the structure and innervation of the myotomes of the lamprey

[ 575 ]

THE STRUCTURE AND INNERVATION OF THEMYOTOMES OF THE LAMPREY

BY A. PETERS AND B. MACKAYDepartment of Anatomy, University of Edinburgh

In lower vertebrates, the trunk musculature is in the form of segmental myotomes,in which the muscle fibres lie parallel to the long axis of the body and are attachedat their ends to connective tissue myosepta. Probably the most primitive arrange-ment of vertebrate somatic musculature is found in Amphioxus, where V-shapedmyotomes are present along the entire length of the body. In cyclostomes, thissimple segmental structure has been lost in the pre-otic region, but is present alongthe rest of the trunk, where it is not disturbed by the presence of paired fins as isthe case in higher fishes. Other primitive features of the lamprey are the failure ofthe ventral and dorsal roots of the spinal nerves to unite after emerging from thespinal cord, and the absence of myelin sheaths in the peripheral nerves (Peters,1960).Grenacher (1867) showed that the myotomes of the lamprey are built up of a

series of units (Kastchen), each of which contains two types of muscle fibre, parietaland central. This was confirmed by Maurer (1894) and Schiefferdecker (1911), whopointed out that the central muscle of a unit is in the form.of plates rather thanfibres. The peripheral nervous system has been studied by Tretjakoff (1927); andfurther important contributions to the innervation of the myotomes have been madeby Johnston (1908), though his observations were incidental to a study of the cranialnerves. Little can be added to Maurer's (1894) account of the structure of the myo-tomes at the light microscope level, but the mode of innervation of the segmentalmuscle units is not described in these early investigations. It was therefore decidedto re-examine the structure of lamprey myotomes, making a detailed study of theirinnervation, by the use of a silver-staining method, supplemented by cholinesterasestaining and electron microscopy. The results are considered in the light of recentobservations on the structure and function of muscle in the higher vertebrates.

MATERIAL AND METHODS

The trunk musculature of adult specimens of Lampetrafluviatilis, and of ammocoetesof L. planeri, was examined. For silver staining, material was fixed either inalcohol-formol-acetic (50% alcohol, 90 ml.; formol, 5 ml.; glacial acetic acid, 5 ml.),or in 10% formalin, and was stained by a silver nitrate-egg albumen method(Peters, 1958).For the demonstration of sites of cholinesterase activity, small blocks of tissue

were fixed for 1 hr. in 10% formalin, and frozen sections cut at 30-60/t. These werestained either with a modification of Koelle's (1950) acetylthiocholine technique,or with the azo-dye method of Lewis (1958); both procedures gave good results.For electron microscopy, small pieces of tissue were fixed for 1 hr. at 40 C. in 1%

576 A. Peters and B. Mackayosmic acid adjusted to pH 7-2 with a veronal acetate buffer (Palade, 1952). The tissuewas then washed in 10% alcohol, dehydrated, and embedded in 1:8-methyl-butylmethacrylate. Thin sections were cut with a glass knife, using a Porter-Blummicrotome, and examined in a Metropolitan-Vickers, E.M. 6, electron microscope.

RESULTS

Structure of the myotomesIn the lamprey, each myotome is folded behind the one in front, and in horizontalsections a myotome lies obliquely with its medial border anterior relative to itslateral border. Thus, in a transverse section through the animal, parts of three tofive myotomes are visible, the most anterior of which lies next to the skin, withthose posterior to it lying successively nearer to the midline (Text-fig. 1 A).

Silver-stained sections show that a myotome is composed of a series of hori-zontally arranged units, stacked one above the other. Each unit extends through-out the myotome from its lateral to medial, and cephalic to caudal extents, andusually contains six horizontal layers of muscle, though sometimes only five arepresent (Text-fig. 1B).The muscle of the dorsal and ventral layers of each unit differs from that of the

central layers in its morphology and innervation, and is composed of a series oflongitudinally oriented muscle fibres (parietal muscle fibres), each 20-30/e thick,lying side by side. Around the lateral aspect of the unit, where the myotomeabuts on the skin, these two layers are continuous with each other, but elsewherethe central layers are exposed and project beyond the limits of the parietal layers(PI. 1, fig. 2).

