The neuronal control of locomotion in the earthworm.

Biol. Rm. (1976),51,pp. 25-52 BRC PAH 51-2

T H E NEURONAL CONTROL OF LOCOMOTION I N THE EARTHWORM BY C. R. GARDNER Department of Physiology and Medical Biochemistry, University of Cape Town (Received 24 March 1975; Revised 15 August 1975) CONTENTS I. Introduction . . . . . . . . 11. Types of movement . . . . . (I) Peristaltic creeping . . . . . . . (2) Rapid escapemovements . . . . 111. The nervous system . . . . . . . (I) Anatomy . . . . . . . . (2) Morphology and physiology . . . . (a) Senseorgans . . . . . . (a) Neuronal circuitry of the non-giant-fibre systems . (c) Neuronal circuitry of the giant-fibre systems (d) Neuromuscularjunctions . . . . . . . IV. The origin of the rhythmic nature of peristalsis . . . V. The r61e of the subepidermal nerve plexus VI. The control of rapid escape movements . . VII. Neurotransmitters . . . . . . . . (I) Monoamines . . . . . . (2) Acetylcholine . . . . . . . . (3) Gamma-aminobutyricacid . . . . . . . VIII. The pharmacology of earthworm locomotion . (I) Monoamines . . . . . . . . (2) Acetylcholine IX. Summary . . . . . . X. References . . . . . . .

.

.

.

.

.

. .

. . . .

.

.

. . . . .

. . . .

I. INTRODUCTION

The earthworm is a much studied species, and in recent years much work has been carried out with regard to the mechanism of its movement. Though there are several descriptions of the modes of movement, their neuronal control and the functional organization of the nervous system are less well understood. Nevertheless an understanding of the mechanisms involved is important to comparative neurophysiology since the earthworm probably stands midway in the evolution from non-myelinated to myelinated neurones and from simple nerve nets to organized reflex systems. This account is restricted, in general, to the species Lumbricus tewestrk L. in comparison with other annelids.

C. R. GARDNER

26 11.

TYPES OF MOVEMENT

(I) Peristaltic creeping If an earthworm is observed as it moves over a rough, dry surface it will be seen that progression is effected by a succession of waves of contraction and elongation, assisted by protrusion and retraction of bristles (setae) on the lateral and ventral surfaces of the body. This type of locomotion is usually described as normal creeping or peristaltic creeping and was first described by Friedlander (1894). Peristalsis was classically thought to be brought about by co-ordinated reciprocal contractions of the longitudinal and circular muscle bands that make up the bulk of the body wall (Chapman, 1950). The coelomic fluid maintains the turgidity of the earthworm and has been referred to as the ‘skeleton’ in this species (Seymour, 1970). Pressure transmission is also a function of the coelomic fluid, transmitting the effects of contraction of one set of muscles to the remaining muscles of the body wall. However, the segments of the earthworm are separated by septa which are quite rigid (Newell, 1950),and, being effectively impenetrable to coelomic fluid (Laverack, 1963) would bulge to some degree under pressure and limit pressure-transmission from one segment to the next. Thus, the septa play an important role in locomotion, allowing different pressures in different parts of the body. Wells (1969)considers that “They act as transverse braces and also check the tendency, which the hydrostatic skeleton of a terrestrial worm must always show, to flow into the lowest part of the body under the influence of gravity”. It has been suggested that the presence of septa allows a worm to keep up vigorous muscular performance over a longer period of time (Clark, 1962;Manton, 1961)although Wells (1969)questioned the interpretation of the data. Early views of the mechanism of peristaltic movement can be represented by the description of Gray & Lissmann (1938) (See Gray, 1968). In the normal resting condition both longitudinal and circular muscles of all segments are partially relaxed. Forward movement begins with contraction of the circular muscles of the anterior segments and a wave of contraction then passes backwards over the circular muscles of the body. When this wave has passed over the front half (6070 segments), the circular muscles at the extreme anterior end of the body relax and the longitudinal muscles begin to contract. A wave of contraction of longitudinal muscles then passes backwards and, having also passed over the front half of the body, is replaced by another wave of contraction of circular muscles of anterior origin. At any one time several adjacent segments exhibit a maximum state of contraction of their longitudinal muscles, with increase in body diameter, and together they form a ‘foot’ or ‘point d’appui’ which remains stationary relative to the ground and against which active muscles of adjacent segments exert tension. The segments immediately anterior to the ‘foot’ are undergoing contraction of their circular muscles and extend forward relative to the ground due to increased coelomic pressure against the ‘foot’ which is attached to the ground by the setae (Newell, 1950).Those segments immediately posterior to the ‘foot’ are undergoing contraction of longitudinal muscles and exerting backwards pull on it, but posterior slip is prevented by the setae which are

The neuronal control of locomotion in the earthworm

27

aligned backwards. In this region coelomic pressure need only be low since the tail is dragged passively forward. Then, as relaxation of the longitudinal muscles in the ‘foot’ region begins, these segments begin to move forward with increasing speed until this relaxation (circular muscle contraction) is complete. During contraction of longitudinal muscles the velocity of the segment decreases to rest at maximum contraction when a ‘foot’ is again formed. Each segment therefore moves forward in a series of 2-3 cm ‘steps’ with peristaltic waves passing over the body at 7-10 per minute (Gray & Lissmann, 1938). Locomotory contractile waves do not always pass more than halfway down the body, and in this case the entire remaining tail is dragged passively forward (Yapp,

1956).

Recent observations by Seymour (1969, I 971) whilst simultaneously recording electromyograms from both longitudinal and circular muscles together with the coelomic pressure, generally confirm this theory of “strictly reciprocally contracting longitudinal and circular musculature surrounding the coelomic hydrostatic skeleton )’ (Seymour, 1971),but he found it to be not always applicable. Simultaneous contraction of both muscle groups was sometimes observed and, also, changes in coelomic pressure in a segment were sometimes caused by active shortening of the adjacent segments and not by the muscular activity in that segment itself.

(2) Rapid escape movements One is apt to consider the earthworm as rather sluggish, but, if an attempt is made to withdraw a worm from its burrow, a very different impression will be gained; the worm can withdraw into its burrow with great rapidity. When an earthworm is stimulated by touching one end, it responds by simultaneous contraction of the entire longitudinal musculature resulting in rapid body shortening. This is characteristic of the escape reactions of many annelids although lacking in those species which do not possess giant nerve fibres (Bullock Sc Horridge, 1965). Since the work of Friedlander (1895) it has been known that the giant nerve fibres are responsible for mediating this rapid escape movement and this was confirmed by Bovard (1918a,b) who also showed that giant fibres were not involved in peristalsis. Rapid escape movements are followed by peristaltic creeping away from the stimulus. 111. THE NERVOUS SYSTEM

(I) Anatomy It is proposed merely to outline here the main structural features of the nervous system of the earthworm as full descriptions can be found elsewhere (Prosser, 1934; Stephenson, 1930; Bullock & Horridge, 1965; Staubesand, Kuhlo & Kersting, 1963). The main nerve trunk, i.e. the ventral nerve cord, lies close to the inner median surface of the body wall, and runs from segment 4 to the rear end of the earthworm. It swells slightly to form a ganglionic enlargement in each segment. In a typical segment (any one from the fourth to the penultimate) three paired segmental nerves originate, one of each pair on each side of the ventral nerve cord, and pass outwards

28

C. R. GARDNER

to the body wall. The anterior pair (segmental nerve I) arises a little behind the anterior septum of the segment, in front of the ganglionic enlargement and at some distance from the next pair. The second and third pairs (segmental nerves I1 and 111) arise close together as a ‘double nerve’ from the ganglionic enlargement. Nerve I11 on each side gives off, near its origin, a small septal nerve which innervates the posterior septum of the segment (Hess, 1 9 2 5 ~ ) . The anterior cerebral ganglion is a bi-lobed structure lying on the dorsal wall of the pharynx in segment 3. This gives rise to two nerves that pass forward to the prostomium, dividing and branching. The nerves, which end in the prostomial epidermis, enlarge to form nodule-like thickenings a short distance before entering the sub-epidermal nerve plexus. The nerves supplying segments I and 2 arise from the circum-oesophageal commissures which surround the oesophagus in segment 3. Segment 4 contains the sub-oesophageal ganglion, which represents the fused ganglia of segments 3 and 4, and the innervation of both segments stems from this ganglion. In the caudal segment Hess ( 1 9 2 5 ~found, ) in most specimens, six pairs of nerves but sometimes only five pairs. The typical arrangement was that of two successive body segments within the one caudal segment. Soon after leaving the cord each segmental nerve divides into two branches. The larger passes ventrally into the longitudinal muscle layer, then turns medianwards and runs between the longitudinal and circular muscle layers almost to the mid-dorsal line, giving off branches which pass somewhat obliquely to the epidermis. The smaller branch also sinks into the longitudinal muscle layer but then enters the circular muscle layer to run ventrally. It ends at the base of the epidermis near the mid-ventral line, giving off branches to the epidermis in the same way as the dorsal rami. The whole arrangement thus forms a nerve ring which is incomplete in the mid-dorsal and midventral lines. The ventral nerve cord in Lumbrim is single but its structure indicates that it is formed by the fusion of paired cords. The neuronal components of the cord are well protected by a sheath having an inner thick layer of connective tissue, a layer of muscle which is mainly longitudinal, a thinner layer of connective tissue and finally an outer peritoneum. Unlike the system in Hirudinae (Scriban & Autrum, 1932)the entire cord is medullary, having cell bodies throughout its length. In transverse section the cord can be seen to consist of an inner fibrous core and an outer cellular layer. The bulk of the fibrous core is the central neuropile with no nerve cell bodies and little glial component. It consists predominantly of nerve fibres with no obvious plane of orientation. No Schwann cells are present and all fibres are non-myelinated (de Robertis & Bennett, 1955; Coggeshall, 1965). The cell bodies mainly lie ventrally and laterally and are loosely packed with many glial cells (Levi, Cowden & Collins, 1966; Zimmermann, 1967). The boundary between the cellular layer and the neuropile is well defined by a layer of neuroglia (Staubesand et al., 1963). It is of interest to compare this organization with that of the ganglionic chain in Himdo which is enclosed in a blood vessel (Nicholls &van Essen, 1974).The surrounding blood provides a ready supply of nutrients and some protection from physical


29

damage as well as maintaining a constant environment around the nervous system. It is probably for these reasons that there is little sheath and fewer larger glial cells in the ganglia of Hirudo, and that the leech ganglion is avascular whereas that in the earthworm is vascular (Gray & Guillery, 1963; Coggeshall, 1965; Coggeshall & Fawcett, 1964). (2) Morphology and physiology (a) Sense organs On the epidermal surface there are aggregates of sensory cells forming small ‘bumps’ on the surface (Langdon, 1895). Both sensory cells and supporting cells are present. The sensory cells are of two types; widely distributed multiciliate cells with cilia passing vertically through the cuticle, and less abundant uniciliate cells, also with vertically orientated cilia (Knapp & Mill, 1971b). Since the cilia pass through the cuticle and into the external environment these authors suggest that the former may be tactile receptors whilst the latter, being less widely distributed, may be chemoreceptors. Also in the epidermis are single sensory cells (Langdon, 1895; Smallwood, 1926) which have many cilia lying parallel with the surface of the epithelium at the base of the cuticle. These are possibly proprioceptors (Knapp & Mill, 1971b). Tactile and pressure receptors in the body wall have been described by Gunther (1971c). Free nerve endings have been observed in the epidermis (Langdon, 1895) and may be related to the exteroceptors of Gunther (1971c) with cell bodies in the ventral nerve cord. The stimuli to which these cells respond remain to be clarified. Dawson (1920) described cells in or between the muscle layers and along the course of nerves which may have a sensory function. Some of these cells are similar to the intermuscular neurones of possibly proprioceptive function described by Gunther (I 971c). Photoreceptor cells exist singly, scattered in the epidermis, unlike the aggregation of photoreceptors in the so-called ‘eye’ of the leech (Hesse, 1897). They are half the height of the epidermis and have a refractile inclusion called a retinella (Hess, 1925b; Stephenson, 1930). Their axons enter the peripheral plexus of nerves but have not been followed far. The distribution of these cells parallels the sensitivity of the body wall to light (Hess, 19qb). Recent electron-microscopic studies of these cells have shown the photoreceptive inclusion to be a central labyrinthic intracellular cavity filled with microvilli, similar to the photoreceptor of the leech (Rohlich, Aros & Viriigh, 1970).

