J. Cell Set, 7, 263-271 (1970) 263Printed in Great Britain

NEUROMUSCULAR JUNCTIONS IN THE BODY

WALL MUSCLES OF THE EARTHWORM,

LUMBRICUS TERRESTRIS LINN.

P. J. MILL AND M. F. KNAPPDepartment of Zoology, University of Leeds, England

SUMMARY

The fine structure of the neuromuscular junctions in the body wall muscles of the earthworm13 described. The segmental nerves send branches into the muscle layers. Axons in the nervebranches contain numerous synaptic vesicles and contact is established between these axonsand muscle fibres or muscle tails; the latter may extend for a considerable distance from themuscle fibre. The cleft between the axolemma and sarcolemma is 85-120 nm wide and con-tains basement membrane material. At intervals small aggregations of electron-dense materialare attached to the axonal membrane and synaptic vesicles are associated with these. Thesarcolemma bears rather larger masses of dense material and is also specialized extracellularly.

INTRODUCTION

The earthworm's body wall muscles are innervated by the segmental nerves. Thereare about 300 motor fibres in the segmental nerves of a typical segment of the earth-worm Pherettma communissima (Ogawa, 1939); these innervate at least 120000 musclefibres. The axons give off many branches to the muscles and their endings are den-dritic in form in fully grown worms but bud-like in young worms. According toRetzius (1892 a), finely branching motor fibres ramify in the muscles of Lumbricus,terminating as free endings which are like knotted threads in appearance. His figuresindicate both multiterminal and polyneuronal innervation. Smallwood (1926) in aninvestigation of Lumbricus terrestris, noted that in addition to fine branching nervefibres with knob-like endings, which he considered to be sensory, there is anothertype of ending characterized by a cluster of branches, but also with knob-like terminals;he thought that these were motor in function. They appear from his drawings to besimilar to the 'en grappe' type of motor ending found in vertebrate slow musclefibres (Gray, 1957). Different staining methods were used to demonstrate these 2 typesof ending; both were multiterminal. Thus the morphological evidence points to theconclusion that in oligochaetes a relatively small number of motoneurons branchprofusely to innervate a large number of muscle fibres.

Likewise, in the polychaetes Nereis and Harmothoe only a small number of motoraxons leave the segmental ganglia (Smith, 1957; Horridge, 1959) and it has beensuggested that the information in these axons is relayed to the muscles by peripheralmotoneurons. However, the available evidence indicates that this is not the case andthat branching of the axons occurs to provide multiterminal and possibly also poly-

264 P. J. Mill and M. F. Knapp

neuronal innervation (Horridge, 1959; Dorsett, 1963, 1964). The 'en grappe' type ofendings have been described in polychaetes by Retzius (18926) and Dorsett (1963,1964).

It is evident that certain muscles at least are capable of both slow and fast contrac-tions (Nicol, 1948; Horridge, 1959; Wilson, i960) and this may be effected either byhaving a dual innervation, as in Crustacea for example, or by having 2 different typesof muscle fibres. The former situation is indicated by the available evidence, bothmorphological (Dorsett, 1964; Mill & Knapp, 1970) and physiological (Nicol, 1948,1951; Horridge, 1959; Wilson, i960). Dorsett (1963) has suggested that if the nerveendings found in Lumbricus by Retzius and Smallwood do represent 2 different typesof motor ending these may provide an anatomical basis for the slow and fast systems ofmuscular contraction.

MATERIAL AND METHODS

Preparations of the body wall muscles of Lumbricus terrestris Linn, and their accompanyingnerves were obtained as described in a previous paper (Mill & Knapp, 1970). Fixation for 1 h in2-5 % glutaraldehyde, buffered with sodium cacodylate, was followed by washing in the bufferand post-fixing in osmium tetroxide (buffered with veronal acetate) for a further hour. Materialwas subsequently washed in the veronal-acetate buffer, dehydrated in graded ethanols andembedded in Epon. Sections were cut on a Huxley microtome and mounted on carbon-coatedgrids. Contrast was improved by staining with uranyl acetate and lead citrate before examina-tion in an AEI EM6B electron microscope. Further details are given in Mill & Knapp (1970).

RESULTS

In each segment of the earthworm the ventral cord gives rise to 3 pairs of nerves,each of which contains motor fibres. The nerves penetrate the body wall muscle nearto the ventro-lateral pair of chaetae and then continue dorsally and ventrally betweenthe longitudinal and circular muscle layers. Branches to the muscles are given off andthese further subdivide so that the nerves observed in electron micrographs include avery variable number of axons. The axons in many of the small nerves contain synapticvesicles but in addition vesicles may occur in some fibres around the periphery of thelarger nerve branches or even of the main segmental nerves themselves. Muscle fibresare closely associated with the axons in which synaptic vesicles are found. Sometimesthe juxtaposition is with the main body of the muscle fibre, but more often it is withmuscle tails, which are devoid of contractile elements. They may extend for a con-siderable distance from the body of the muscle fibre before making contact with anerve (Fig. 5). The muscle tails differ from those which connect muscle fibres toconnective tissue (Mill & Knapp, 1970) in the absence of fibrillar bundles; there is nocharacteristic specialization of the sarcoplasm. Multiple synapses on a single musclefibre have not been observed but the fibres are very long and the work of the lightmicroscopists indicates that there are a number of neuromuscular junctions along thelength of each muscle fibre (Retzius, 1892a; Smallwood, 1926; Dorsett, 1963, 1964).However, muscle tails from more than one fibre have been seen associated with a singleaxonal ending Ghal cells are not abundant in earthworm nerves and are absent from

