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

NEUROMUSCULAR JUNCTIONS IN THE BODY WALL MUSCLES OF THE EARTHWORM, LUMBRICUS TERRESTRIS LINN.

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

SUMMARY The fine structure of the neuromuscular junctions in the body wall muscles of the earthworm 13 described. The segmental send branches into the muscle layers. in the branches contain numerous synaptic vesicles and contact is established between these axons and muscle fibres or muscle tails; the latter may extend for a considerable distance from the muscle 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 material are attached to the axonal membrane and synaptic vesicles are associated with these. The sarcolemma 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. There are 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 muscle fibres. 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 to Retzius (1892 a), finely branching motor fibres ramify in the muscles of Lumbricus, terminating as free endings which are like knotted threads in appearance. His figures indicate both multiterminal and polyneuronal innervation. Smallwood (1926) in an investigation of Lumbricus terrestris, noted that in addition to fine branching nerve fibres with knob-like endings, which he considered to be sensory, there is another type 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 be similar to the 'en grappe' type of motor ending found in vertebrate slow muscle fibres (Gray, 1957). Different staining methods were used to demonstrate these 2 types of ending; both were multiterminal. Thus the morphological evidence points to the conclusion that in oligochaetes a relatively small number of motoneurons branch profusely to innervate a large number of muscle fibres. Likewise, in the polychaetes Nereis and Harmothoe only a small number of motor axons leave the segmental ganglia (Smith, 1957; Horridge, 1959) and it has been suggested that the information in these axons is relayed to the muscles by peripheral motoneurons. However, the available evidence indicates that this is not the case and that 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 of endings 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 by having a dual innervation, as in Crustacea for example, or by having 2 different types of muscle fibres. The former situation is indicated by the available evidence, both morphological (Dorsett, 1964; Mill & Knapp, 1970) and physiological (Nicol, 1948, 1951; Horridge, 1959; Wilson, i960). Dorsett (1963) has suggested that if the nerve endings found in Lumbricus by Retzius and Smallwood do represent 2 different types of motor ending these may provide an anatomical basis for the slow and fast systems of muscular contraction.

MATERIAL AND METHODS Preparations of the body wall muscles of Lumbricus terrestris Linn, and their accompanying nerves were obtained as described in a previous paper (Mill & Knapp, 1970). Fixation for 1 h in 2-5 % glutaraldehyde, buffered with sodium cacodylate, was followed by washing in the buffer and post-fixing in osmium tetroxide (buffered with veronal acetate) for a further hour. Material was subsequently washed in the veronal-acetate buffer, dehydrated in graded ethanols and embedded in Epon. Sections were cut on a Huxley microtome and mounted on carbon-coated grids. 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 near to the ventro-lateral pair of chaetae and then continue dorsally and ventrally between the longitudinal and circular muscle layers. Branches to the muscles are given off and these further subdivide so that the nerves observed in electron micrographs include a very variable number of axons. The axons in many of the small nerves contain synaptic vesicles but in addition vesicles may occur in some fibres around the periphery of the larger nerve branches or even of the main segmental nerves themselves. Muscle fibres are closely associated with the axons in which synaptic vesicles are found. Sometimes the juxtaposition is with the main body of the muscle fibre, but more often it is with muscle 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 a nerve (Fig. 5). The muscle tails differ from those which connect muscle fibres to (Mill & Knapp, 1970) in the absence of fibrillar bundles; there is no characteristic specialization of the sarcoplasm. Multiple on a single muscle fibre have not been observed but the fibres are very long and the work of the light microscopists indicates that there are a number of neuromuscular junctions along the length 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 single axonal 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 of naked 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 and neuromuscular junctions occur between the exposed axons and the adjacent muscle cells. 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 material which is often more concentrated in the middle, so that a denser line is seen along the length of the cleft. The axons are packed with synaptic vesicles which are 30-70 nm in diameter and have moderately electron-dense contents. Other vesicles, 80-150 nm in diameter, with an electron-dense core, are found in smaller numbers. Other axonal inclusions in the region are mitochondria and small quantities of glycogen. In the region of the neuromuscular junctions the axolemma is generally somewhat blurred and indistinct. However, intermittent and very small aggregations of dense material do occur in places on the inner side of the axonal membrane. In Fig. 2 the synaptic vesicles appear to be closely associated with these electron-dense regions. The sarcolemma exhibits very distinct specialization. It is very prominent, with a clear trilaminate, unit-membrane structure, the inner lamina of which appears more dense than the outermost one. A collection of electron-dense material within the muscle cells is closely apposed to the sarcolemma. Extracellularly an electron-dense line lies parallel 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. These fibrils are at an angle of 50-700 to the sarcolemma (Figs. 3, 4). Careful examination of the 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'structures seen 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 vesicles and the vesicles with an electron-dense core. When this occurs the axolemma and sarcolemma are much closer (10-20 nm) than in the junctions described above, but no specialization of either membrane has been observed.