(b) Neuronal circuitry of the non-giant-$bre systems Afferent fibres passing into the nerve cord are of three types: (I) Fine fibres (0.10.3 pm diameter) from the epidermal sense organs. They enter in all segmental nerves and most divide in a T-shaped manner (Retzius, 1892)and their long, mainly ipsilateral, ascending and descending axons generally terminate within 1-2 segments of entry (Ogawa, 1939; in Pheretima, Gunther, 1971b). They travel in five nerve bundle on each side of the nerve cord (Gunther, 1971b). Numerous evenly-spaced terminal processes of these axons are arranged on the medial surface, directed towards the neuropile (Ogawa, 1939) in regions where contact could be made with small motoneurones, interneurones or giant-fibre systems. (2) Larger fibres (10pm diameter) from

30

C. R. GARDNER

intermuscular neurones enter the cord more dorsally and terminate ipsilaterally or contralaterally. These may be from proprioceptive cells (Gunther, 1971 b, c). (3) Fibres of intermediate size from the periphery with cell bodies in the ventral ipsi1ater;li nerve cord. These fibres have collaterals in the ventral neuropile. The cells are either unipolar (about 16 cells per ganglion) with their major fibre in the third segmental nerve of the same or preceding ganglion, or bipolar, sometimes even tripolar (2 cells per ganglion), with one fibre in the third segmental nerve of the same ganglion and the other(s) in the first segmental nerve of the same or (and) the following ganglion. The morphology of these neurones suggests that they may have exteroceptive function (Gunther, 1971b, c) similar to sensory neurones of like morphology in Himdo (Nicholls & Baylor, 1968). Prosser (1935) demonstrated that nerve fibres within the segmental nerves respond to tactile, chemical and photic stimulation of the epidermis with a series of slow repetitive potentials and estimated their conduction velocity at 0.04-0*08m/s. These responses may have been generated in the fine exteroceptive fibres. When studied in this way, sensory input to any segment was found to be from sensory receptors in that or the adjacent segments and the receptive fields of fibres in each segmental nerve greatly overlapped. Further studies have confirmed that the earthworm is sensitive to tactile, chemical and proprioceptive stimuli (Laverack, 1960, 1961; Mill & Knapp, 1967; Knapp & Mill, 1968~2,b) and to light (Hess, 1925b). However, Mill & Knapp (1967), recording from nerves cut between the recording site and the ventral nerve cord, showed that sensory input was segmental, the first segmental nerve receiving fibres from the anterior part of its own segment and the double nerve from the posterior part. Chemical and tactile sensory input is via segmental nerves I and I11 whilst proprioceptive input is via all three segmental nerves (Knapp & Mill, 1 9 6 8 ~ ) . A proportion of the impulses recorded by Prosser (1935) were ‘efferent sensory’ impulses (Mill & Knapp, 1967; Knapp & Mill, 19683) which result from axon reflexes in bipolar neurones (Gunther, 1971c) in the case of touch receptors. Their r61e is uncertain. They may be a by-product of the unpolarized network of sensory fibres or, alternatively, they may be of functional importance, being able to modify the pattern of impulses arriving at the synapses (Mill & Knapp, 1967). Efferent neurones not involved directly in the giant-fibre reflex are unipolar cells in the ventral and lateral nerve cord whose axons give off collaterals in the dorsal neuropile and then leave the cord through all segmental nerves, predominantly contralaterally. There are about 50 of these cells in each ganglion (Gunther, 1971b). In earlier studies these cells were probably grouped together with the type 3 afferent cells previously described (Retzius, 1892; Smallwood, 1930; Prosser, 1934; Ogawa, 1939). Prosser (1935) demonstrated that the motor field affected by stimulation in one segment, Iike the sensory field, covers adjacent segments, though the responses in these segments were weaker. Subsequently it has been shown that the motor fibres of the first segmental nerve innervate approximately the anterior two-thirds of the same segment and a small portion of the anterior segment while those of the ‘double nerve’ innervate approximately the posterior two-thirds of the same segment and a small portion of the segment posterior to it (Drewes & Pax, 1 9 7 4 ~ )The . conduction

The neuronul control of locomotion in the earthworm

31

velocity of these small motor fibres was observed to be 0.2-0-6m/s (Gunther, 1972) which is similar to the value (0.4 m/s) obtained by Horridge & Roberts (1960) although giant motoneurone responses were not distinguished by the latter authors. It seems probable that the small motoneurones are important in generating peristalsis. Early descriptions of interneurones within the nerve cord suggested the existence of small cells with local processes and larger cells with a span of up to four segments in each direction (Zawarzin, 1925) but no fibres travelling for longer distances within the nerve cord (Stephenson, 1930). However, Gunther (1971b) found three types of interneurone which together make up almost 90% of the total cell number in each ganglion. The first type and by far the most common consists of small cells with short axons in the ipsilateral neuropile. The cells of the second type are larger and are also numerous (approximately 280 per ganglion) but less so than the small interneurones (400 per ganglion). These appear regularly and symmetrically on both sides of the cord and their ipsilateral or contralateral axons run mostly longitudinally and often the length of a segment. These two groups may correspond to those of previous descriptions. However, Gunther (1971b) also found a third group of up to six ‘polysegmental interneurones ’ per segment with axons running longitudinally for at least 30 segments. The conduction velocity of slow impulses along the nerve cord is 0-4-0.6 m/s (Roberts, 1967) which conforms to the transmission speed normally found in small unmyelinated fibres. However, it is difficult to assess the number of synapses involved in such a pathway and thus to what degree transmission in the cord is affected by the delay and modification of the signal in passing across synapses. Synapses are axo-axonic. There are few, if any, axo-somatic synapses within the nerve cord (Bullock, 1951;de Robertis & Bennett, 1955; Gunther, 1971 b; Gunther & Schurmann, 1973) other than some on the soma of the giant fibre. It remains possible that sensory cells contact with small motoneurones directly or via the different types of interneurone. However, little is known of the degree or diversity of the interrelationships of different components of the nervous system. The work of Gunther (1971b) has shown that there is a much greater degree of morphological regularity of the many neural elements of the cord than previously thought. Thus, there is a morphological basis for such systematic and organized reflexes as have been suggested in the propagation of peristaltic waves of motion. There are fewer cells within the ganglia of Hirudo (about 800 in Lumbricus (Gunther, 1971b) and 350 in Hirudo (Coggeshd & Fawcett, 1964)) and they are more amenable to study, especially with electrophysiological techniques. In the leech a high degree of specificity has been observed in terms of function and synaptic contacts (Nicholls & van Essen, 1974) even when modified by discrete lesions of the nerve cord (Jansen, Muller & Nicholls, 1974).There are 7 pairs of central sensory cells (Nicholls & Baylor, 1968), 14 pairs of excitatory motoneurones and 3 pairs of inhibitory motoneurones (Stuart, 1970; Nicholls & Purves, 1970) in each ganglion, except those at either end of the nerve cord. Three of the pairs of sensory cells respond to light touch of the corresponding ipsilateral segment and have been called ‘touch cells’. There are two pairs of ‘pressure cells’ and two pairs of ‘noxious cells’ which respond to more severe

32

C. R.GARDNER

stimuli such as pinching or cutting the skin. Sensory and motor fields are discrete and show a constant position and size from segment to segment. Motor fields of different neurones innervating any one segment are arranged in a ‘quilt pattern’ (Nicholls & Baylor, 1968;Stuart, 1970;Gerschenfeld, 1973). Such regular organization of neurones is a general characteristic of invertebrate nervous systefis (Kandel & Kupfermann, 1970) and it is hoped that future studies will clarify the detailed organization of the nervous system of earthworms.

Neuronal circuitry of the giant-Jibre systems In the dorsal part of the ventral nerve cord of earthworms and other oligochaetes lie three giant fibres, heavily sheathed and separated from the rest of the cord. They extend through the whole of the body from cerebral ganglion to pygidium (Nicol, 1948). A similar large longitudinal axon with a cell body in each ganglion has recently been shown in Hirudo (Frank, Jansen & Rinvik, 1975). Two smaller giant fibres lie ventrally in Lumbricus (Stough, 1926; Smallwood & Holmes, 1927) but their synaptic connexions and physiological r81e are not well understood (Smallwood, 1930; Gunther, 1971b; Gunther & Schurmann, 1973). It is interesting that there are synaptic connexions between the ventral giant fibres and many small axons which form its ‘sheath’ (Coggeshall, 1965; Gunther, 1971b). The dorsal giant fibres consist of one medial fibre flanked by two smaller lateral fibres. They are relatively small in the anterior and posterior segments; the median fibre reaches its maximum diameter (about 75 pm in Lumbricus) in the first or second quarter of the body, the lateral pair reach maximum diameter (about 5opm) in the third quarter (Stephenson, 1930; Bullock & Horridge, 1965).The median fibre appears to start in the cephalic portion of the suboesophageal ganglion (Stough, 1926). The laterals are said to decussate here and enter the circum-oesophageal connectives, but they could not be followed for any great distance towards the brain (Bullock & Horridge, 1965). Injection of fluorescent dye into single giant fibres showed that they are segmental interneurones, conventional except for their size. Each giant axon has only one soma, with one nucleus, in each segment. The lateral and medial giant cells do not have direct connexions (Mulloney, 1970; see also Schurmann & Gunther, 1973). In each segment the giant fibres are interrupted by oblique septa which represent the cell boundaries of each segmental unit. The septa of the median fibre are near the intersegmental boundaries whilst those of the lateral fibres occur in the middle of each ganglion (Stough, 1926; Mulloney, 1970). Early observations with the electron microscope demonstrated the membrane structure of the septa but there were differences in the descriptions as to whether small vesicles occurred only on the ‘presynaptic’ side of the septum (de Robertis & Bennett, 1955; Issidorides, 1956; Hama, 1959,1961). Recently the septal structure has been described in detail and shown to be more complex than originally thought and it possibly includes several of the different structures previously described (Oesterle & Barth, 1973). Further to this, in adult worms, as many as 50% of septa in the median fibres and 20% of those in lateral fibres may be missing, so that the giant fibres are partially syncytial (Gunther, 1971a). Moreover it seems likely that the septal junctions have an electrotonic transmission (c)

The neuronul control of locomotion in the earthworm

33

mechanism (Bullock, 19453; Kao & Grundfest, 1957), and this, together with syncytial development, allows the rapid conduction along the giant fibres. The large longitudinal fibre in the leech has no septa (Frank et al., 1975) and this may represent a further stage in the evolutionary development of giant fibres. It remains possible that there is also a chemical mechansim in coexistence with the electrotonicmechanism (Antonov, 1964; Oesterle & Barth, 1973; Christoffersen & Miller, 1973).

Fig. I. The giant neurones in one ganglion of the ventral nerve cord of the earthworm, Lwnbrinrs tmestris (after Giinther & Walther, 1971).The median giant fibre (MG) and the lateralgiant fibres (LG) are represented in trans%erse section at several positionswithin the ganglion. There is a single cell body for each fibre (SM, SL) in each ganglion of the nerve cord. The giant interneurone (IG) with its cell bodies (SIG) makes contact with collaterals which originate from theventral surfaceof the median giant fibre. Note the electrotonicjunctionof the two lateral giant fibres. Three pairs of giant motoneuronesare associated with the median giant fibre (GMM) and one pair with the lateral giant fibres (GML) and their axons leave the nerve cord in the contralateral segmental nerves (I, 11,111). A more detailed description of the interconnexions of these giant neurones, and their connexions with other neurones in the nerve cord can be found in the text.

