in cross-section and seems to be more or less uniformly distri-buted throughout the fibre. Individual units of this material range from 1 to5/x in diameter and presumably constitute single muscle fibrils. In theremainder of the muscle-fibres, the fibrillar material appears in cross-sectionas large clumps separated by irregular canals. The canals often contain a finegreen line in the Masson's stain, which may be sarcolemma. In this part of the


muscle it is difficult to determine the outline of individual muscle-fibresbecause there is very little extracellular space and the peripheral boundingmembrane seems of the same type as that lining the internal canals.

Longitudinal sections further indicate the presence of two different kindsof muscle-fibres, as seen in fig. 3, A. Fibres showing the punctate fibrillardistribution in cross-section are seen at the right of the figure to have shortstriation intervals. The fibres with the large fibrillar clumps are to the leftin the figure and have very long striation intervals.

In fig. 3, B, c, fresh teased muscle-fibres are shown in phase contrast. Asingle fibre with long striation interval is shown in fig. 3, B to have two kindsof alternating dark bands composing the striations. One band is dense andforms a continuous structure across the width of the fibre. The other bandalso extends across the full width of the fibre, but is more diffuse. Withouta polarized light study it is not possible to say conclusively which of these twodark regions is the A band and which is the Z band. However, judging fromother phase contrast muscle studies (Hess, 1961a) it seems that the densecontinuous line is most probably the Z band and it will be considered so here.As the two kinds of bands are evenly spaced, with the distance between densebands equal to that between diffuse bands, one can measure sarcomere lengthon the basis of the interval between like bands; it is about 12/u. This fibretype has the large clump-like fibrillar distribution in cross-section. Fig. 3, cshows a fibre with short striation intervals of 2 to 3 JX. Here the dark transversebands appear fragmented and do not extend unbroken across the width of thefibre. This type of fibre with the short broken striations has the punctateappearance of the fibrils in transverse view.

Innervation of accessory flexor muscle

Efferent axons. The accessory flexor muscle is innervated by two efferentnerve-fibres, a thick one and a thin one, branching from the leg nerve andenclosed in a connective tissue sheath as seen in figs. 1 and 4, A. The thickfibre averages about 14^ in diameter and forms a characteristic 'Y' configura-tion when it sends a branch as the stem of the 'Y' ventrally across the flexormuscle to join the proximal head of the accessory flexor. The thin nerve-fibreaverages 2 to 3/x in diameter and its branching from the leg nerve is muchless regular than for the thick fibre. It sometimes accompanies the thick fibre

FIG. 2 (plate). A, fresh preparation of the proximal head of the accessory flexor muscle (afp)in the same orientation as seen in fig. 1. The elastic strand (es) is seen to pull the looselypacked 'Fibrillenstruktur' fibres out from the main muscle mass. The white area (en) is a bitof connective tissue attached to the efferent nerves just as they enter the muscle. The nervesare not in the plane of focus. A section through the muscle at plane 'p: is seen in the picturebelow.

B, transverse section taken through the accessory flexor muscle at the level indicated aboveby line 'p'. Stained with Masson's trichrome. 'Fibrillenstruktur' muscle-fibres are seen atthe right. Note the punctate distribution of the fibrillar material. These fibres are welldefined by a distinct outer limiting membrane and are loosely packed. The 'Felderstruktur'fibres are seen to the left, and show the fibrillar material clumped into larger irregular unitsseparated by wide channels.

M. j . coiii:\


branch along the proximal limb of the 'Y' (fig. 4, A) and other times it runsalong the distal limb. The initial branching of the thin fibre often occurs deep-within the leg nerve and is not readily observed. As the two efferent fibresrun toward the proximal accessory flexor head, they start simultaneous andrepeated branching about 3 mm after leaving the leg nerve and enter themuscle as a tangle of many nerve twigs (fig. 4, D). These twigs continuebranching profusely as they enter the muscle and turn to run proximally anddistally along its length.

After giving off the branch to the proximal head, the thick and thin fibresrun distally within the leg nerve almost the entire length of the meropodite toenter the posterior surface of the accessory flexor distal head where they bothterminate with profuse branching (fig. 1).