DISCUSSION The myoneural junctions described in this paper are thought to be at or near the region of synaptic contact between the motor and the muscle, on the grounds that the axons are packed with vesicles which, in both appearance and size, closely resemble synaptic vesicles observed at neuromuscular junctions in all the major phyla. The earthworm junctions are similar in many respects to those of vertebrate twitch and slow striated muscles, in which the synaptic cleft is of the order of 40-50 nm wide (Table 1) and basement membrane material is interposed between pre- and post- synaptic membranes. The axolemma and sarcolemma show localized thickenings in vertebrate striated muscles, and synaptic vesicles are associated with those of the axolemma (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 axolemma and sarcolemma are usually equally thickened. In the nematode Ascaris the cleft is 50 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 specialization of 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 is a 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 junction differs from the usual invertebrate situation in not having a close junction less than 20 nm wide and free of basement membrane. The existence of a closer junction cannot be entirely excluded, but extensive searches have failed to reveal one. The specializa- tion of axonal and muscular membranes, and in particular the association of vesicles with the axolemmal thickening lends support to the view that these are true synaptic junctions. In Ascaris a muscle arm extends from the muscle cell to the nerve cord, where myoneural contact is established (Rosenbluth, 1965). However, in arthropods and vertebrates it is usual for the main body of the muscle fibre to be innervated directly by an axon. The earthworm appears to represent an interesting intermediate con- dition since, although some neuromuscular junctions occur between the axon and muscle fibre as in higher groups, in other instances muscle tails may extend for some distance from the fibre before making contact with a nerve. A considerable amount of physiological evidence is now available, particularly from the Arthropoda, to suggest that innervation of invertebrate muscle fibres is often polyneuronal. However, no morphological criteria have been found to enable the physiologically distinct types of to be distinguished from each other in the electron microscope. Peterson & Pepe (1961) found only one morphological type of neuromuscular junction on a stretch receptor muscle in the crayfish, although it is known to receive both excitatory and inhibitory innervation. Also inhibitory axons innervate the stretch receptor sensory and the inhibitory synapses on this neuron have essentially the same structure as excitatory synapses described previously by other authors. The neuromuscular synapses on fast and slow muscle fibres of the accessory flexor muscle of Cancer are also all of one type, although they are innervated by both excitatory and inhibitory (Cohen & Hess, 1967). Polyneuronal innervation is indicated in the earthworm, since miniature excitatory and inhibitory junction potentials have been recorded in the body wall muscles and, on very 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 capable of 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 body wall (Heumann & Zebe, 1967; Rosenbluth, 1968; Mill & Knapp, 1970). However, the structure of obliquely striated muscle fibres is such that variations in, say, sarcomere length and width, such as are found in some arthropod muscles, would be difficult to detect. Whether or not such differences occur in annelids, it is almost certain that the innervation is polyneuronal. If this is so then the neuromuscular junctions described in this paper may represent 2 or more physiologically distinct types of synapse which cannot be distinguished morphologically on the available information. On the other hand, junctions with different physiological properties may also show structural differences, in which case those described here must be examples of just one of the types of junction present in this muscle.

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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 muscle tails contain a considerable amount of glycogen (gl). At the arrow the sarcolemma is cut obliquely and a lattice-like pattern is present, x 22500. Figs. 2-4. Higher-power electron micrographs showing details of the apposition betwen the axolemma and sarcolemma. In Figs. 2, 4 aggregations of dense material are attached to the axolemma and in some cases synaptic vesicles appear to be closely associated with this material (arrows). Larger dense masses are found on the inner surface of the sarcolemma and in these regions extracellular specialization of the muscle membrane occurs. In some of the vesicles with an electron-dense core (vc) the surface of 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 which synaptic vesicles (sv) occur, x 18500. Neuromuscular junctions in earthworm 271