0

Each of the central layers is 40-60g thick, and appears to be a continuous sheetof muscle, for in sections stained by Wilder's reticulin method, no connective tissuepartitions can be seen subdividing the sheets internally, although a thin layer ofconnective tissue is present between adjacent layers of a unit. That each of thecentral layers is a complete sheet can be seen clearly in transverse sections throughammocoete myotomes, but in similar sections of an adult lamprey the centralplates are sometimes broken up (PI. 1, fig. 4), probably as a consequence of shrinkageduring preparation. Furthermore, it is possible to obtain horizontal sections passingthrough a single central layer over the whole extent of a myotome, and these showno apparent subdivision of the sheet into component fibres. Therefore each centrallayer can probably be considered as a single muscle plate.While the central muscle plates of each unit have only a thin network of connec-

tive tissue between them, each parietal muscle fibre is surrounded by a thick layerof connective tissue which binds it firmly to adjacent parietal fibres in its own andthe neighbouring unit (PI. 1, fig. 4), and at the edge of a unit is often continuouswith the subcutis of the skin. Thus, though the central plates may sometimesbecome separated during processing of the tissue, each layer of parietal fibres alwaysremains intact.While the parietal muscle fibres are well formed and clearly visible in the adult

lamprey, in a 10 cm. ammocoete they are less distinct and form only a thin layer(2-38# thick) on the outside of a unit. However, the thickness of the parietal layers

Structutre and innervation of myotomes of the lamprey 577

Posterior

Anterior

A

Central 7 7Parietal 7ZDCZ

Central -I;C

B Myoseptum

Text-fig. 1. Diagrams showing the arrangement, and composition, of lamprey myotomes. A, Por-tion of the body has been cut away to show the arrangement of the myotomes (M1-M7).These are W-shaped, and are folded obliquely one behind the other. The spinal cord (S) liesabove the notochord (N), and the ventral (V) and dorsal (D) nerve roots emerge alternatelyfrom each side of the spinal cord. Each ventral root divides into dorsal and ventral ramiwhich run on the medial surface of the myotomes; some of the nerve fibres from the ventralroots pass into the myotomes, as shown in the horizontal section, to innervate the parietalmuscle fibres. B, Enlargement of parts of two myotomes (M1 and M2 in A), showing thestructure of the muscle units which make up the myotomes. Each unit extends throughthe whole width of a myotome and consists of (usually) four central plates bounded, exceptat the medial and myoseptal surfaces, by a single layer of parietal muscle fibres. (See text.)

578 A. Peters and B. Mackayincreases as the animal grows, so that in the adult each parietal layer approachesthe thickness of a central muscle plate (P1. 1, fig. 3).

After silver staining, the myofibrils of the parietal muscle fibres appear thickerand more widely separated than those of the central muscle plates (P1. 1, fig. 3).(This is clearly indicated in the diagrams of Maurer (1894) and Schiefferdecker(1911).) In electron microscope sections, these differences in thickness of the myo-fibrils are not apparent, but the myofibrils of the parietal muscle (P1. 3, fig. 11) aremore widely separated by sarcoplasm than those of the central plates, and theparietal muscle sarcoplasm contains relatively larger numbers of mitochondria(P1. 2, fig. 9; P1. 3, fig. 11). In addition, the sarcoplasm of both types of musclecontains electron dense, homogeneous bodies (P1. 2, fig. 9; P1. 3, fig. 11), and theseare more numerous in the parietal muscle where they are often concentrated underthe sarcolemma (P1. 3, fig. 11).Both parietal fibres and central plates are attached to the myosepta by myo-

tendinous junctions which do not appear to differ in structure from those describedin other vertebrates by Schwarzacher (1959) and Muir (1961). At a myotendinousjunction, the sarcolemma displays numerous invaginations (P1. 2, fig. 10, J) fromwhich emerge the collagen fibrils of the myoseptum, while the myofilaments appearto be attached to the inner surface of the invaginated sarcolemma. The parietalfibres taper as they approach a myoseptum (P1. 1, fig. 2), so that the area occupiedby their myotendinous junctions is not extensive; the invaginations of their junctionsare, however, deeper than those of the central muscle plates which end bluntly bymyotendinous junctions extending through the entire width of the myotome fromits lateral to medial extent.