Neuromuscular junctions in earthworm 265

many of the smaller nerve branches, which consequently are composed entirely ofnaked axons. Neuromuscular junctions may be found on all sides of these small nerves(Fig. 1). In other cases the ghal cells form an incomplete sheath around the nerve andneuromuscular junctions occur between the exposed axons and the adjacent musclecells.

Between the axons and the muscle cell is a cleft, generally 85-120 nm wide(Figs. 1-5). It contains moderately electron-dense basement membrane materialwhich is often more concentrated in the middle, so that a denser line is seen along thelength of the cleft. The axons are packed with synaptic vesicles which are 30-70 nmin diameter and have moderately electron-dense contents. Other vesicles, 80-150 nmin diameter, with an electron-dense core, are found in smaller numbers. Other axonalinclusions in the neuromuscular junction region are mitochondria and small quantitiesof glycogen. In the region of the neuromuscular junctions the axolemma is generallysomewhat blurred and indistinct. However, intermittent and very small aggregations ofdense material do occur in places on the inner side of the axonal membrane. In Fig. 2the synaptic vesicles appear to be closely associated with these electron-dense regions.The sarcolemma exhibits very distinct specialization. It is very prominent, with a cleartrilaminate, unit-membrane structure, the inner lamina of which appears more densethan the outermost one. A collection of electron-dense material within the musclecells is closely apposed to the sarcolemma. Extracellularly an electron-dense line liesparallel to the muscle membrane and about 20 nm from it. Fine fibril-like structures,about 27 nm long, extend from the membrane to just beyond the dense line. Thesefibrils are at an angle of 50-700 to the sarcolemma (Figs. 3, 4). Careful examination ofthe region marked by an arrow in Fig. 1 reveals a lattice-like pattern where the sarco-lemma of a muscle tail is cut obliquely. This suggests that the 'fibril-like'structuresseen in Figs. 3, 4 may be sections through parallel ridges which extend in 2 directions,approximately at right angles to each other, over the outer surface of the sarcolemma.Rarely muscle tails wrap around isolated axons, which contain both synaptic vesiclesand the vesicles with an electron-dense core. When this occurs the axolemma andsarcolemma are much closer (10-20 nm) than in the junctions described above, butno specialization of either membrane has been observed.

DISCUSSION

The myoneural junctions described in this paper are thought to be at or near theregion of synaptic contact between the motor axon and the muscle, on the grounds thatthe axons are packed with vesicles which, in both appearance and size, closely resemblesynaptic vesicles observed at neuromuscular junctions in all the major phyla.

The earthworm junctions are similar in many respects to those of vertebrate twitchand slow striated muscles, in which the synaptic cleft is of the order of 40-50 nmwide (Table 1) and basement membrane material is interposed between pre- and post-synaptic membranes. The axolemma and sarcolemma show localized thickenings invertebrate striated muscles, and synaptic vesicles are associated with those of theaxolemma (Birks, Huxley & Katz, i960). A much narrower cleft of 10-20 nm is

Neuromuscular junctions in earthworm 267

reported for insect, crustacean and cephalopod neuromuscular junctions (Table 1),with no basement membrane material between the membranes, and the axolemmaand sarcolemma are usually equally thickened. In the nematode Ascaris the cleft is50 nm wide (Rosenbluth, 1965) and in the ctenophore Leucothea, 15-20 nm wide(Horndge, 1965). In neither of these animals does there appear to be any specializationof the membranes.

In a number of muscles there are 2 types of cleft associated with the contact zones.In vertebrate smooth muscle they are 180 and 20 nm in width respectively, and base-ment membrane material intervenes in the former (Caesar, Edwards & Ruska, 1957).In skeletal muscle of the blowfly larva one type has a cleft of 50-100 nm which con-tains basement membrane material, and there is a thickened sarcolemma. The other isa tighter junction, 15 nm wide, from which the basement membrane material is excluded,and both membranes are thickened (Osborne, 1967).

With the possible exception of Ascaris, the earthworm neuromuscular junctiondiffers from the usual invertebrate situation in not having a close junction less than20 nm wide and free of basement membrane. The existence of a closer junction cannotbe entirely excluded, but extensive searches have failed to reveal one. The specializa-tion of axonal and muscular membranes, and in particular the association ofvesicles with the axolemmal thickening lends support to the view that these are truesynaptic junctions.