The sheath surrounding the giant fibres is composed of concentric lamellae of supporting membrane, similar in appearance to vertebrate myelin (Taylor, 1940). Unlike vertebrate myelm, however, interlamellar sheath cell cytoplasm separates the lamellae so that the inner surfaces of the lamellar membranes do not fuse (Bullock & Horridge, 1965). There are no nodes of Ranvier (Coggeshall, 1965) and saltatory conduction is unlikely, but it has been suggested that the sheath plays an important role in the high conduction velocity of the giant fibres (Bullock, 1945b). 3

B R E 51

34

C. R. GARDNER

The connections between the giant fibres and other neurones are summarized in Fig. I . The afferent connexions of the median giant fibre first make chemical synaptic contact with a giant interneurone ventral to the fibre (Gunther & Walther, I ~ I )which , is in electrical contact with the process from the segmental cell body to the median giant fibre (Giinther & Schurmann, 1973). The giant interneurone receives numerous afferent fibres from the neuropile. There are also two branches (collaterals), one leaving the ventral surface of the giant axon between the first pair of segmental nerves, and the other just posterior to the third pair (Mulloney, 1970; Gunther, 1971a). No processes were observed passing into the segmental nerves, contrary to earlier findings (Ogawa, 1939, in Pheretima comrnunissima). Efferent connexions of the median giant fibre in each segment are of two types; a specific efferent system involving giant motoneurones, and contacts with small segmental motoneurones. Three pairs of giant motoneurones connect with the median giant fibre. The axons of each pair make contact with one of the three branches of the giant fibre and then leave the cord contralaterally, each in one of the three segmental nerves (Gunther & Walther, 1971). The synapse between giant fibre and giant motoneurone is probably electrotonic (Gunther, 1972; Gunther & Schurmann, 1973). The median giant fibre is connected with small cells, presumably small motoneurones, via the giant interneurone. The ‘giant to giant interneurone’ junction is thus probably not polarized and transmits afferent and efferent impulses. The synapses between these motoneurones and the giant interneurone are chemical (Gunther & Schurmann, 1973). The axons of the giant motoneurones in the segmental nerves can be separated from those of the small motoneurones on the basis of conduction velocity and amplitude of action potentials (Gunther, 1972; Drewes & Pax, 1974b). The afferents of the lateral giant fibre are not separated from the fibre by an interneurone. The ventral, contralateral cell body of each fibre is multipolar and the neurite passing to the giant axon also branches (Mulloney, 1970; Gunther & Schiirmann, 1973). Some synaptic contacts occur on all of its processes. These connexions have been partly identified as with sensory fibres from the epidermis, although these constitute a surprisingly small proportion, and with multisegmental axons from the main fibre bundles or giant interneurones (Gunther & Schurmann, 1973). These authors assumed that most fierents were segmental interneurones of the nerve cord, as the nerve endings contained large granular vesicles characteristic of their cell bodies and not of the primary sensory cells (Myhrberg, 1972). Each lateral giant fibre also has two processes leaving its ventral surface segmentally, just posterior to the first and just anterior to the second segmental nerves (Mulloney, 1970; Gunther, 1971a). Fewer connexions between these and fibre bundles in the neuropile have been described (Gunther & Schurmann, 2973). The lateral fibres also have collaterals from the ventral surface anterior to the intersegmental septum, one pair of branches of which make electrotonic connexion (Mulloney, 1970; Gunther & Schurmann, 1973). The efferent connections of the lateral giant fibre system are via one pair of giant motoneurones which contact the fibres at their segmental bridge. Afterwards each axon divides into two branches which leave the cord on their contralateral side through


35

the third nerve of the same ganglion and the first nerve of the next (Gunther & Walther, 1971). The lateral giant fibre system also has efferent chemical connexions with thin arborizations which probably belong to small motoneurones (Gunther & Schurmann, 1973).

( d ) Neuromuscular junctions The longitudinal and circular muscles of the body wall are obliquely striated but their fine structural organization is fundamentally similar to that of cross-striated muscle fibres (Hanson, 1957; Kawaguti & Ikemoto, 1959; Ikemoto, 1963; Staubesand & Kersting, 1964; Nishihara, 1967; Heumann & Zebe, 1967; Knapp & Mill, 1971a; Mill & Knapp 1 9 7 0 ~ )In . Pheretima communissima observation of membrane potentials on longitudinal muscle fibres demonstrated a diffusely innervating inhibitory neuromuscular mechanism as well as an excitatory mechanism, possibly also diffusely innervating (Hidaka, Ito, Kuriyama & Tashiro, 1969b) but in Lumbricus Drewes & Pax (19743) did not demonstrate any inhibitory mechanism in longitudinal muscle although there may be such a mechanism in the circular muscle (Drewes & Pax, 1971). In Pheretima the excitatory transmitter probably increases the membrane permeability to sodium and potassium whilst the inhibitory transmitter increases chloride conductance (Hidaka et al., 1969b). The mean membrane potential of longitudinal muscle fibres in Lumbricus was estimated at 47.9 mV (Drewes & Pax, 1 9 7 4 ~and ) - 36.6 mV (Chaichenko & Klevets, 1972) but the ionic mechanism of the recorded excitatory junction potentials has not yet been studied in this species. Chang (1969) recorded a resting membrane potential of - 37 mV in Pheretima hawayana which is maintained by sodium and chloride ions whilst potassum ions are less important. Membrane potentials of somatic muscles in earthworms and leeches are generally low, about - 35 mV (Gerschenfeld, 1973). Whilst Mill & Knapp (197ob) identified only one morphological type of neuromuscular junction on longitudinal muscle, other studies have shown two (Smallwood, 1926; Nishihara, 1967; Rosenbluth, 1972). The last author showed in Lumbricidae one type resembling the cholinergic neuromuscular junctions of vertebrate skeletal muscle and a second type resembling adrenergicjunctions in vertebrate smooth muscle. A similar differentiation of neuromuscular junctions into these two types has been shown in Hirudo (Yaksta & Coggeshall, 1973). These two types may represent different transmissions controlling separate neuromuscular functions. Direct electrical activation of circular muscle in Pheretimu evoked only slow-rising contractions which may involve a sodium spike in muscle fibres (Tashiro, 1971). However, the longitudinal muscles responded with an initial fast-rising phasic contraction related to muscle-spike generation and a following tonic contraction not so related (Hidaka, Kuriyama & Yamamoto, 1969) but similar to ‘catch’ contractions in other invertebrate species (Tashiro & Yamamoto, 1971). These authors postulated that depolarization of the muscle membrane released calcium ions from the plasma membrane to generate the phasic contraction and from the sarcoplasmic reticulum to generate the tonic contraction. The membrane of the muscle fibres in Pheretima are more permeable to sodium and

-

3-2

36

C. R. GARDNER

potassium ions than mammalian or amphibian muscle membranes and the high permeability to sodium ions is probably controlled by the extracellular concentration of calcium ions (Hidaka, Ito & Kuriyama, 1969).

IV. THE ORIGIN OF THE RHYTHMIC NATURE OF PERISTALSIS

Friedlander (I 894) initially proposed that the peristaltic waves of muscular contraction were transmitted by sequential reflex activity, the ‘pull’ from an active segment initiating an intrasegmental reflex in the next segment and so resulting in transmission of a wave of contraction along the entire body. However, other investigators showed that this was not the whole story (Garrey & Moore, 1915; Bovard, 19184 b). Bovard concluded that the spread of peristalsis involved some flow of impulses within the nerve cord, independent of the chain-reflex effect (see von Holst, 1933). Further evidence against chain reflexes being the sole mechanism was found by Collier (1939a, b). The addition of weights at different stages during peristalsis in isolated preparations suspended longitudinally never broke the cycle. However, he also found that after transection of the circular muscle, the rhythm still persisted. This does not necessarily argue that the contraction of the circular muscle is not, in the intact worm, the cause of subsequent contraction of the longitudinal muscle as the constant tension applied by the recording lever to the longitudinal muscle may substitute for the lengthening caused by the contraction of the circular muscle. However, the postulate that peripheral stretch receptors are necessary is supported by the observation that when elicited by very weak stimulation, contractile waves do not pass along the entire length of the body (Yapp, 1956). Thus, the transmission of locomotory waves of contraction may be the result of decremental transmission within the nerve cord (Roberts, 1967) reinforced by stretch reflexes. This mechanism does not explain the continuing rhythmic nature of the peristaltic contractions, If the rhythm is generated within the nervous system (neurogenic) then which element is responsible for its generation? Although sense organs may respond with rhythmic impulses (Prosser, 1935)the frequency of this rhythm is much greater than that of the peristalsis. Furthermore, two different stimuli, which both cause rhythmic impulses in segmental nerves, do not always lead to augmented peristalsis, but may lead to inhibition (Prosser, 1935; Collier, 1939a, b). Spatial summation of asynchronous rhythmic discharges in different sensory fibres (see Moore, 1922) into a slower synchronous rhythm in the nerve cord seems unlikely without further processing within the nerve cord, nor was it observed by Roberts (1967). Stimulation of segmental nerves with single or repetitive shocks does not evoke rhythmic contractions in either circular or longitudinal muscles (Botsford, 1941; Horridge & Roberts, 1960; Drewes & Pax, 1971, I974b; Gardner & Cashin, 1975) although a ‘staircase’ enhancement of responses or biphasic responses were observed. The muscles of the earthworm’s body wall do show spontaneous discharges (Hidaka, Ito & Kuriyama, 1969; Drewes & Pax, 1971; Chaichenko & Klevets, 1972), but this may sometimes be explained by the effects of the physiological salt solutions used in the


37

studies (Drewes & Pax,1 9 7 4 ~ )However, . the somatic muscles of Pheretima hawayana are particularly active, showing spontaneous discharges at 2-8 Hz (Chang, 1969). On the basis of these data it seems probable that the rhythm of peristalsis is generated by the ventral nerve cord, either spontaneously, being modified by sensory input, or in response to sensory input. The nerve cord shows spontaneous rhythmic activity (Gray & Lissmann, 1938; Bullock, 1 9 4 5 ~ Vereschagin ; & Sytinskii, 1960) even when isolated (Gray & Lissmann, 1938) although these authors were not convinced that the rhythmic activity of the isolated cord could be equated with the rhythm of peristalsis. When the nerve cord is in .situ the rhythmic activity within the nerve cord was directly related to peristaltic muscular activity. The r61e of the nerve cord in generating the rhythm of peristalsis has been put in doubt because isolated strips of body wall, in the absence of the nerve cord, contract rhythmically when exposed to 5-hydroxytryptamine (5HT) (Gardner & Cashin, 1975). This observation is significant in the light of the presence of 5HT in neurones in the ventral nerve cord (Rude, 1966; Myhrberg, 1967; Kerkut, Sedden & Walker, 1967), some of which send axons to the periphery (Myhrberg, 1967; Gardner, 1975). Thus, the origin of the rhythm of peristalsis is still not certain and further study must determine whether it can be explained by rhythmicity in one component of the nervous system (i.e. in ‘generator neurones’ similar to those found in other invertebrates (see Kandel & Kupfermann, 1970) or whether a more complex mecfianism generates regular waves of contraction of the body wall during peristalsis.

V. THE ROLE OF THE SUBEPIDERMAL NERVE PLEXUS There is an extensive subepidermal nerve plexus in the earthworm (Smallwood, 1926; Prosser, 1934; Ogawa, 1939) but its physiologicalsignificanceremains uncertain. It is a region of much branching of sensory and motor fibres but synaptic junctions may not be present (Myhrberg, 1972), even though cells occur within the plexus (Smallwood, 1926). Lengths of body wall free of nerve cord do not usually exhibit spontaneous waves of contraction (Collier, 1 9 3 9 ~ Andersson ; & Fange, 1967; Gardner & Cashin, 1975) although Mennicke (1925) observed rhythmic activity in about half of such preparations. Also such lengths of wall between intact sections do not conduct waves if passive stretch is prevented (Garrey & Moore, 1915; Bovard, 1 9 1 8 ~ although ) the contrary has been claimed (Hess, 1 9 2 5 ~ )Isolated . strips from some levels (e.g. caudal) do exhibit conducted, spontaneous waves (Prosser, 1950) and sometimes show clearcut responses to bright light or tactile stimulation. It thus seems possible that there is some kind of direct neuronal connexion between receptors and muscles, possibly involving the subepidermal plexus. However, such connexions may not be important in peristaltic creeping as the observed responses were weak and infrequent.