Innervation of the myotomesIn the lamprey, the pairs of dorsal and ventral nerve roots emerge alternately

from the spinal cord (Text-fig. 1 A) and remain separate throughout their peripheralcourse. The peripheral nerves are unmyelinated, but each axon is surrounded by asheath which is one Schwann cell thick (Peters, 1960). At this level, most of thenerve fibres fall within the size range 3 to 6,u, though some may be as large as 10tin diameter. The dorsal roots run through myosepta to the skin, and branches tothe myotomes have not been seen. Each ventral root divides into dorsal and ventralrami which course over the inner surface of the myotomes (Text-fig. 1A), givingbranches which either enter the myotomes to run between the muscle units, orsweep round the edges of the myotomes to enter the myosepta on each side. Thecourse of these nerves is most easily followed in horizontal sections.

In large ammocoetes, it can be seen that one, and less frequently two, nerve fibresfrom the ventral roots enter the myotomes between each of the muscle units, so thata nerve fibre is sandwiched between every adjacent pair of parietal muscle layers.Nerve fibres in this position proceed laterally, and give branches which innervateboth layers of parietal muscle fibres. They do not remain within the same myotome,however, for having innervated the medial half or two-thirds of a pair of parietalmuscle layers in the myotome, a nerve then crosses the anterior myoseptum toenter the myotome in front, again coming to lie between and to supply two adjacentparietal muscle layers in the lateral portion of that myotome (Text-fig. 2). Some-

Structure and innervation of myotomes of the lampreytimes a nerve may cross over into yet another myotome to innervate parietalmuscle fibres at the extreme lateral margin of this myotome. The distribution of someof these nerve fibres is shown in Text-fig. 2, which is based on the paths of nervefibres in a 10 cm. ammocoete of L. planeri. The distribution of these nerve fibres isthe same in adults of L.fiuviatilis, but while the nerves give offrelatively few branchesin this ammocoete, in the adult the branches are more numerous and they dividefrequently to give off fine collaterals which produce an extensive network betweenadjacent muscle units (P1. 1, fig. 5). Some of these fine branches appear to terminatein expansions on the surface of the parietal muscle fibres, but no well-defined end-plates have been observed in the silver-stained sections.The nerve branches which sweep round the edge of a myotome and enter the

myosepta are best observed in transverse sections, where it can be seen that they

Notochord

PosteriorAtro

Mo ~~~M3 M2 M4Text-fig. 2. Diagram to show the distribution of the nerves which enter the myotomes and run

between the muscle units. Nerves from the ventral roots (Vg-V4) enter the correspondingmyotomes (M1-M4) and innervate parietal muscle fibres in the medial part of this myotomebefore crossing the anterior myoseptum to pass into the lateral part of the myotome in front.Sometimes one nerve may innervate parietal muscle fibres in three different myotomes; forexample, V4 innervates M4, M3 and Ma. This diagram is based on reconstructions of the pathsof nerves in a 10 cm. ammocoete of L. planeri.

form an extensive network over the ends of the central muscle plates of the units.They do not extend beyond the edges of the central plates, however, so that whileeach muscle unit is surrounded by nerve networks on all sides, no nerve fibres arefound within the units.The two histochemical procedures used to demonstrate sites of cholinesterase

activity give similar results on lamprey myotomal muscle. In the frozen sectionillustrated in PI. 1, fig. 6, which has been cut in a parasagittal plane and stainedwith the acetylthiocholine technique, the muscle units are outlined by zones ofstain. Considerable diffusion has occurred, but it is possible to recognize that theenzyme is confined to the myoseptal margins of the units, and to the parietal fibresall along their length; the bulk of the central plates is free from stain. When diffusion

579

A. Peters and B. Mackayis minimized by reducing the incubation time (P1. 2, fig. 7) it is clear that most ofthe staining along the parietal fibres can be attributed to the nerve fibres runningbetween the units (P1. 2, fig. 7, N). With the azo-dye method, it is possible to reducenerve staining considerably, and these preparations show small areas of intensestaining at intervals along the parietal fibres. The supposition that they representneuromuscular junctions is supported by electron microscopy (PI. 3, fig. 11).

