J. Cell Set, 7, 263-271 (1970)
Printed in Great Britain
263
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 nerves send branches into the muscle layers. Axons in the nerve
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. T h e cleft between the axolemma and sarcolemma is 85-120 nm wide and contains 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 earthworm Pherettma communissima (Ogawa, 1939); these innervate at least 120000 muscle
fibres. The axons give off many branches to the muscles and their endings are dendritic 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 contractions (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 examination 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 considerable 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
connective tissue (Mill & Knapp, 1970) in the absence of fibrillar bundles; there is no
characteristic specialization of the sarcoplasm. Multiple synapses 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 neuromuscular junction 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 sarcolemma 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 axon 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 postsynaptic 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 basement 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 contains 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 specialization 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 condition 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 synapse 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 neuron 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 neurons (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, Kuriyama & 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.
REFERENCES
ANDERSSON-CEDERGREN, E. (1959). Ultrastructure of motor end plate and sarcoplasmic components of mouse skeletal muscle fibre as revealed by three-dimensional reconstructions from
serial sections. J. Ultrastruct. Res. (Suppl.) 1, 1-191.
BIRKS, R., HUXLEY, H. E. & KATZ, B. (i960). The fine structure of the neuromuscular junction
of the frog. J. Physiol., Lond. 150, 134-144.
CAESAR, R., EDWARDS, G. A. & RUSKA, H. (1957). Architecture and nerve supply of mammalian
smooth muscle tissue. J. btophys. btochem. Cytol. 3, 867-877.
COHEN, M. J. & HESS, A. (1967). Fine structural differences in 'fast' and 'slow' muscle fibres of
the crab. Am. J. Anat. 121, 285-304.
DORSETT, D. A. (1963). The motor axon terminations of annelids. Nature, Lond. 198, 406.
DORSETT, D. A. (1964) The sensory and motor innervation of Nereis. Proc. R. Soc. B 159,
652-667.
EDWARDS, G. A. (1959). The fine structure of a multiterminal innervation of an insect muscle.
J. btophys. bwchem. Cytol. 5, 241-243.
GRAY, E. G (1957). The spindle and extrafusal innervation of a frog muscle. Proc. R. Soc. B
146, 416-430.
GRAZIADEI, P. (1966). The ultrastructure of the motor nerve endings in the muscle of cephalopods. J. Ultrastruct. Res. 15, 1-13.
HEUMANN, H.-G. & ZEBE, E. (1967). Uber Feinbauund Funktionweise der Fasern aus dem Hautmuskelschlauch des Regenwurms, Lumbricus terrestris L. Z. Zellforsch. mikrosk. Anat. 78,
131-150HIDAKA, T., ITO, Y., KURIYAMA, H. & TASHIRO, N. (1969). Neuromuscular transmission in the
longitudinal layer of somatic muscle of the earthworm. J. exp. Bwl. 50, 417-430.
HORRIDGE, G. A. (1959). Analysis of the rapid responses of Nereis and Harmothoe (Annelida).
Proc. R. Soc. B 150, 245-262.
HORRIDGE, G. A. (1965). Non-motile sensory cilia and neuromuscular junctions in a ctenophore independent effector organ. Proc. R. Soc. B 162, 333-350.
KARLSSON, U. L. (1962). Specialised contact regions of the myoneural motor junction of the
frog, jth Int. Congr. Electron Microsc, Pennsylvania, vol. 2 (ed. S. S. Breese), p. 114. New York
and London. Academic Press.
MILL, P. J. & KNAPP, M. F. (1970). The fine structure of obliquely striated body wall muscles
in the earthworm, Lumbricus terrestris Linn. jf. Cell Sci. 7, 231-261.
NICOL, J. A. C. (1948). The function of the giant axon of Myxicola ivfundtbulum. Can. J. Res.
26, 212-222.
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 different
ages. Sci. Rep. T6hoku Univ., Ser. IV, 13, 395-488.
OSBORNE, M. P. (1967). The fine structure of neuromuscular junctions in the segmental muscles
of the blowfly larva. J. Insect Physiol. 13, 827-833.
Neuromuscular junctions in earthworm
PETERSON,
269
R. P. & PEPE, F. A. (1961). Fine structure of inhibitory synapses in the crayfish.
jf. biophys. btochem. Cytol. 11, 157-169.
G. & HESS, A. (1966). Differences in internal structure and nerve terminals of the slow
and twitch muscle fibres in the cat superior oblique. Anat. Rec. 154, 243-252.
RETZIUS, G. (1892a). Das Nervensystem der Lumbricinen. Biol. Unters. N.F. m, 1-16.
RETZIUS, G. (18926). Ueber Nervenendigungen an den Parapodienborsten und iiber die Muskelzellen der Gefasswande bei den polychaten Annulaten. Biol. Fb'ren. Forh., Stockholm 3,
85-89ROSENBLUTH, J. (1965). Ultra8tructure of somatic muscle cells in Ascaris lumbricoides. II.
Intermuscular junctions, neuromuscular junctions and glycogen stores. J. Cell Biol. 26,
579-591ROSENBLUTH, J. (1968). Obliquely striated muscle. IV. Sarcoplasmic reticulum, contractile
apparatus and endomysium of the body muscle of a polychaete, Glycera, in relation to its
speed. J. Cell Biol. 36, 245-259.
SMALLWOOD, W. M. (1926). The peripheral nervous system of the common earthworm,
Lumbncus terrestris. J. comp. Neurol. 42, 35-55.
SMITH, D. S. (i960). Innervation of the fibnllar flight muscle of an insect: Tenebrio mohtor
(Coleoptera). J. biophys. biocliem. Cytol. 8, 447-466.
SMITH, J. E. (1957). The nervous anatomy of the body segments of nereid polychaetes.
Phil. Tram. R. Soc. Ser. B 240, 135-196.
WILSON, D. M. (i960). Nervous control of movement in annelids. J. exp. Biol. 37, 46-56.
PILAR,
(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 numerous synaptic vesicles (sv) some larger vesicles with electron-dense cores (vc), mitochondria (m) and a small amount of glycogen (gl). A cleft containing basement membrane 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) (approximately 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