38

C. R. GARDNER VI. THE CONTROL OF RAPID ESCAPE MOVEMENTS

It is well established that the giant fibres are concerned with rapid escape movements in earthworms (Bovard, 1918b; Stough, 1930; ten Cate, 1938; Rushton, 1945). Eccles, Granit & Young (1933) showed that a stimulus above threshold induced an all-or-none response consisting of two impulses, one travelling faster than the other. The faster impulse was generated by the median giant fibre and had a conduction velocity of 17-25 mls (10-12 "C). The slower impulse (7-12 m/s) was generated by the lateral fibres. The observation that only one impulse stems from both lateral giant fibres indicates that the transverse electrotonic connections between them (Wilson 1961; Mulloney, 1970; Gunther & Schurmann, 1973) have physiological function. The existence of two fibres which act as one may be a result of fusion of a paired cord system during evolutionary development and is frequently observed in invertebrates. Another notable example is the electrotonicjunction coupling the two 'colossal' cells of Retzius in the ganglion of the leech (Hagiwara & Morita, 1962). The action potentials of the giant fibres are extremely large and Rushton & Barlow (1943) were able to record them from the external surface of the earthworm. Kao & Grundfest (1957) found the resting potential of giant axons to be about - 70 mV and the action potentials 80-100 mV. The spike lasted only I ms at 20 "C, with no hyperpolarization at the end of the action potential. Stough (1930) believed the giant fibres to be polarized and that the median fibre conducted only from anterior to posterior and the laterals conducted in the opposite direction. However, Eccles et al. (1933) showed that both fibres could conduct in both directions. This was confirmed by Bullock (1945b) who showed that the functional polarity in the intact animal presumably depended on the anatomical arrangements of the sensory connexions. No structural differences of the giant fibres have been found in anterior and posterior regions of the worm to account for this functional polarity (Mulloney, I 970; Gunther & Schiirmann, 1973). Different characteristics of accommodation and facilitation have been observed at 'giant fibre-to-motoneurone' junctions (Roberts, 1966) but similar differences were not observed for ' sensory-to-giant fibre' junctions (Roberts, I 962b). Regional differences in sensory input were shown by Rushton (1946). The presence of two separate systems of giant fibres is difficult to explain as their functions seem similar, although they have opposite effects on the setae in the same segment (Rushton, 1946). The giant fibres conduct impulses at a 'supernormal' rate when conditioned by 2-5 previous impulses (Bullock, 1951). This facilitation is independent of stimulus frequency, but fatigue occurs with continuing stimulation. The physiological significance of this phenomenon is not clearly understood. It is interesting that stretching the giant fibre does not affect its conduction velocity (Bullock, Cohen & Faulstick, 1950) and thus normal locomotion, which probably involves changes in the length of the nerve cord (Staubesand et al., 1963), would not affect the mediation of the escape response by the giant fibres. The median giant fibre is one element in an efferent-afferent synaptic circuit producing after-discharge and repetitive firing (Amassian & Floyd, 1946; Kao,


39

1956; Kao & Grundfest, 1957; Christoffersen & Miller, 1973). Synaptic contact between the median giant fibre and a giant interneurone in each segment (Gunther & Walther, 1971) may be involved in a neural circuit responsible for this repetitive activity. This activity may enable the earthworm to grade its rapid escape response, which might be important in such a burrowing animal normally subjected to much sensory input from the periphery, most of which would not require a maximum withdrawal response (Roberts, 1 9 6 2 ~ ) . When one end of an earthworm is repeatedly prodded, the rapid contractions of the longitudinal muscle, extensive at first, get progressively smaller and finally cease altogether, although normal locomotory movements continue. This rapid fatigue is characteristic of ‘startle responses’ in many invertebrates. Roberts (1960, 1962b, 1966) showed that this is due to accommodation at ‘sensory-to-giant’ junctions and thought that the function of the phenomenon is to prevent the animal from being dominated by its escape response. However, fatigue at ‘giant-to-motor ’ junctions was also observed and the possibility that a partial state of fatigue exists under normal circumstances was suggested. This mechanism was regarded as a safety mechanism such that the muscular mechanism should not be fatigued when required for peristalsis (Roberts, 1962b). A facilitatory mechanism also exists at the ‘giant-to-motor junctions prior to fatigue (Roberts, 1966) and this may function to intensify the escape response, to maintain a proportion of ‘giant to-motor ’ junctions in a functional state at any moment or as a gating mechanism such that only the most important and severe stimuli actually elicit a motor response. Recent observations have shown that there are specific and separate motor pathways for the median and lateral fibre systems (Gunther & Walther, 1971) and that their electrophysiological characteristics are different (Gunther, 1972). Transmission at the ‘median giant fibre-to-giant motoneurone’ junction was always I :I with no facilitation and little fatigue and, in contrast to the observations of Roberts (1962b), neuromuscular fatigue was indicated to be mainly responsible for the vanishing of the escape response in the median fibre system. The ‘lateral giant fibre-to-giant motoneurone’ junction transmits in a I :I fashion but only after facilitation, and is easily fatigued. The myoneural junction of this system shows no facilitation and much less fatigue (Gunther, 1972), and is similar to the giant efferent pathways in polychaetes (Horridge, 1959; Wilson, 1960). The physiological significance of these differences are yet to be explained. VII. NEUROTRANSMITTERS

(I) Monoamines With the development of quantitative methods for assay of catecholamines their presence was shown in the tissues of worms (Lumbiczls and Nephtys) (Ostlund, 1954; von Euler, 1961; Clark, 1966). Several authors have found noradrenaline (NA) (1-0-1.6 ,ug/g) and dopamine (DA) (2-0-3-0 ,ug/g) to be present in the ventral nerve cord (Rude, 1969; Ehinger & Myhrberg, 1971; Gardner & Cashin, 1975). Higher levels of DA than NA suggest that DA is a neurotransmitter and not just a precursor

40

C. R. GARDNER

of NA. Little adrenaline has been detected in the nerve cord (Myhrberg & Rosengren, 1967). Histochemical studies using fluorescence have located primary catecholamines in epidermal sensory cells in several annelids (mainly dopamine) and in one pair of cell bodies in each segmental ganglion at the second segmental nerve in Lumbrincs (possibly noradrenaline) (Ehinger & Myhrberg, 1971; Myhrberg, 1967; Rude, 1966; Kerkut et al., 1967; Teichmann & Aros, 1966; Clark, 1966 (in Ne$hthys); Plotnikova & Govyrin, 1966) and also in Allolobophora caliginosa (Gardner & Cashin, 1975). The fluorescent sensory cells send fibres into the ventral nerve cord and account for about 10% of all sensory nerve fibres in the segmental nerves. The possibility that both NA and DA may exist in the Same neurone was discussed by Ehinger & Myhrberg (1971). Electron-microscope studies have confirmed this view when the presence of granular vesicles in nerve terminals is used as the main criterion (Myhrberg, 1971, 1972). It is interesting that there are no catecholamines in the nerve cord of Hirudo (Kerkut et al., 1967) although they are present in the perioesophaged ganglia (Ehinger, Falck & Myhrberg, 1968). Bieger & Hornykiewicz ( 1 9 7 2 ~suggested ) that NA and DA may have a r61e in the sensory modulation of locomotion, by an action within the nerve cord, as they enhance the peristaltic contractions caused by tension in isolated sections of earthworm. We have confirmed these findings (Gardner & Cashin, 1975). Whilst touch, stretch (Collier 1 9 3 9 ~ )or illumination of such preparations after dark adaptation (own observations) may elicit similar responses it has been suggested that the monoaminergic sensory cells are concerned with the reception of photic or tactile stimuli (Bieger & Hornykiewicz, 1972b). Chromaffin cells were shown in the ventral nerve cord of Lumbricus herculeus (Gaskell, 1914) and Lumbricus terrestris (Lancaster, 1939) and it was supposed that these cells contained adrenaline. However, 5-hydroxytryptamine (5HT) also gives the chromaffin reaction and, by use of histochemical tests, Bianchi (1962, 1967) showed that nerve cells in Eiseniafoetida and Octalasium complanatum which gave a chromaffin reaction contained a substance similar to 5HT. As in other invertebrates (see Gerschenfeld, 1973) there are large amounts of 5HT (7-10,uglg) in the nerve cord (Welsh & Moorhead, 1960; Myhrberg & Rosengren, 1967; Rude, 1969). There are 5HT-containing cell bodies in the ventral nerve cord (Rude, 1969; Myhrberg, 1967; Kerkut et al., 1967; Gardner & Cashin, 1975) which could be interneurones (Rude, 1969; Myhrberg, 1972) or motoneurones (Rude, 1969; Myhrberg, 1967; Gardner, 1975) or both. Recent autoradiographic observations in Nereidae have shown uptake of 3H-5HT into neuromuscular junctions (DhainautCourtois & Dhainaut, 1974). Neuromuscular junctions with dense-core vesicles in Hirudo give 5HT-like histochemical reactions and take up 5HT but not NA (Yaksta & Coggeshall, 1973). These findings support the hypothesis that the neuromuscular junctions with granular vesicles (Rosenbluth, 1972) contain 5HT. Some neurones in the nerve cord of Hirudo also contain 5HT. Six such neurones in each ganglion have been found generally (see Gerschenfeld, 1973; Sakharov, 1970) although a seventh has recently been shown (Marsden & Kerkut, 1969).The ‘colossal’


41 cells of Retzius are of particular interest. There is little doubt that they contain gHT. The amine is probably present in granulated vesicles and the fluorescent spectrum produced by treatment with formaldehyde is very similar to that produced by gHT in albumin film (Rude, Coggeshall & van Orden, 1969). Furthermore, the amine present in isolated Retzius cells has similar fluorescent and chromatographic properties to gHT (Rude et al., 1969). Thin varicose fibres containing gHT are intermingled with muscle cells of the longitudinal circular and dorso-ventral layers and Ehinger et al. (1968) proposed that they were the fibres of the Retzius cells. However, Gerschenfeld (1973, p. 13) states that “it is not known whether Retzius neurones actually release gHT through their endings and precisely where these endings are located”.

(2) Acetylcholine Acetylcholine (ACh) has long been implicated as a neuromuscular transmitter in earthworms (Mennicke, 1925; Wu, 1939; Bacq, 1947). ACh causes contraction of the isolated body wall and this is potentiated by anticholinesterases(Bacq & CoppCe, 1937; Wu, 1939; Andersson & Fange, 1967). Muscle responses to electrical stimulation were similarly potentiated (Botsford, 1941). A rBle for ACh at synapses in the ventral nerve cord is suggested by the presence of acetylcholinesterase on the border between the neuropile and neuronal layers and scattered in the neuropile (Vigh-Teichmann & Goslar, 1969). However, the enzyme is localized on neuroglial membranes (Dhainaut-Courtois & Dhainaut, 1974, in N e r e i h ; Teravainen, 1969, in Lumbrieus) and not on post-synaptic membranes and it is not certain if its action is to hydrolyse released neurotransmitter. Teravainen (1969) described ‘ventral giant cells’ which could be cholinergic, but it is not certain if they are the cell bodies of the giant fibres or the giant motor neurones. The Retzius cells respond to ACh suggesting a possible transmitter r61e for this substance in the nerve cord of the leech (Kerkut & Walker, 1967).