Cholinesterase is confined to the myoseptal margins of the central muscle plates(P1. 1, fig. 6; PI. 2, fig. 7). Part of this reaction may be due to a myotendinousresponse as at most vertebrate neuromuscular junctions (see Gerebtzoff, 1959), butit may be observed in PI. 2, fig. 8, that small circumscribed zones of intense staining(indicated by arrows) are present towards the periphery of the stained region. Thesemay well be the sites of motor end-plates, since electron microscopy shows thatthe end-plates (P1. 2, fig. 10, E) are situated just short of the actual edge of the plate,at the sides of the myotendinous junctions (P1. 2, fig. 10, J).The Schwann cell sheath is absent at myoneural junctions, and at this point the

basement membrane on the outside of the sheath blends with that of the sarcolemma(P1. 3, fig. 12, L). Apart from this common layer derived from the two basementmembranes, the axolemma and sarcolemma come into close proximity and areseparated by a distance of only 500-700 A. (P1. 3, fig. 12; PI. 4, fig. 13). Theaxoplasm of a nerve ending contains numerous vesicles, 250-500 A. in diameter(PI. 3, fig. 12; PI. 4, figs. 13, 14, V), and a higher density of mitochondria than isfound elsewhere in the axoplasm (P1. 3, fig. 12; P1. 4, fig. 14, M). These features alsocharacterize the neuromuscular junctions in other animals (e.g. Robertson, 1957).The parietal muscle is innervated by the nerves running between the muscle

units, and in electron microscope sections the neuromuscular junctions on a parietalmuscle fibre are found on the side of the fibre adjacent to the neighbouring muscleunit (P1. 3, fig. 11). A neuromuscular junction typical of a parietal muscle fibre isillustrated in PI. 3, fig. 12, and the Schwann cell sheath can be seen to be absentin two regions, E1 and E2, although the subjacent sarcolemma shows none of thecomplex infoldings that characterize the subneural apparatus of higher vertebrates(Couteaux, 1955). Sometimes a single nerve fibre may form neuromuscular junctionson two adjacent parietal muscle fibres, as in the case of the nerve illustrated inPI. 3, fig. 11, which forms neuromuscular junctions at the sites indicated by arrows,although at the magnification of the micrograph it is not possible to see them clearly.

In their morphology, most of the myoneural junctions on the central muscleplates resemble those of the parietal fibres where the subneural apparatus is eitherabsent or poorly developed (PI. 3, figs. 11, 12). At the myoneural junctions illu-strated in PI. 4, fig. 13, the sarcolemma adjacent to the nerve shows only one smallinvagination (arrow). In the few instances in which a well-defined subneural appara-tus has been observed at the neuromuscular junction on a central plate, it has thesame form as that described at reptilian (Robertson, 1956) and human (De Harven &Coers, 1959) neuromuscular junctions. The sarcolemma is thrown into numerousinvaginations which are lined by the sarcolemmal basement membrane, while theaxolemma does not enter these invaginations but bridges over them. However, inthe lamprey these invaginations are more irregular in their arrangement than thoseof either the reptilian or human myoneural junction where they are evenly spaced.

580

Structure and innervation of myotomes of the lamprey 581In the lamprey, axons terminating on either parietal or central muscle usually liein shallow depressions of the sarcolemma, but occasionally on a central plate thenerve fibre approaching a neuromuscular junction lies in a well-defined synapticgutter (P1. 2, fig. 10).From this description it is clear that there is considerable variation in the struc-