Efferent nerve expansions. As seen in fig. 4, D the thick and thin efferentfibres branch profusely upon entering the proximal accessory flexor head.Numerous varicosities and expansions are seen along the nerve-fibres and atfirst view these were thought to be artifacts commonly found in methyleneblue staining. However, under closer inspection, it is seen that many of thenerve expansions occur at the ends of branches rather than as characteristicbeading along the length of the nerve-fibre. Under high magnification, asseen in fig. 4, B, E, short branches are observed to come off a long fibre andthese branches end as well-defined terminal expansions averaging 10 to 15^,in their longest dimension. These expansions at nerve terminals look quitedifferent from the usual tapering free motor endings described in crustaceanmuscle (Mangold, 1905; van Harreveld, 1939; Hoyle, 1957). They give theappearance of an efferent nerve terminal apparatus.

Sections of the proximal accessory flexor head were cut and stained withsilver according to the technique of Weiss (1934). As seen in fig. 4, c, F,definite structured expansions occur along a nerve-fibre where it is in contactwith muscle. These expansions often appear slightly granular and correspondroughly in size to the expansions seen with methylene-blue staining. Theygive the impression of a specialized terminal structure.

However, the most convincing evidence for an expanded efferent terminalapparatus in this muscle comes from phase contrast observation on freshteased material. Such observations show the occurrence of two definite typesof specialized enlargements associated with nerves as seen in fig. 5. Fig. 5, Ashows an efferent nerve-fibre apparently terminating on a muscle-fibre by

FIG. 3 (plate). All figures from proximal head of accessory flexor muscle.A, longitudinal section showing the broad striation type muscle-fibres on the left and the

narrow striation muscle-fibres on the right. Stotler protargol silver stain.B, fresh teased preparation showing a portion of a single muscle-fibre with long striation

intervals viewed in phase contrast. This type of fibre shows the 'Felderstruktur' organizationof the fibrillar material in cross-section.

c, fresh teased preparation of a single short striation muscle-fibre seen in phase contrast.Note the irregular broken nature of the transverse bands and the short interval between them.This kind of fibre has the 'Fibrillenstruktur' type of organization in the sarcoplasm.


forming a rectangular plaque-like structure 20 to 3 o /x long and 1 o to 15 /x wide.These plaques are filled with dense granules and have a few large, clear,vacuolar areas.

The second type of expansion associated with efferent nerve is seen in freshmaterial under phase contrast in fig. 5, c. A nerve-fibre merges into a grape-like cluster of spheres. Each sphere is 8 to 10 JU, in diameter and the entiremass of spheres ranges from 30 to 40 \L in diameter.

If teased preparations of this muscle are stained for cholinesterase accordingto the technique of Koelle (1951, 1955) the two types of expansions can beseen. Fig. 5, B shows a teased preparation incubated in an acetyl-thiocholinesubstrate and viewed under phase contrast. The definite rodlets and granulesoccupying the terminal are similar to those seen with phase microscopy in thefresh material (compare fig. 5, A and B). Fig. 5, D shows a 'grape cluster' typeof expansion viewed from above, after incubation in a butyryl-thiocholinesubstrate and stained for cholinesterase. Fine granules may be seen at theperiphery on the right side of the structure and dark clumps of material appearnear the centre. In general, the 'plaque' enlargements are more clearly seen ifincubated in acetyl-thiocholine and the 'grape cluster' structures seem to showup better if incubated in butyryl-thiocholine. The lack of cleanly differentiatedintensely darkened areas after staining for cholinesterase leaves it open toquestion whether or not these regions do indeed contain cholinesterase. Allthat can be said at present is that the structures are not readily evident withbright field microscopy before treatment with the cholinesterase techniqueand that they can be seen following Koelle's procedure. This may be due tonon-specific density changes induced by the fixation and staining processes.The material is included here simply as another piece of evidence substantiat-ing the existence of these specialized structures.

DiscussionThe striking morphological differentiation of two muscle-fibre types in the

proximal accessory flexor head is remarkably similar to the two kinds of

FIG. 4 (plate). A, methylene-blue stained whole mount of the thick and thin efferent nervefibres to the proximal head of the accessory flexor muscle showing the typical 'Y1 configurationwhere the thick fibre sends a branch toward the muscle. Proximal is to the left and ventral(toward the muscle) is to the top.

B to F are taken from proximal head of accessory flexor muscle.B, a part of D at higher magnification, showing an efferent nerve-fibre giving off branches

which end in definite terminal expansions.c, longitudinal section showing dense black efferent nerve with definite expansion along its

length.D, whole mount stained with methylene blue, showing efferent innervation just after the

nerves enter the muscle. Note the appearance of terminal expansions particularly in lowerright corner even at this low magnification.