(3) Gamma-aminobutyric acid Gamma-aminobutyric acid (GABA) may function as the neurotransmitter at inhibitory neuromuscular junctions on the longitudinal muscles of the body wall in Pheretima communissim (Ito, Kuriyama & Tashiro, 1969, 1970; Hidaka, Ito, Kuriyama & Tashiro, 1969a, b) but a similar mechanism has not been found in Lumbricus terrestris (Drewes & Pax, 1974b) although an inhibitory mechanism may exist in the innervation of the circular muscle (Drewes & Pax, 1971). The GABA antagonist, picrotoxin, enhanced contractions of the body wall and bioelectrical activity of the nervous system (Vereschagin & Sytinskii, 1960) but GABA does not inhibit AChinduced contractions of the body wall of Lumbricus (Koidl, 1974) or of the marine annelid Abarenicola pacifica (Florey & Florey, 1965). Qualitative evidence has been found for the presence of GABA in the nervous tissues of N e r k (Dhainaut-Courtois & Dhainaut, 1974; Dhainaut-Courtois, Caridroit & Biserte, 1969) but paper chromatographic methods demonstrated little GABA in the nervous system of Eisenia, although

42

C. R. GARDNER

GABA-transaminase, which catabolizes GABA, was found in a similar concentration to that in mouse brain (Pasantes, Tapia & Massieu, 1962). It is possible that GABA is not the normal substrate for this enzyme in the worm. In contrast to this, Osborne (1971) found high concentrations of GABA in the cerebral ganglia of Lumbricus, but subsequent observations have again found very little GABA in Lumbricus or Hirudo (Koidl, 1974). The chemical transmitter which produces inhibitory post-synaptic potentials in ‘touch’ cells in the leech (Baylor & Nicholls, 1969)) remains to be identified. VIII. THE PHARMACOLOGY OF EARTHWORM LOCOMOTION

(I) Monoamines The biosynthesis and degradation of the monoamines show some similarities to mammalian systems. I found that isolated nerve cords incubated with 14C-DAstore it and convert a portion to 14C-NAwhich is also retainedin the nerve cord (unpublished), suggesting the presence of dopamine-/3-hydroxylase. Both the catecholamines and 5 H T may be catabolized by monoamine-oxidases as inhibitors of these enzymes increase the concentrations in the nerve cord (Myhrberg, 1967; Gardner & Cashin, I 975) and sensitize isolated sections to exogenous monoamines (Bieger & Hornykiewicz, 1972a, b ; Gardner & Cashin, 1975). The greater increase in 5HT than in catecholamine concentration which we observed after a monoamine-oxidase inhibitor may indicate an alternative means of catabolism of NA and DA, perhaps by catechol0-methyl transferase. After inhibition of monoamine-oxidase Bieger & Hornykiewicz (19724 b) found that DA was most and 5HT least potent in enhancing peristalsis and that NA was now inhibitory, whereas we found that gHT was most potent and NA and DA both inhibited in 20% of applications (Gardner & Cashin, 1975). These differences could stem from different methods of pretreatment. Relaxation of isolated body wall by DA has been observed whilst NA caused a contraction (Anderson & Fiinge, 1967). Our observation of a reduction of responses of cordless body wall to stimuli which activate nerve endings, but not muscles directly (Gardner & Cashin, 1975) and to ACh application(Bieger & Hornykiewicz, I 972 a) suggest a depression of neuromuscular transmission by the catecholamines similar to that in mammals (Jenkinson, Stamenovie & Whitaker, 1968; Capetola, Ferko & Calesnick, 1974). This effect appears to be separate from their neurotransmitting r61e in the nerve cord and may represent a hormonal or neurosecretory effect (see Ito, Kuriyama & Tashiro, 1970). In contrast, the biosynthesis and degradation of gHT in Himdo are unlike those in mammals. Only one of two enzymes necessary for 5HT synthesis in mammals may be present in the leech (Coggeshall, Dewhurst, Weinrich & McCaman, 1972;Hildebrand, Barker, Herbert & Kravitz, 1971). Furthermore, there is no mine-oxidase activity in the ventral nerve cord or muscles (Della Corte & Nistri, 1974; Yaksta & Coggeshall,

1973).

The DA receptors in the nerve cord of Lumbricus are similar to those in mammals, being stimulated by apomorphine and piribedil (Saka & Tsuji, 1934; Bieger & Hornykiewicz, 1972a, b ; Gardner & Cashin, 1975) but the usefulness of this prepara-


43

tion as a model of mammalian DA receptors was limited by difficulty in demonstrating specific receptor blockade. sHT contracted isolated body wall, as observed by Anderson & Fange (1967), but was not the only amine to do so. No mention was made with respect to rhythmic contractions. In sections of bodywall with intact nerve cord, gHT consistently enhanced tension-induced peristalsis (Bieger & Hornykiewicz, 1972a ; Gardner & Cashin, 1975) and we showed that inactive cordless body wall contracted rhythmically to gHT under these conditions. I did not consistently observe this effect with NA, DA or ACh (Gardner, 197s). Thus, gHT may have a r81e in the motor system during peristalsis. Both gHT and ACh depress the tonic more than the phasic contraction in directly stimulated longitudinal muscle in Pheretima communissima (Hidaka et al., 1969; Tashiro & Yamamoto, 1971). It was postulated that this effect was due to a release of calcium ions from the plasma membrane and sarcoplasmic reticulum by the depolarization produced by electrical stimulation or ACh. Having a similar action to ACh, sHT may also depolarize the muscle membrane, but no rhythmic activity was evoked by gHT in these preparations. However, these experiments were conducted under isometric conditions and the stimulus of constant tension, which is provided by isotonic conditions, may be required for rhythmic activity. The action of gHT on the musculature of the body wall of the leech again contrasts with that of the earthworm. Single muscle fibres in the longitudinal dorsal muscle have been studied with intracellular microelectrodes. ACh depolarized the membrane or evoked an increased frequency of spontaneous excitatory junction potentials. 5 HT decreased the frequency of these junction potentials (Walker, Woodruff & Kerkut, 1968). sHT relaxes the dorsal muscle and reduces contractions induced by ACh (Schain, 1961). It thus seems possible that 5HT is an inhibitory neurotransmitter in the leech although inhibitory post-synaptic potentials have not been recorded from muscle fibres (Walker et al., 1968; Washizu, 1967). Thus, whilst gHT may have a motor function in annelids it has different effects on the muscles of different members of the group. This is further emphasized by the action of sHT in sabellid worms. It relaxes dorsal longitudinal muscle strips but does not antagonize contractions induced by ACh (Alvarez, del Castillo & Sanchez, 1969). The storage mechanisms for the monoamines are similar to those in mammals. We found that all the monoamines were depleted by reserpine but only the catecholamines and not gH T were depleted by 6-hydroxydopamine. Furthermore, the releasing agent, dexamphetamine, could enhance peristalsis as long as the nerve cord was present. Para-chloramphetamine, which releases gHT to a greater extent than catecholamines in mammals, enhances peristalsis when the nerve cord is present and has a SHT-like effect on the body wall alone. This provides pharmacologicalevidence for the presence of SHT-containing nerve-endings in the periphery. In support of this, depletion of catecholamines does not affect peristalsis but it is abolished if gHT is also depleted (Gardner & Cashin, 1975). This further suggests that gHT may be a final link in the neural control of peristalsis. In all studies of monoamine systems in the earthworm the concentrations of

44

C. R. GAFWNER

pharmacological agents, in common with endogenous monoamine levels, are high by comparison with those used in mammalian studies but similar to those required in other invertebrate species (Dahl & Rosengren, 1966). 5HT may also be an inhibitory transmitter at a synapse on the Retzius cell body in the ventral nerve cord of the leech. The Retzius cells are inhibited when gHT is applied by dilution in the environment or by iontophoresis (Kerkut & Walker, 1967). This effect was caused by altered permeability of the cell membrane to chloride ions and the receptors on Retzius cells have some similarities to 5HT receptors in mammals (Smith & Walker, 1973). (2)Acetylcholine The ACh receptors in the earthworm’s body wall, generally in the longitudinal muscle, have been much studied but there are discrepancies between the observations. Mennicke (1925)suggested that the receptors were ‘muscarinic’ in nature, being blocked by atropine and not by curare. However, subsequent studies have demonstrated blockade (Haluk & Baysal, 1972),partial blockade (Wu, 1939;Andersson & F h g e , 1967)or no blockade (Gardner & Cashin, 1975)with atropine. The receptors may not be ‘nicotinic’ in nature, as tubocurarine and gallamine have not been consistently shown to block responses to ACh (Andersson & Fange, 1967;Gardner & Cashin, 1975)although blockade has been observed (Baysal, 1967;Haluk & Baysal, 1972). Tubocurarine does block miniature excitatory junction potentials and the identical effects caused by ACh on neuromuscular junctions of Pheretima communissima (It0 et al., 1970).Nicotine at first causes contraction, then relaxation of the body wall and blocks the effects of ACh (Wu, 1939), which might suggest a ‘nicotinic’ receptor. Also piperazine depolarizes the muscle membrane of Pheretima hawayana. This effect is unlike the well known action of this drug in Ascaris lumkicoides (Del Castillo, de Mello & Morales, 1964)and is blocked by atropine (Chang & Bruno, 1970). Thus, it may not be possible to apply the receptor classifications appropriate for mammals to invertebrate species. In contrast to the dorsal muscle of the leech, which has nicotinic receptors (Chang & Gaddum, 1933; McIntosh &Perry, 19go),both the lack of sensitivity and these difficulties in comparing receptor types have limited the use of the body wall of the earthworm for the assay of ACh. Contractions of the body wall induced by ACh are single, swift and tonic in nature, but not rhythmic (Andersson & F h g e , 1967;Wu, 1939;Gardner & Cashin, 1975) and are therefore different from those induced by gHT under isotonic conditions. The roles of ACh and gHT during locomotion require further investigation. ACh depolarizes Retzius cells in the ganglion of the leech (Kerkut & Walker, 1967). However, the receptors do not fall clearly into the muscarinic or nicotinic classifications (Woodruff, Walker & Newton, 1971). IX. SUMMARY

I. The two types of movement in the earthworm, peristalsis and rapid escape, are described. Peristalsis or normal locomotion is brought about by waves of contraction of antagonistic longitudinal and circular muscles. Movement is with respect to a point


45

which is fixed by contact of the setae and pressure on the walls of the burrow. The important role of the coelomic fluid as a ‘fluid skeleton’ is stressed. Rapid escape movements are mediated by the dorsal giant fibres of the ventral nerve cord and consist of simultaneous contracture of all the longitudinal muscles of the body wall. 2. The anatomy of the nervous system is outlined and its cellular components and organization discussed in detail. Several morphological types of sensory receptor are related to the reception of tactile, chemical, proprioceptive and photic stimuli and the organization of sensory input is outlined. Within the nerve cord the interneurones and dorsal giant fibres are described. The mechanism of impulse transmission across the giant fibre septa is reviewed, and while it is concluded that the major means of transmission is electrotonic it is possible that there is also a chemical mechanism. There is still a lack of knowledge of specific synaptic connexions within the nerve cord, with the exception of the giant motor system. This contrasts with detailed knowledge of synaptic connexions in the leech nervous system. The differences between the specific giant motor system, which is similar to those in other invertebrate nervous systems, and the small motoneurones can be equated with the possibly different rBles of the two systems in rapid escape and peristalsis respectively. Neuromuscular junctions are described. 3. Hypotheses on the origin of the rhythm of peristalsis are discussed, and it is concluded that there are two possible sources. Either there is a generator mechanism in the ventral nerve cord or a peripheral mechanism which may involve sensory to motor connexions via the sub-epidermal nerve plexus. 4. The characteristics of the giant-fibre reflex are discussed with respect to the escape response. Facilitation of giant-fibre conduction velocity and giant-to-motor unctions, repetitive giant-fibre discharge and fatigue of several synaptic junctions are related to control and fatigue of the escape response in the whole worm. The functional polarity of the escape response in Lumbricus is noted. 5. Evidence is presented for the existence and transmitter rBles of noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5HT) and acetylcholine (ACh) in Lumbriw. NA and DA are not present in the ventral nerve cord of Hirudo.Gammaaminobutyric acid (GABA) seems unlikely to be a transmitter in Lumbricus or Hirudo although it may be in Pheretima. There is little evidence of inhibitory mechanisms in Lumbriw. NA and DA are primarily sensory transmitters, possibly released from terminals in the nerve cord of cells responsive to photic and tactile stimuli. 5HTcontaining cells in the nerve cord send processes into the neuropile of the cord and to the periphery and gHT may be the transmitter released by some interneurones in the cord and by some motoneurones. ACh seems established as a transmitter at neuromuscular junctions of annelids but the r6les of ACh and 5HT in peristaltic muscle contractions of Lumbriw need further clarification. 6. The mechanisms of synthesis, storage and catabolism of monoamines in annelids are compared with those of mammals. Strong similarities are noted for Lumbricus but not for Hirudo. The pharmacological typing of receptors derived from studies of mammals may apply to the ACh receptors in the musculature of the leech body wall