ture of lamprey myoneural junctions, particularly in the arrangement of the sarco-lemma, which may sometimes form a well-defined subneural apparatus, but morefrequently shows very few or no invaginations. A gradation between these twoextremes is often seen in electron microscope sections through the site of innervationof a central muscle plate, where many individual myoneural junctions may bepresent in one section. These junctions also vary in other ways. Sometimes, as inPI. 4, fig. 14, the axon is small and packed with vesicles, while in other cases, as inPI. 4, fig. 13, a large axon is bare over a small area and the vesicles are confined tothat region. The myoneural junction in P1. 4, fig. 13, is one of three formed by thesame large axon (diameter 10et) on the side of one muscle plate. All three junctionshave the same form and are separated from each other by a distance of 15-20,.Thus, in contrast to the parietal muscle fibres, a section through the site of inner-vation of a central muscle plate may show different forms of neuromuscular junctions,and as many as seven different points of innervation have been observed on a singlemuscle plate in the same electron microscope section. This would seem to indicatethat despite the apparent continuity of each central sheet, it is innervated alongthe whole length of its myoseptal margin.

DISCUSSION

The muscle units in Lampetra fluviatilis and in L. planeri have the same structure,and this appears to be constant in the lamprey, since in addition to the abovespecies it has been described by Grenacher (1867) for Petromyzon marines, byJohnston (1908) for P. dorsatus, and by Peachey (1960b) for P. branchialis. In hisstudies on L. fluviatilis, Maurer (1894) considered that the parietal muscle wasformed by an anastomosing network of fibres, a view supported by Schiefferdecker(1911), but we are unable to confirm this finding as both light and electron micro-scopic preparations show that the parietal muscle is composed of individual fibres.A more difficult concept is the organization of the muscle of the central layers of

a unit into individual sheets. Proof of this must depend on the failure to demon-strate any vertical fibrous partitions or sarcolemmal sheets within the centralmuscle layers, and since these have not been observed in our preparations we mustconclude that each central layer consists of a single, continuous sheet of muscle,invested in a separate sarcolemmal sheath, with no subdivision into smaller com-ponents. The only other instance known to us of vertebrate muscle occurring insheets is in Amphioxus, where Grenacher (1867) described the myotomes as beingbuilt up of muscle plates; this has recently been supported by Peachey (1960 a) fromelectron microscope studies.The two types of muscle in the lamprey have been shown to differ in their mode

of innervation, the parietal muscle fibres being innervated along their length,while the central plates are innervated at both ends in the myoseptal regions.

A. Peter and B. MackayJohnston (1908) states that the fine terminal branches of the nerves to the parietalmuscle fibres are 'moderately slender, slightly varicose fibres which show no signsof any special end organ'. A similar conclusion is reached by Tretjakoff (1927),although he shows some of the terminal branches of the nerves ending in knob-likeexpansions. Our own preparations show no such varicosities of these fine terminalbranches and no structures that could be termed nerve terminals are visible in them.As pointed out by Johnston (1908), although it is clear that the distribution of onenerve fibre to two and sometimes three myotomes appears to be the rule, no expla-nation for such a distribution is apparent.The central muscle plates appear to be innervated by the nerves which sweep round

the edges of the myotomes into the myosepta, and the myoneural junctions on thesecentral plates lie at the edges of their myoseptal margins. Johnston (1908) alsodescribes the myoseptal nerves branching to form end-plates in this region. A similarform of innervation occurs in the myotomes of a number of other lower vertebrates(Mackay & Peters, 1961), in which the muscle fibres are also innervated at both ends.The site and form of the endings may, however, vary somewhat: in Xenopustadpoles, for instance (Mackay, Muir & Peters, 1960), the neuromuscular junctionsare related to the sarcolemma within or between the invaginations at the myo-tendinous junction, and not to its side as in the lamprey.The rather diffuse staining obtained after the histochemical demonstration of

cholinesterase in lamprey muscle may be a consequence of the absence of a well-developed subneural apparatus such as is found in the higher vertebrates (e.g.Robertson, 1956; De Harven & Coers, 1959). In electron microscope sections, asubneural apparatus is often not seen at the myoneural junction, but this may bebecause the apparatus is poorly developed so that many sections do not pass throughsarcolemmal infoldings. Absence of a subneural apparatus has been described inthe neuromuscular junctions of leg muscles of the wasp (Edwards, Ruska & DeHarven, 1958), and in vertebrate smooth muscle (Caesar, Edwards & Ruska, 1957).When present, however, the subneural apparatus in the lamprey resembles that ofhigher vertebrates in its basic structure, though the infoldings are relatively fewand are usually more shallow and irregular in their arrangement.The reason for the existence oftwo types of muscle in the lamprey is obscure, though