E, a part of D at high magnification, showing an efferent nerve-fibre ending in an expan-sion resembling an arrow-head.

F, another longitudinal section showing a loop of nerve-fibre with a definite structuredexpansion believed to be an efferent nerve junction.

E to F stained with the Weiss silver technique.


striated muscle-fibres described in the vertebrates. Kruger (1949) and hisgroup have devoted considerable study to the comparative histology of extra-fusal muscle-fibres in frogs and birds. He divides striated muscle-fibres intotwo groups; those in which the muscle fibrils are distinct and give a punctateappearance in cross-section ('Fibrillenstruktur'), and those muscle-fibres inwhich the fibrils are grouped together in clumps and present an areal or diffusepattern in transverse view ('Felderstruktur'). He presents evidence sub-stantiating the hypothesis that muscle-fibres with' Fibrillenstruktur' are thosewhich produce a 'fast' or twitch-like response while the 'Felderstruktur' fibresare those of the 'slow' or tonic system. Further histological work has con-firmed this concept (Gray, 1957, 1958; Hess, 1960a). Work by Kuffler andVaughan Williams (1953) presents evidence that there are two functional typesin frog extrafusal muscle-fibres. The so-called 'fast' muscle-fibre gives atwitch-like response, and the 'slow' muscle-fibre produces graded contrac-tions. The latter, together with its innervation, has often been referred to asthe small motor system. The recent combined histological and functionalstudy of Peachey and Huxley (1962) has conclusively demonstrated in the frogthat 'Fibrillenstruktur' fibres are of the 'twitch' type while the 'Felderstruk-tur' is associated with slow muscle-fibres.

Gray (1957, 1958) and Hess (1960 a, b) also point out that there are twodistinct kinds of motor-nerve terminations on the different vertebrate muscletypes. The 'fast' or 'Fibrillen' muscle-fibres are innervated by classical motorend-plates which are often referred to as the 'en plaque' type of termination.There is generally only one such terminal per muscle-fibre. The slow or'Felderstruktur' fibres, on the other hand, are innervated by nerve-fibresterminating in small round grape-like masses which have been termed 'engrappe' motor endings. A single slow muscle-fibre may have up to 13such endings (Hess, 1960a). Hess also demonstrated the presence of the twomuscle-fibre types and endings in chicken (1961&) and guinea-pig (1961a) andhas suggested a correlation with 'fast' and 'slow' type muscle-fibres.

The same two kinds of muscle-fibres and motor terminations have also beenobserved in the intrafusal fibres of the amphibian muscle-spindle (Gray, 1957).Boyd (1962) described two types of muscle in the mammalian muscle-spindlebut raises some question as to whether they fit into Kruger's classification.

FIG. S (plate). All figures are from the proximal head of the accessory flexor muscle.A, fresh teased preparation in phase contrast showing an efferent nerve-fibre merging with

an expanded 'plaque'. Note granules, vacuoles, and large dark clumps in the terminalexpansion.

B, preparation similar to A seen in phase contrast after staining for cholinesterase in anacetyl-thiocholine substrate. Note the definite structured aspect of the terminal expansion.

C, 'grape cluster' efferent nerve expansion seen in profile in fresh teased preparation, phasecontrast. Note nerve-fibre coming from the left and entering a cluster of spheres which isbelieved to be part of a nerve terminal region.

D, teased preparation showing 'grape cluster' expansion seen from above after staining forcholinesterase in butyryl-thiocholine substrate and viewed in bright field illumination. Thenerve enters the terminal region from above and large dark masses are seen in the lower leftportion of the structure while small granules are seen along the periphery at the right.


The muscle types found in the crustacean accessory flexor muscle systemcorrespond in many ways to the two kinds of vertebrate muscle-fibresdescribed above. In the accessory flexor fibre with short striations, the Z discextends broken across the fibre and the sarcoplasm is divided into smallregular longitudinal units giving a rather uniform punctate appearance intransverse section. This crustacean fibre type therefore has many of themajor structural characteristics of vertebrate'Fibrillenstruktur' muscle-fibres.The unbroken appearance of the Z disc in the long striation muscle-fibre andthe grouping of fibrillar material into large clumps closely resembles thestructural specialization of the vertebrate' Felderstruktur' muscle-fibre. Com-parison of figs. 2 and 3 in this oaper with figs. 1 and 2 in the paper by Hess(1961a) shows a remarkable similarity between the two kinds of muscle-fibresfound in the crustacean system and the two kinds of muscle-fibres found in theextra-ocular muscles of the guinea-pig. The strong correlation in the verte-brates between structure and function in striated musculature (Ginsborg,i960; Peachey and Huxley, 1962) leads one to believe that a similar functionalrelationship may exist for the two distinct types of fibres found in the acces-sory flexor muscle. Experiments are in progress to test this hypothesis andit appears that the 'Fibrillenstruktur' crustacean fibres may be 'fast' whilethose with 'Felderstruktur' may be 'slow', just as in the vertebrates.