46

C. R. GARDNER

(nicotinic) and to the DA receptors in the nerve cord of Lumbricus,but not to the ACh receptors, either on the Retzius cells in the leech ganglion or in the musculature of the earthworm body wall. X. REFERENCES V. (1969). Pharmacological responses of the dorsal ALVAREZ, M. C., DEL CASTILLO,J. & SANCHEZ, longitudinal muscle of Sabellastmte magnifca. Comparative Biochemistry and Physiology 29, 93 1-42. AMASSIAN, V . E. & FLOYD, W. F. (rg46). Repetitive discharge of giant nerve fibres of the earthworm. Nature, London 157,412-3. ANDERSON, R. & FANGE, R. (1967).Pharmacologic receptors of an annelid (Lumbricus terrestris). Archives Internationales de Physiologie et de Biochimie 7 5 , 461-8. ANTONOV, V . F. (1964). T h e effect of monoiodoacetate and temperature on the conduction of a nerve impulse through the septa of the earthworm giant axom. Biophysics 9, 194-203. BACQ,2. M. (1947). L'acCtylcholine et l'adrhnaline chez les invertbbrks. Biological Reviews of the Cambridge Philosophical Society 22, 73-91. BACQ,2. M. & COPPBE,G. (1937). Reaction des vers et des mollusques a I'hsbrine. Existence de nerfs cholinergiques chez les vers. Archives Intermtionales de Physiologie et de Biochimie 45, 31-24. BAYLOR,D. A. & NICHOLLS, J. G. (1969). Chemical and electrical synaptic connections between cutaneous mechanoreceptor neurones in the central nervous system of the leech. Journal of Physiology 203,592409. BAYSAL, F. (1967). The effects of certain agents on the isolated muscle of the earthworm (Lumbricus terrestris). Ankara Universitesi T i p Fakultesi Mecmuasi 20, 1-5. BIANCHI,S. (1962). Sur la prksence d'un mathriel fluorescent dans les cellules nerveuses des ganglions du systbme nerveux central de quelques Oligochhtes. Recherches histochimiques et d'histospectographie de fluorescence. Annales d'Histochimie, Suppl. 2, 143-5. BIANCHI,S. (1967). T h e amine secreting neurones in the central nervous system of the earthworm (Octalasium camplcmatum) and their possible neurosecretory role. General and Comparative Endocrinology 9, 343-8. BIEGER,D. & HORNYKIEWICZ, 0. ( 1 9 7 2 ~ )Dopamine . in the earthworm, Lumbricus terrestris, enhancement o f rhythmic contractile activity. Journal of Neuropharmacology XI, 745-8. BIEGER,D. & HORNYKIEWICZ, 0. (1g72b). Dopamine, noradrenaline, 5-hydroxytryptamine and sensory stimulation in Lumbricus terrestris. Abstract 129, 5th International Congress of Pharmacology, San Francisco. BOTSFORD, E. F. (1941).T h e effect o f physostigmine on the responses of earthworm body wall preparations to successive stimuli. Biological Bulletin. Marine Biologic~lLaboratory, Woods Hole 80, 299-3 I 3. BOVARD, J. F. ( 1 9 1 8 ~ )T. h e transmission of nervous impulses in relation to locomotion in the earthworm. University of California Publication in Zoology 18, 1 0 3 3 4 . BOVARD, J . F. (Igr8b). T h e function of giant fibres in earthworms. University of California Publication in Zoology 18, 135-44. BULLOCK, T . H . ( 1 9 4 5 ~ )Problems . in the comparative studies of brain waves. YaleJournal of Biology and Medicine 17, 657-79. BULLOCK, T . H. (19456). Functional organization of the giant fibre system of Lumbricus. Journal of Neurophysiology 8, 55-71. BULLOCK, T . H . (1951).Facilitation of conduction rate in nerve fibres. Journal of Physiology 114,89-97. BULLOCK, T. H., COHEN,M. J. & FAULSTICK, D. (1950). Effect of stretch on conduction in single nerve fibres. Biological Bulletin. Marine Biological Laboratory, Woods Hole 99, 320. BULLOCK, T. H. & HORRIDGE, G. A. (1965). Annelida. In Structure and Function in the N m o u s Systems of Invertebrates, vol. I San Francisco : Freeman. CAPETOLA, R., FERKO, A. P. & CALESNICK, B. (1974). T h e inhibitory action of dopamine on the rat anterior tibialis muscle preparation. Archives Intermtionales de Phurmacodynamie et de Therapie 207, 107-13. CHAICHENKO, G. M. & KLEVETS, M . Y . (1972).Electrical potentials in somatic muscles of the earthworm Lumbricus terrestris. Zhurnal Evoliutsionnoi Biokhimii i Fiziologii 7, 43 2-3. CHANG,Y . C. (1969). Membrane potential o f muscle cells from the earthworm Pheretima hawayana. American Journal of Physiology 216, 1258-65. CHANG, Y . C. & BRUNO,2. (1970). T h e effect of piperazine on toad and earthworm muscle membrane potentials. Pharmacology 4, 143-5 I . CHANG,H. C. & GADDUM, J. M . (1933). Choline esters in tissue extracts. Journal of Physiology 7 9 , 255-85.


47

CHAPMAN,G. (1950). On the movement of worms. Journal of Experimental Biology 27, 29-39. CHRISTOFFERSEN, G.R. J. & MILLER,L. A. (1973). Two types of excitatory activity recorded from the median giant fibre of the earthworm. Acta Physiologa’ca ScandiMoica &I, 425-7. CLARK,M. E. (1966). Histochemical localization of monoamines in the nervous sytem of the polychaete Nephtys. Proceedings of the Royal Society of London, Series B 165,30835. CLARK, R. B. (1962). On the structure and function of polychaete septa. Proceedings of the Zoological Society, London 138,543. COGGESHALL, R. E. (1965). A h e structural analysis of the ventral cord and associated sheath of Lumbricus terrestris. Journal of Comparative Neurology 125, 393-8. COGGESHALL, R. E., DEWHURST, S. A., WEINREICH, D. & MCCAMAN,R. E. (1972). Aromatic acid decarboxylase and choline acetylase activities in a single identified 5HT containing cell of the leech. Journal of Neurobiology 3, 259-65. COGGESHALL, R. E. & FAWCETT, D. W. (1964). The fine structure of the central nervous system of the leech Hirudo medicinalis.Journal of Neurophysiology 27, 229-89. COLLIER, H.0.J. ( 1 9 3 9 ~ )Central . nervous activity in the earthworm. I. Responses to tension and to tactile stimulation. Journal of Experimental Biology 16,286-99. COLLIER, H.0. J. (19396). Central nervous activity in the earthworm. 11. Properties of the tension reflex. Journal of Experimental Biology 16,300-12. DAHL,E. & ROSENGREN, L. (1966). Neuronal localization of dopamine and 5-hydroxytryptamine in some Mollusca. Zeitschrift f a r ZelIforschung und Mikroskopische Anatomie 7 1 , 489-98. DAWSON, A. B. (1920). The intermuscularnerve cellsof the earthworm.Joumo1 of Comparative Nacrology 32 1 5 5 7 1 . DE ROBERTIS, E. D. & BENNETT, H. S. (1955). Some features of the submicroscopic morphology of synapses in frog and earthworm. Journal of Biophysical and Biochemical Cytology I, 47-58. DEL CASTILLO, J., DE MELLO,W. C. & MORALES,T. (1964). Mechanism of the paralyshg action of piperazine in Ascaris muscle. British Journal of Pharmacology 22,463-77. DELLA CORTE, L. & NISTRI,A. (1974). Amine oxidase activity in tissue of the leech ( H i d medicinalis). British Journal of Pharmacology 52, 1 2 9 ~ . DHAINAUT-COURTOIS, N., CARIDROIT, M. & BISERTE, G. (1969). Sur la prbsence de la N,N‘-dhbthyl-5hydroxytryptamine (bufotbnine) et de l’acide y-aminobutyrique dam le systbme nerveux d’une Annelide Polychbte. Comptes Rendw de Sdances de la So&& de Biologie et de sesfiliales 163,1563-7. DHAINAUT-COURTOIS, N. & DHAINAUT,A. (1974). Gamma-aminobutyric acid, bufotenin and acetylcholine. Subcellularlocalizationin the nervous system of annelida (Nereidae). General and Comparative Endmhology 22, 354. DREWES, C. D. & PAX,R. A. (1971). Mechanical responses of the body wall muscle of the earthworm, Lumbricus terrestris, to segmental nerve stimulation. Canadian Journal of Zoology 49, 1527-34. D m , C. D. & PAX, R. A. ( 1 9 7 4 ~ ) Neuromuscular . physiology of the longitudinal muscle of the earthworm, Lumbricus terrestris. I. Effects of different physiological salines. Journal of Experimental Biology 60,445-52. DREWES, C. D. & PAX,R. A. (19746). Neuromuscular physiology of the longitudinal muscle of the earthworm, Lumbricus terrestris. 11.Patterns of innervation.Journa1 of Experimental Biology 60,45347. DREWES, C. D. & PAX,R. A. (1974~).Neuromuscular physiology of the longitudinal muscle of the earthworm, Lumbricus terrestris. 111. Mapping of motor fields. Journal of Experimental Biology 60, 49-75. ECCLES, J. C., GRANIT,R. & YOUNG, J. Z. (1933). Impulses in the giant nerve fibres of earthworms. Journal of Physiology 33,23-5. EHINGER, B., FALCK, B. & MYHRBERG, H. E. (1968). Biogenic amines in Hirudo medicinalis. Histochemie 15,140-9.

EHINGER, B. & MYHRBERG, H. E. (1971). Neuronal localization of dopamine, noradrenaline and 5 hydroxytryptamine in the central and peripheral nervous sytem of Lumbrim terrestris (L.). Histochemie 28, 265-75. FLOREY, E. & FLOREY, E. (1965). Cholinergic neurones in the Onychophora: A comparative study. Comparative Biochemistry and Physiology 15, 12536. FRANK, E., JANSEN, J. K. S. & RINVICK,E. (1975). A multi-somatic axon in the central nervous system of the leech. Journal of Comparative Neurology 159,1-13. FRIEDLANDER, B. (1894). Beitriige zur Physiologie des Centralnervensystems und des Bewegmgsmechanismus der Regenwiirmer. Pfliigers Archiv: European Journal of Physiology 58, 168-207. FRIEDLANDER, B. (I895). uber die RegenerationherausgeschnitterTheile des Centralnervensystemsvon Regenwijrmer. Zeitschrift fur wissenschaftliche Zoologie 60, 249-83. GARDNER, C. R. (I975). A role for monoamines in the control of locomotion in the earthworm, LztmbZinrs terrestris. South African Medical Journal 49. 269.