the differences in morphology and innervation between these two types indicate thatthey subserve different physiological roles. The parietal fibres have relatively moresarcoplasm, more nuclei, and more extensive innervation than the central muscleplates, and these characteristics are also shown by muscle fibres in higher verte-brates which have been demonstrated to have a tonic or postural function. On theother hand, the structure of the central muscle plates is more akin to that of thetetanic or twitch fibre of higher vertebrates. (For a review of this subject, seeMackay, 1961.)Some information is available on the other commonly occurring cyclostome,

Myxine, where two varieties of muscle fibre have also been described (Maurer, 1894;Cole, 1907). Again the myotomes are built up of muscle units, but fibres correspond-ing to the lamprey parietal fibres are confined almost entirely to a single layer on theventral aspect of a unit. The second form of muscle in Myxine, forming the bulk ofthe units and corresponding to the central plates of the lamprey, is described as

582

Structure and innervation of myotomes of the lampreyoccurring in fibres rather than sheets. These two types of muscle in Myxine havevery recently been studied electrophysiologically by Andersen & Jansen (personalcommunication), who reach the tentative conclusion that the two histological typesmay have functional properties corresponding to the fast and slow muscle fibresof the frog (Kuffler, 1953).

SUMMARY

The segmental myotomes in the lamprey are built up of a series of horizontallyarranged muscle units, stacked one above the other. Each unit extends throughoutthe entire horizontal plane of a myotome, and usually consists of six layers; fourcentral muscle plates are bounded dorsally, laterally and ventrally by a singlelayer of closely packed parietal muscle fibres.

Cholinesterase activity is found along the length of the parietal muscle fibres,whereas it is confined to the myoseptal margins of the central plates. The parietalfibres are innervated at points along their length by nerves which enter the myotomesbetween the muscle units to form a plexus which thus lies between adjacent layers ofparietal muscle. Having supplied the parietal fibres in the medial portion of onemyotome, a nerve crosses a myoseptum to enter the myotome in front, and mayeven reach a third myotome, supplying parietal muscle fibres in the lateral portionsof the second and third myotomes. The central muscle plates are innervated at bothmyoseptal margins by ventral root fibres that enter the myosepta. Nerves do notpenetrate into the individual muscle units.The sites of innervation are confirmed by electron microscopy, which shows that

at the myoneural junctions the folds of the sarcolemma are shallow and irregular intheir arrangement. At such junctions, the nerve fibres lose their sheaths and theaxoplasm contains numerous mitochondria and vesicles.

It is suggested that the central muscle plates may have a twitch and the parietalfibres a tonic function.

We wish to thank Prof. G. J. Romanes for his interest and advice in the courseof this work. The electron microscope, which is on permanent loan from the Well-come Foundation, was maintained by Mr G. Wilson.

REFERENCESCAESAR, R., EDWARDS, G. A. & RusKA, H. (1957). Architecture and nerve supply of mammalian

smooth muscle tissue. J. Biophys. Biochem. Cytol. 3, 867.COLE, F. J. (1907). A monograph on the general morphology of the myxinoid fishes, based on a

study of Myxine. Part II. The anatomy of the muscles. Trans. Roy. Soc. Edinb. 45, 683.CoUTEAUx, R. (1955). Localization of cholinesterase at nerve-muscle junctions. Int. Rev. Cytol.

4, 335.DE HARVEN, E. & COERS, C. (1959). Electron microscope study of the human neuro-muscular

junction. J. Biophys. Biochem. Cytol. 6, 7.EDWARDS, G. A., RUSKA, H. & DE HARVEN, E. (1958). Electron microscopy of peripheral nerves

and neuromuscular junctions in the wasp leg. J. Biophys. Biochem. Cytol. 4, 107.GEREBTZOFF, M. A. (1959). Cholinesterase. Oxford: Pergamon Press.GRENACHER, H. (1867). Beitrage zur haheren Kenntniss der Muskulatur der Cyclostomen und

Leptocardier. Z. wiss. Zool. 17, 577.JOHNSTON, J. B. (1908). Additional notes on the cranial nerves of Petromyzonts. J. comp.