It should be pointed out, however, that transitional stages between the twostructural extremes do appear (fig. 2, B) and the clean separation of thesemuscle-fibres into two categories may be an over-simplification. This pointhas also been made by Boyd (1962) for vertebrate muscle.

The appearance of large, well-differentiated expansions associated with theefferent nerves innervating the accessory flexor muscle is unlike the fine taper-ing motor endings commonly described for crustacean muscle (see Wiersma,1961, for review). Maynard and Maynard (i960) have described two kindsof efferent nerve terminations on muscle-fibres of the muscle receptor organsin the lobster. These endings, as seen with cholinesterase staining techniques,seem to consist of delicate arborizations and appear unlike the large structuresdescribed here. The electron microscopic studies of Peterson and Pepe(1961 a, b) on the muscle-receptor organs in the crayfish also indicate that theefferent nerve endings on the receptor muscles are in the neighbourhood of1 to 2/n and probably are similar to the fine tapering crustacean motor ter-minations previously seen with the light microscope. The expanded struc-tures associated with efferent nerve terminations described here appear toform some type of specialized nerve terminal apparatus in the accessory flexormuscle. The functional role played by these structures as well as their preciserelationship to the axon terminals and muscle membrane await furtherinvestigation.

This investigation was supported by PHS research grant B-1624 from theNational Institute of Neurological Diseases and Blindness, U.S. Public HealthService.


The technical assistance of Mr. Herbert Swick is much appreciated. Heaided this work under the sponsorship of the National Science FoundationUndergraduate Research Participation Program.

Mrs. Ruth Day of the Hallmark Fisheries, Charleston, Oregon, has gener-ously provided crabs for this work and her aid is gratefully acknowledged.

ReferencesALEXANDROWICZ, J. S., 1951. Quart. J. micr. Sci., 92, 163.BARTH, G., 1934. Z. wiss. Zool., 145, 576.BOYD, I. A., 1962. Phil. Trans., Ser. B, 245, 81.COHEN, M. J., i960. Anat. Rec, 137, 346.

1963- J- comp. Biochem. and Physiol., 8, 223.GINSBORG, B. L., i960. J. Physiol., 150, 707.GRAY, E. G., 1957. Proc. roy. Soc. B, 146, 416.

1958. J. Anat. (Lond.), 92, 559.VAN HARREVELD, A., 1939. J. comp. Neurol., 70, 267.HESS, A., 1960a. Amer. J. Anat., 107, 129.

19606. Anat. Rec, 139, 173.1961a. J. cell. comp. Physiol., 58, 63.19616. J. Physiol., 157, 221.

HOYLE, G., 1957. Comparative physiology of the nervous control of muscular contraction.Cambridge (University Press).

KOELLE, G. B., 1951. J. Pharmacol., 103, 153.1955. J. Pharmacol, exp. Ther., 114, 167.

KROGER, P., 1949. Anat. Anz., 97, 169.KUFFLER, S. W., and VAUGHAN WILLIAMS, E. M., 1953. J. Physiol., rai, 289.MANGOLD, E., 1905. Z. allg. Physiol., 5, 135.MASSON, P., 1929. J. tech. Meth. and Bull. int. Ass. Med. Mus., 12, 75.MAYNARD, E. A., and MAYNARD, D. M., i960. J. Histochem. Cytochem., 8, 376.PEACHEY, L. D., and HUXLEY, A. F., 1962. J. Cell Biol., 13, 177.PETERSON, R. P., and PEPE, F. A., 1961a. Amer. J. Anat., 109, 277.

19616. J. biophys. biochem. Cytol., I I , 157.STOTLER, W. A., 1951. Anat. Rec, 109, 387.WEISS, P., 1934. J. exp. Zool. 68, 393.WIERSMA, C. A. G., 1961. In: The Physiology of Crustacea, vol. II, edited by T. H. Waterman.

N.Y. (Academic Press).

muscle fibres and efferent nerves in a crustacean receptor … · 2006. 5. 26. · muscle fibres...

Documents