In Ascaris a muscle arm extends from the muscle cell to the nerve cord, wheremyoneural contact is established (Rosenbluth, 1965). However, in arthropods andvertebrates it is usual for the main body of the muscle fibre to be innervated directlyby an axon. The earthworm appears to represent an interesting intermediate con-dition since, although some neuromuscular junctions occur between the axon andmuscle fibre as in higher groups, in other instances muscle tails may extend for somedistance from the fibre before making contact with a nerve.

A considerable amount of physiological evidence is now available, particularlyfrom the Arthropoda, to suggest that innervation of invertebrate muscle fibres is oftenpolyneuronal. However, no morphological criteria have been found to enable thephysiologically distinct types of synapse to be distinguished from each other in theelectron microscope. Peterson & Pepe (1961) found only one morphological type ofneuromuscular junction on a stretch receptor muscle in the crayfish, although it isknown to receive both excitatory and inhibitory innervation. Also inhibitory axonsinnervate the stretch receptor sensory neuron and the inhibitory synapses on thisneuron have essentially the same structure as excitatory synapses described previouslyby other authors. The neuromuscular synapses on fast and slow muscle fibres of theaccessory flexor muscle of Cancer are also all of one type, although they are innervatedby both excitatory and inhibitory neurons (Cohen & Hess, 1967).

Polyneuronal innervation is indicated in the earthworm, since miniature excitatoryand inhibitory junction potentials have been recorded in the body wall muscles and, onvery rare occasions, both could be recorded from the same cell (Hidaka, Ito, Kuri-yama & Tashiro, 1969). In addition it has been shown that annelid muscles are capableof both fast and slow contraction (Nicol, 1948; Horridge, 1959; Wilson, i960),

268 P. J. Mill and M. F. Knapp

although only one morphological type of muscle fibre has been recognized in the bodywall (Heumann & Zebe, 1967; Rosenbluth, 1968; Mill & Knapp, 1970). However, thestructure of obliquely striated muscle fibres is such that variations in, say, sarcomerelength and width, such as are found in some arthropod muscles, would be difficult todetect. Whether or not such differences occur in annelids, it is almost certain that theinnervation is polyneuronal. If this is so then the neuromuscular junctions describedin this paper may represent 2 or more physiologically distinct types of synapse whichcannot be distinguished morphologically on the available information. On the otherhand, junctions with different physiological properties may also show structuraldifferences, in which case those described here must be examples of just one of thetypes of junction present in this muscle.

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CAESAR, R., EDWARDS, G. A. & RUSKA, H. (1957). Architecture and nerve supply of mammaliansmooth muscle tissue. J. btophys. btochem. Cytol. 3, 867-877.

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652-667.EDWARDS, G. A. (1959). The fine structure of a multiterminal innervation of an insect muscle.

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KARLSSON, U. L. (1962). Specialised contact regions of the myoneural motor junction of thefrog, jth Int. Congr. Electron Microsc, Pennsylvania, vol. 2 (ed. S. S. Breese), p. 114. New Yorkand London. Academic Press.

MILL, P. J. & KNAPP, M. F. (1970). The fine structure of obliquely striated body wall musclesin the earthworm, Lumbricus terrestris Linn. jf. Cell Sci. 7, 231-261.

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NICOL, J. A. C. (1951). Giant axons and synergic contractions in Branchiomma vesiculosum.J. exp. Bwl. 28, 22-31.

OGAWA, F. (1939). The nervous system of earthworm (Pheretima communisstma) in differentages. Sci. Rep. T6hoku Univ., Ser. IV, 13, 395-488.

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(Received 17 September 1969—Revised 27 January 1970)

270 P. J. Mill and M. F. Knapp

Fig. i. A small nerve which contains a number of axons. Within the axons are numer-ous synaptic vesicles (sv) some larger vesicles with electron-dense cores (vc), mito-chondria (m) and a small amount of glycogen (gl). A cleft containing basement mem-brane material separates the axons from a muscle fibre (mf) and the muscle tails (mt)of other fibres. The sarcolemma of the contact zone is highly specialized. The muscletails contain a considerable amount of glycogen (gl). At the arrow the sarcolemma iscut obliquely and a lattice-like pattern is present, x 22500.

Figs. 2-4. Higher-power electron micrographs showing details of the appositionbetwen the axolemma and sarcolemma. In Figs. 2, 4 aggregations of dense materialare attached to the axolemma and in some cases synaptic vesicles appear to be closelyassociated with this material (arrows). Larger dense masses are found on the innersurface of the sarcolemma and in these regions extracellular specialization of the musclemembrane occurs. In some of the vesicles with an electron-dense core (vc) the surfaceof the core appears to be irregular, with small projections. Fig. 2, x 28000; Figs, 3, 4,x 38000.

Fig. 5. A muscle tail (mt) extends for some distance from a muscle fibre (mf) (approxi-mately 5"4/im in this case) to make contact with a nerve containing axons in whichsynaptic vesicles (sv) occur, x 18500.

Neuromuscular junctions in earthworm 271