Neurol. 18, 569.

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584 A. Peters and B. MackayKOELLE, G. B. (1950). Histochemical differentiation oftypes of cholinesterases and their localisation

in the tissues of the cat. J. Pharmacol., Baltimore, 100, 158.KUFFLER, S. W. (1953). The two skeletal nerve-muscle systems in frog. Arch. exp. Path. Pharmak.

220, 116.LEWIS, P. R. (1958). A simultaneous azo-dye coupling technique suitable for whole mounts.

Quart. J. micr. Sci. 99, 67.MACKAY, B. (1961). Studies on the morphology, innervation and growth of skeletal muscle fibres.

Ph.D. Thesis, University of Edinburgh.MACKAY, B. & PETERS, A. (1961). Terminal innervation of segmental muscle fibres. In Electron

Microscopy in Anatomy. (Editors, J. D. Boyd, F. R. Johnson and J. D. Lever.) London:Edward Arnold.

MACKAY, B., MUIR, A. R. & PETERS, A. (1960). Observations on the terminal innervation ofsegmental muscle fibres in amphibia. Acta anat. 40, 1.

MAURER, F. (1894). Die Elemente der Rumpfmuskulatur bei Cyclostomen und Hoheren Wirbel-tieren. Morph. Jb. 21, 473.

MUIR, A. R. (1961). Observations on the attachment of myofibrils to the sarcolemma at themuscle-tendon junction. In Histochemistry of Cholinesterase, p. 182. Basel: Karger.

PALADE, G. E. (1952). A study of fixation for electron microscopy. J. exp. Med. 95, 285.PEACHEY, L. (1960a). Proceedings of the First International Congress of Electron Microscopy (Delft).PEACHEY, L. (1960b). Personal communication.PETERS, A. (1958). Staining of nervous tissue with protein-silver mixtures. Stain Tech. 33, 47.PETERS, A. (1960). The structure of peripheral nerves of the lamprey. J. ultrastructure Res. 4,349.ROBERTSON, J. D. (1956). The ultrastructure of a reptilian myoneural junction. J. Biophys.

Biochem. Cytol. 2, 369.ROBERTSON, J. D. (1957). Some aspects of the ultrastructure of double membranes. In Ultra-

structure and Cellular Chemistry of Neural Tissue, vol. II, 1. (H. Waelsch, editor.) New York:Hoeber-Harper.

SCHIEFFERDECKER, P. (1911). Untersuchungen uber die Rumpfmuskulatur von Petromyzonfluviatilis in Bezug auf ihren Bau und ihre Kernwehrhaltnisse, uber die Muskelfaser als Solcheund uber das Sarkolemm. Arch. mikr. Anat. 78, 472.

SCHWARZACHER, H. G. (1959). Vber die Lange und Anordnung der Muskelfasern in menschlichenSkeletmuskeln. Acta anat. 37, 217.

TRETJAKOFF, D. (1927). Das periphere Nervensystem des Flussneunauges. Z. wiss. Zool. 129, 359.

EXPLANATION OF PLATESThese figures are of adult specimens of L. fluviatilis.

PLATE 1Fig. 1. Transverse section of part of a myotome (M) adjacent to the notochord (N); the myotome

is composed of a series of muscle units (U). Silver stain, x 40.Fig. 2. Parasagittal section of part of a myotome. The parietal muscle fibres (P) taper as they

approach the myoseptum (S) and terminate short of the central muscle plates (C) which endbluntly. Silver stain, x 110.

Fig. 3. Transverse section of part of a myotome. The myofibrils of the parietal muscle fibres (P)are more widely separated than the finer myofibrils of the central muscle plates (C). Silverstain, x 120.

Fig. 4. Transverse section ofmuscle units stained by Wilder's reticulin method. The parietal musclefibres (P) extend around the lateral surfaces of the muscle units where these come in contactwith the skin (on the left). In contrast to the central muscle plates (C) the parietal musclefibres are bound tightly together by connective tissue. In this section the central muscleplates are fragmented, so that the arrangement of the parietal fibres is more apparent. x 90.