48

C. R. GARDNER

GARDNER, C. R. & CASHIN, C. H. (1975). Some aspects of monoamine function in the earthworm, Lumbricus terrestris. Neuropharmacology 14, 493-500. GARREY,W. E. & MOORE, A. R. (1915). Peristalsis and coordinationin the earthworm. AmericanJournal of Physiology 39, 139-48. GASKELL, J. F. (1914).The chromaffin system of annelids and the relation of this system to the contractile vascular system in the leech, Hirudo medicinalis. Philosophical Transactions of the Royal Society of London, Series B 305, 153-211. GERSCHENFBLD, H. M. (1973). Chemical transmissionin invertebrate central nervous systems and neuromuscular junctions. Physiological Reviews 53, 1-1 19. GRAY,H. G. & GUILLERY, R. W. (1963). An electron microscopical study of the ventral nerve cord of the leech. Zeitschrift fur Zellforschung und Mikroskopische Anatomie 60, 826-49. GRAY,J. (1968). Animal Locomotion. London. Weidenfeld and Nicolson. J. W. (1938). Locomotory reflexes in the earthworm. Journal of Experimental GRAY,J. & LISSMANN, Biology 15, 506-17. GUNTHER, J. (1971a). Der cytologische Aufbau der dorsalen Riesenfasem von Lumbricus terrestris L. Zeitschrift fiir evissenrchaftliche Zoologie 183,5 1 7 0 . GUNTHER, J. (1971b). Mikroanatomie des Bauchmarks von Lumbrim terrestris L. Zeitschrift fur MOTphologie der Tiere 70, 141-82. G ~ ~ ~ HJ. E (1971 R , c). On the organization of enteroceptive afTerents in the body segments of the earthworm. Verhandlungen der Deutschen Geselkchaft fur Zoologie 64, 261-5. G-ER, J. (1972). Giant motor neurons in the earthworm. Comparative Biochemistry and Physiology 4% 96773. G~THER J. , & S C ~ R M A NF.NW. , (1973). Zur Feinstruktur des dorsalen Riesenfasersystems im Bauchmark des Regenwurms. Zeitschrzft fiir Zellforschung und Mikroskopische Anatomie 139, 369-96. GUNTHER, J. & WALTHER, J. B. (1971). Functionelle Anatomie der dorsalen Riesenfaser-Systeme von Lumbricus tmestris L. Zeitschrift fiir Morphologie der Tiere 70,253-80. HAGIWARA, S. & MORITA, H. (1962). Electrotonictransmissionbetween two nerve cells in leech ganglion. Journal of Neurophysiology 25, 7 2 1 3 1 . F. (1972). The effect of some substances on the cholinergic responses of earthHALUK,V. & BAYSAL, worms. Turk Hqiyen ve Temrbi Biyolqji Dergisi 31,5-13. HAMA,K. (1959). Some observations on the fine structure of the giant nerve fibres of the earthworm. Journal of Biophysical and Biochemical Cytology 6, 6 1-6. HAMA, K. (1961). The fine structure of some electrical synapses. Science of the Living Body 12,72-84. HANSON, J. (1957). The structure of the smooth muscle fibres in the body wall of the earthworm. Journal of Biophysical and Biochemical Cytology 3, I I 1-22. HESS,W. N. (1925~).Nervous system of the earthworm, Lumbricuc terresi*is.Journal of Morphology 40, 235-59. HESS,W. N. (1925b). Photoreceptors of the earthworm, Lumbricus terrestnk, with special reference to their distribution, function and structure. Jownal of Morphology 41,63-95. HESSE,R. (1897). Untersuchungen iiber die Organe der Lichtemfindung bei niederen Thieren. 111. Die Sehorgane der Hirudineen. Zeitschrift fiir wissmchaftliche Zoologie 6 ~671-7707. , HEUMANN, H. G. & ZEBE,E. (1967). Uber Feinbau und Funktionsweise der Fasem aus dem Hautmuskelschlauch des Regenwurms, Lu&cus terreshis L. Zeitschrift fur ZeQforschungund Mikroskopische Anatomie 78, 131-50. HIDAKA, T., ITO,Y. & KURIYAMA, H. (1969). Membrane properties of the somatic muscle (obliquely striated) of the earthworm. Journal of Experimental Biology 50, 387-403. HIDAKA, T., ITO,Y., KURIYAMA, H. & TASHIRO, N. (19690). Effects of various ions on the resting and active membrane of the somatic muscle of earthworm. Journal of Experimental Biology 50,405-15. HIDAKA, T., ITO,Y., KURIYAMA & TASHIRO, N. (19696).Neuromuscular transmissionin the longitudinal layer of somatic muscle in the earthworm. Jownal of Ejcperintental Biology 50, 417-30. HIDAKA, T., KURIYAMA, H. & YAMAMOTO, T. (1969). The mechanical properties of the longitudinal muscle in the earthworm. Journal of Expm'mental Biology 50,431-43. HILDEBRAND, J. G., BARKER, D. L., HERBERT, E. & KRAVITZ, E. A. (1971).Screening for neurotransmitters : a rapid neurochemical procedure. Journal of Neurobiology 2, 23 1-46. HORRIDGE, G. A. (1959). Analysis of the rapid responses of Ner& and Hannothoe (Annelida). Proceeding of the Royal Society of London, Series B 150, 245-62. HORRIDGE, G. A. & ROBERTS, M. B. V. (1960). Neuromuscular transmission in the earthworm. Nature, London 186,650. IKEMOTO, N. (1963). Further studies in electron microscopic structure of the oblique striated muscle of the earthworm. Eisenia foetida. BiologicalJournal of Okayama University 9, 81-126.


49

ISSIDORIDES, M. (1956).Ultrastructure of the synapse in the giant axons of the earthworm. Experimental Cell Research IT, 423-36.

ITO,Y., KURIYM, H. & TASHIRO, N. (1969). Effects of GABA and picrotoxin on the permeability of the longitudinal muscle of the earthwormto various anions.Journa1 of Experimental Biology 51,36375. ITO,Y . KURIYAMA, H. & TASHIRO, N. (1970). Effects of catecholamineson the neuromuscularjunction of the somatic muscle of the earthworm. Journal of Experimental Biology 54, 167-86. JANSEN, J. K. S., MULLER,K. J. & NICHOLLS,J. G. (1974).Persistent modification of synaptic interactions between sensory and motor nerve cells following discrete lesions in the central nervous system of the leech. Journal of Physiology 242,289-305. JFNKINSON, D. H., STAMENOVIC, B. A. & WHITAKER, B. D. L. (1968). The effect of noradrenaline on the end plate potential in twitch fibres of the frog. Journal of Physiology 195, 743754. KANDEL,E. R. & KUPFERMA",I. (1970). The functional organization of invertebrate ganglia. Amccal Revia~sof Physiology 32, 193-258. KAo, C. Y.(1956).Basis for after-discharge in the median giant axonof the earthworm. Science,N m York 123, 803. KAO,C. Y.& GRUNDPHST, H. (1957).Postsynaptic electrogenesisin septate giant axons. I. Earthworm median giant axon. Journal of Neurophysiology 20, 553-73. KAWAGUTI, S. & IKEMOTO, N. (1959).Electron microscopic patterns of earthworm muscle in relaxation and contraction induced by glycerol and adrenosine-triphosphate. Biological Journal of Okuymna University 5, 57-72. KERKUT,G. A., SEDDEN, C. B. & WALKER, R. J. (1967). Cellular localization of monoamines by fluorescence microscopy in Hirudo medicinalis and Lumbricus terrestris. Comparative Biochemistry and Physiology 21,687-90. KERKTJT, G. A.&WALKER, R. J. (1967).The action of acetylcholine,dopamine and 5-hydroxytryptamine on the spontaneous activity of the cells of Retzius of the leech, Hirudo medicinalis. British Journal of Phamcology 30, 644-54. KNAPP,M. F. & MILL, P. J. (1968~).Chemoreception and efferent sensory impulses in Lumbricus terrestris Lim. Comparative Biochemistry and Physiology 25, 523-8. KNAPP,M. F. & MILL,P. J. (19683).Efferent sensory impulses in the earthworm, Lumbricus terrestris Linn. Journal of Physiology 197,83-4~. KNAPP,M. F. & MILL,P. J. (1971a). The contractilemechanism in obliquely striated body wall muscle of the earthworm, Lumbricus terrestris.Journal of Cell Science 8,413-25. KNAPP,M. F. & MILL,P. J. (1971b). The fine structure of ciliated sensory cells in the epidermis of the earthworm, Lumbricus terrestris. Tissue and Cell 3,623-36. KOIDL,B. (1974). The GABA content of the central nervous system of Crustacea and Annelida: a comparison. Journal of Comparative Physiology ~,49-55. LANCASTER, S . (1939).Nature of the chromaffin cells in certain annulates and arthropods. Transactions of the American Microscopical Society 58,904. LANGDON, F. E. (1895). The sense organs of Lumbricw agricola. Journal of Morphology 11, 193234. LAVERACK, M. S. (1960). Tactile and chemical perception in earthworms. I. Responses to touch, sodium chloride, quinine and sugars. Comparative Biochemistry and Physiology I, 155-63. LAVERACK, M. S. (1961).Tactile and chemical perception in earthworms. 11. Responses to acid pH solutions. Comparative Biochemistry and Physiology 2,22-34. LAVERACK, M. S. (1963). Nervous System. In The Physiology of Earthworms. Oxford and London. Pergamon Press. G. H. (1966). The microscopic anatomy and ultrastructure of LEVI,J. U., COWDEN, R. R. & COLLINS, the nervous system in the earthworm (Lumbricus)with emphasis on the relationship between glial cells and neurones .Journal of Comparative Neurology 127,489-507. MANTON, S. M. (1961). Experimental zoology and problems of arthropod evolution. In The Cell and the Organism (ed. J. A. Ramsey and V. B. Wigglesworth) Cambridge University Press. MARSDEN,C. A. & KERKUT, G. A. (1969). Fluorescent microscopy of the 5HT- and catecholaminecontaining cells in the central nervous system of the leech Hirudo medicinalis.Comparative Biochemistry and Physiology 31,851-62. MENNICKE, H. (1925).Uber die Wirkung vershiedener Substanzen auf die Muskulatur des Regenwurms. Zeitschrift fur geschictliche Medizin 43,454-62. MCINTOSH, F. C. & PERRY,W. L. M. (1950).Biological estimation of acetylcholine.Methods in Medical Research 3, 78-92. MILL,P. J. & KNAPP.M. F. (1967).Efferent sensory impulses and the innervation of tactile receptors in Allolobophora longa Ude. and Lumbricus terrestris Linn. Comparative Biochemistry and PhysioIogy 23,263764

B R E 51

50

C. R. GARDNER

MILL,P. J. & KNAPP,M. F. (1970a). The fine structure of obliquely striated body wall muscles in the earthworm, Lumbricus tewestris. Journal of Cell Science 7,233-61. MILL,P. J. & KNAPP,M. F. (1970b).Neuromuscularjunctions in the body wall muscle of the earthworm, Lumbrinrs terrestris. Linn. Journal of Cell Science 7, 263-71. MOORE, A. R. (1922). Muscle tension and reflexes in the earthworm. Journal of General Physiology 5, 327-33. MULLONEY, B. (1970).Structure of the giant fibres of earthworms. Science, New York 168,994-6. MYHRBERG, H. E. (1967). Monoaminergic mechanisms in the nervous systems of Lumbricus terrestris. Zeitschrift f u r Zellforschmg und Mikroskupische Anatomie 81,322-43. MYHRBERG, H.E. (1971). Ultrastructural localization of monoamines in the epidermis of Lumbricus terrestris (L.) Zeitschnyt f u r Zellforchung und Mikroskopische Anatomie 117,139-54. MYHRBERG, H. E. (1972). Ultrastructural localization of monoamines in the central nervous system of Lumbricus terrestris (L.) with remarks on neurosecretory vesicles. Zeitschrift f u r Zellforschung und Mikroskopische Anatomie 126,348-62. MYHRBERG, H. E. & ROSENGFCEN, E. (1967).Unpublished observations cited in Myhrberg (1967). NEWELL, G. E. (1950). The role of coelomic fluid in the movements of earthworms. Journal of Experimental Biology 27, 110-21. NICOL,J. A. C. (1948). The giant axons of Annelida. Quarterly Review of Biology 23, 291-323. NICHOLLS, J. G. & BAYLOR, D. A. (1968).Specific modalities and receptive fields of sensory neurones in the CNS of the leech. Journal of Neurophysiology 31, 740-56. NICHOLLS, J. G. & VAN ESSEN,D.(1974). The nervous system of the leech. Scientific Americun 230, 38-48. NICHOLLS, J. G. & PURVFS, D. (1970). Monosynaptic chemical and electrical connections between sensory and motor cells in the central nervous system of the leech. Journal of Physiology 209,647-67. NISHIHARA, H . (1967). Fine structure of the earthworm body wall muscle. Acta Anatomica Nipponicu 42,38-9. OESTERLE, D. & BARTH,F. (1973).Zur Feinstruktur einer elektrischen Synapse. Zeitschrzyt f u r Zellforschung und Mikroskopische Anatomie 136,139-52. OGAWA, F.(1939).The nervous system of the earthworm in different ages. Sciences Report of the Research Institutes, Tohoku University 13,395-488. OSBORNE, N. N. (1971).Occurrence of GABA and taurine in the nervous systems of the dogfish and some invertebrates. Comparative and General Pharmacology 2,433-8. OSTLUND, E. (1954).The distribution of catecholamines in lower animals and their effect on the heart. Acta Physiologica Scandinavica 31,Suppl. 112. PASANTES, H., TAPIA, R. & MASSIEU,G. (1962). Nota acerca de les aminoacidos libres y actividad de aljunas enzimas dependientes de fosfato de piridoxal en la cuerda nerviosa de la lombriz de tierra. Anales, Instituto Biologia (Mexico) 33, 25-33. PLOTNIKOVA, S.I. & GOWRIN,V. A. (1966).(Adrenergicelements in the nervous system of earthworms). Zhurnal Evoliutsionnoi Biokhimii i Fiziologii. 3, 226-33. PROSSER, C. L. (1934).The nervous system of the earthworm. Qruzrterly Rm'ew of Biology 9, 181200.