Fig. 5. Horizontal section through the myotomal muscle of an adult lamprey, showing a nervefibre (N) branching over the surface of the parietal muscle fibres. At the edge of the section,part of a central muscle plate (C) is visible. Silver stain, x 100.

Fig. 6. Parasagittal section of the myotomal muscle stained by the acetylthiocholine method forcholinesterase; the preparation is overstained and some diffusion has occurred. The enzymeis found along the length of the parietal muscle fibres (P), but on the central muscle plates (C)is confined to the regions adjoining the myosepta (M). x 45.

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PETERSxAND.MA.,A:-ST. UCTURE AND INNERVATION OF MYOTOMES OF T~lE LAMPREY


Plate 4

Structure and innervation of myotome8 of the lamprey 585

PLATE 2

Fig. 7. Parasagittal section of the myotomal muscle stained by the acetylthiocholine method.Cholinesterase is present at the ends of the central muscle plates (C) where these adjoin themyosepta (X). The apparent staining of the parietal muscle is due to cholinesterase located inthe nerve fibres (N) that run between the adjacent layers of these fibres. x 100.

Fig. 8. Part of a central muscle plate in the myoseptal region; acetylthiocholine method. Most ofthe cholinesterase activity is the result of a myotendinous reaction (T), but small areas ofintense staining (arrows) are present on the sides of this region. x 610.

Fig. 9. Electron micrograph ofa central muscle plate. The myofibrils (F) are closely packed together,so that there is little sarcoplasm between them. The mitochondria (M) are fewer in number thanin the parietal muscle fibres (P1. 3, fig. 11). Note the osmiophilic bodies (X) between the myo-fibrils. x 8,000.

Fig. 10. Electron micrograph of part of the myotendinous region of a central muscle plate. Anerve ending (E) indents the muscle plate at the side of the myotendinous junction, whereconnective tissue fibrils of the myoseptum (S) are inserted into the invaginations (J) of thesarcolemma. Large numbers of mitochondria (M) are present in the sarcoplasm in the regionof the nerve ending (E). x 5,000.

PLATE 3

Fig. 11. Electron micrograph of parts of two parietal muscle fibres (P1 and P2), one of which (P2)contains a nucleus (Nu). The sarcoplasm between the myofibrils (F) is more extensive thanin the central muscle plates (fig. 9), and contains more mitochondria (M). Between the twoparietal muscle fibres is a nerve (N), packed with vesicles and mitochondria, which formsneuromuscular junctions with two parietal muscle fibres at the places indicated by arrows.Note the osmiophilic bodies (X) between the myofibrils and under the sarcolemma. x 8,000.

Fig. 12. Electron micrograph of a myoneural junction on a parietal muscle fibre (P). The nervefibre (N) is packed with vesicles (V) and mitochondria (M). In two regions (E1 and E2) theSchwann cell sheath (S) is absent, so that the axolemma (Al) is only separated from thesarcolemma (Z) by a homogeneous layer (L) formed from the basement membranes on theoutsides of the axolemma and sarcolemma. Note the absence of a subneural apparatus.x 24,000.

PLATE 4

Fig. 13. Electron micrograph of a myoneural junction (J) on a central muscle plate (C). This isone of three junctions formed by a large nerve fibre (N), 10,u in diameter. The axoplasm of thejunctional region contains a localized concentration of vesicles (V), and the Schwann cellsheath (S) is absent, so that the axolemma (Al) comes into close proximity to the sarcolemma(Z) which has a small invagination (arrow). x 32,000.

Fig. 14. Electron micrograph of a myoneural junction on a central muscle plate (C). At the junctionthe nerve fibre (N) contains numerous vesicles (V) and mitochondria (M), and the Schwanncell sheath (S) is absent so that the axolemma (Al) comes into close proximity to the sarcolemma(Z). The sarcolemma (Z) has numerous invaginations (I) which are irregular in their arrange-ment and are lined by the basement membrane (B) of the sarcolemma. Numerous mitochondria(M1) are situated in the sarcoplasm subjacent to the junctional region. x 33,000.

Anat. 9537

the structure and innervation of the myotomes of the lamprey

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