PROSSER, C. L. (1935). Impulses in the segmental nerves of the earthworm. Journal of Experimental Biology 12, 95-104. PROSSER, C. L. (1950).Nervous systems. In Comparative Animal Physiology, C. L. Prosser, F. A. Brown, D. W.Bishop, T. L. Jahn, and V. J. Wulff, p. 811. Philadelphia: Saunders. RETZIUS, G. (1892).Das Nervensystem der Lumbriciden. Biologische Untersuchungen 3, 1-16. ROBERTS, M. B. V. (1960).Giant fibre reflex of the earthworm. Nature, London 186,167. ROBERTS, M. B. V. (1962a).The giant fibre reflex of the earthworm, Lumbricus ter~estris.I. The rapid response. Journal of Experimental Biology 39, 219-27. ROBERTS, M. B. V. (19626). The giant fibre reflex of the earthworm, Lumbricus terrestris L. 11. Fatigue. Journal of Experimental Biology 39, 229-37. ROBERTS, M. B. V. (1966). Facilitation in the rapid response of the earthworm, Lumbricus terrestris. Journal of Experimental Biology 45, 141-50. ROBERTS, M. B. V. (1967).Slow activity in the nervous system of the earthworm, Lumbricus tewestris. Journal of Experimental Biology 46,571-83. ROHLICH, P., AROS,B. & VIRAGH,Sz. (1970).Fine structure of photoreceptor cells in the earthworm, Lumbrinrs tmestris. Zeitschrift fur Zellforschung und Mikroskopische Anatomie 104,345-57. ROSENBLUTH, J. (I 972). Myoneural junctions of two ultrastructurally distinct types of earthworm body wall muscle. J o u m l of Cell Biology 54, 56679. RUDE,S. (1966).Monoamine containing neurons in the nerve cord and body wall of Lumbricus terrestris. Journal of Comparative Neurology 128, 397-412.


51

RUDE,S. (1969). Catecholamines in the ventral nerve cord of Lumbricus terrestris. Comparative Biochemistry and Physiology 28, 747-52. RUDE,S., COGGESHALL, R. E. & VAN ORDEN,L. S. (1969). Chemical and ultrastructural identification of 5-hydroxytryptamine in an identified neurone. Journal of Cell Biology 41,832-54. RUSHTON, W. A. H. (1945). Motor response from giant fibres in the earthworm. Nature, London 156, 109-10.

RUSHTON, W. A. H. (1946). Reflex conduction in the giant fibres of the earthworm. Proceedings of the Royal Society of London Series B 133, 109-20. RUSHTON, W. A. H. & BARLOW,H. B. (1943). Single fibre response from an intact animal. Nature, London 152,597-8. SAKA,J. & TSUJI, H. (1934). Wirkung des Apomorphim auf Regenwiirmer. TohokuJournal of Experimental Medicine 24, 565-71. SAKHAROV, D. A. (1970). Cellular aspects of invertebrate neuropharmacology. Annual R m k of Pha???IaCOlOgy10, 335-52. SCHAIN,R. J. (1961). Effects of 5-hydroxytryptamine on the dorsal muscle of the leech (Hirudo medicinalis). British Journal of Pharmacology 16,257-61. SCH~~RMA", F. W. & G ~ T H E RJ., (1973). Elektronenmikroskopische Untersuchungen am dorsalen Riesenfasersystem im Bauchmark des Regenwurms (Lumbricus terrestris L.). Zeitschrqt fur Zellforschung und Mikroskopische Anatomie 139,35 1-68. SCRIBAN, 1. A. & AUTRUM, H. (1932). Hirudinae. In Kukenthal's Handbook of Zoology 2 (81, 119-352. SEYMOUR, M. K. (1969). Locomotion and coelomic pressure in Lumhicus terrestris L. Journal of Experimental Biology 51,47-58. SEYMOUR, M. K. (1970). Skeletons of Lumbricus terrestris L. and Arenicola marina (I,.). Nature, London 228,383-5. SEYMOUR, M. K. ( I 971). Coelomic pressure and electromyogram in earthworm locomotion. Comparative Biochemistry and Physiology 40A, 859-64. SMALLWOOD, W. M. (1926). The peripheral nervous system of the common earthworm, Lumbricus terrestris. Journal of Comparative Neurology 42,35-55. SMALLWOOD, W. M. (1930). The nervous structure of the annelid ganglion. Journal of Comparative Neurology 51, 377-92. SMALLWOOD. W. M. & HOLMES.M. T. (1027). , ., The neurofibrillar structure of the giant fibres in Lumbricus terrestris and Eisenia foetida. Journal of Comparative Neurology 43, 327-45. SMITH,P. A. & WALKER,R. J. (1973). Studies on 5-hydroxytryptamine receptors of neurones from Hirudo medicinalis. British Journal of Pharmacology 47, 6 3 3 ~ . STAUBESAND, J. & KERSTING, K. H. (1964). Feinbau und Organization der Muskelzellen des Regenwurms. Zeitschrift fur Zellforschung und Mikroskopische Anatomie 62,416-42. STAUBESAND, J., KUHLO,B. & KERSTING, K. H. (1963). Licht und elektronmikroskopische Studien am Nervensystem des Regenwurms. I. Die Hiillen des Bauchmarkes. Zeitschrift fur Zellforschung und Mikroskopische Anatomie 61,401-33. STEPHENSON, M. D. (1930). The nervous system. In The Oligochaeta. London: Oxford University Press. STOUGH,H. B. (1926). Giant nerve fibres of the earthworm. Journal of Comparative Neurology 40, 409-43 * STOUGH, H. B. (1930). Polarization of the giant nerve fibres of the earthworm. Journal of Comparative Neurology 50, 2I 7-29. STUART,A. E. (1970). Physiological and morphological properties of motoneurones in the central nervous system of the leech. Journal of Physiology 209,627-46. TASHIRO, N. (1971). Mechanical properties of the longitudinal and circular muscle in the earthworm. Journal of Experimental Biology 55, 101-10. TASHIRO, N. & YAMAMOTO, T. (1971). The phasic and tonic contraction in the longitudinal muscle of the earthworm. Journal of Experimental Biology 55, I I 1-22. TAYLOR, W. G. (1940). The optical properties of the earthworm giant fibre sheath as related to fibre size. Journal of Cellular and Comparative Physiology 15, 363-71. TEICHMANN, I. & AROS,B. (1966). Fluorescence microscopic demonstration of catecholamine containing nerve cells and fibres in the central nervous system of invertebrates. Acta Morphologicu Academiae Scientiarum Hungaricae 16,350. TEN CATE,J. (1938). Sur la fonction des neurochordes de la chaine ventrale du ver de terre (Lumbricus terrestris). Archives nierlandaise de Physiologie 23, 136-40. TERAVAINEN, H.(1969). Ultrastructural distribution of cholinesterase actibity in the ventral nerve cord of the earthworm, Lumbricus terrestris. Histochemie 18,177-90. VERESCHAGIN, S.M. & SYTINSKII,I. A. (1960). Effect of GABA and p-alanine on motor activity and bio-electrical activity of annelid ganglia. Translated from Doklady Academii Nauk S.S.S.R. 132, 1213-1 5.

-

52

C. R. GARDNER

VIGH-TEICHMANN, I. & GOSLAR. H. G. (1969).Enzyme histochemical studies of the nervous system. 111. The distribution of some hydrolases in the central nervous system of the earthworm (Eisenia foetida). Histochemie 14,352-465. VON EULER,U. S. (1961).Occurrence of catecholamines in Acrania and Invertebrates. Nature, London 19, 170-1. VON HOLST, E. (1933).Witre versuche zum nervosen Mechanismus der Bewegung beim Regenwurm (Lumbricus terrestris). Zoologische Jahrbiicher 51, 547-88. WALKER,R. J., WOODRUFF, G. N. & KERKUT, G. A. (1968).The effect of acetylcholine and 5-hydroxytryptamine on electrophysiological recording from muscle fibres of the leech. Comparative Biochemistry and Physiology 24, 987-90. WASHIZU, Y. (1967).Electrical properties of leech dorsal muscle. Comparative Biochemism and Physiology 20,641-6. WELSH,J. H.& MOORHEAD, M. (1960).The quantitative distribution of 5-hydroxytryptamine in the invertebrates, especially in their nervous systems. ~ournalof Neurochemistry 6,146-69. WELLS, G. P. (1969). Mechanisms of movement in worms. Proceedings of the Challenger Society IV part I , 36-50. WILSON,D. M. (1960).Nervous control of movement in annelids. Jmrnal of ExperimentulBiology37, 46-56. WILSON,D. M . (1961).The connections between the lateral giant fibres of earthworms. Comparative Biochemistry and Physiology 3, 274-84. WOODRUFF, G. N., WALKER, R. J. &NEWTON, L. C. (1971). The action of some muscarinic and nicotinic agonists on the Retzius cells of the leech. General and Comparative Pharmacology 2, 106-17. Wu, K. S. (1939).The action of drugs, especially acetylcholine, on the annelid body wall (Lumbricus, Arenicola). Journal of Experimental Biology 16,2 5 1-7. YAKSTA,T. & COGGESHALL, E. (1973).Neuromuscular transmitters in a simple nervous system. Texas Reports 011 Biology and Medicine 31, 607. YAPP,W. B. (1956).Locomotion of worms. Nature, London 177, 614-16. ZAWARZIN, A. (1925).Der Parallelismus der Strukturen als ein Grundprinzip der Morphologie. Zeitschrift f u r wissenschaftlche Zoologa'e 124,I 18-212. ZIMMERMANN, P. (1967). Fluoreszenzmikroskopische Studien iiber die Verteilung und Regeneration der Faserglia bei Lumbricus terrestris L. Zeitschrift f u r Zelvorschung und Mikroscopische Anatom'e 81, 190-220.

Nematode locomotion: dissecting the neuronal-environmental loop.

Neuronal mechanisms of human locomotion.

Visual control of locomotion.

Control of biped locomotion.

Minimum toe clearance: probing the neural control of locomotion.

Motor systems, with specific reference to the control of locomotion.

Locomotion control of hybrid cockroach robots.

The control of neuronal Ca2+ homeostasis.

Neurosecretory control of integumental water exchange in the earthworm Lumbricus terrestris L.

Application of circular statistics to the study of neuronal discharge during locomotion.

Intermittent locomotion as an optimal control strategy.

Soil geochemistry and digestive solubilization control mercury bioaccumulation in the earthworm Pheretima guillemi.

Genomic control of neuronal demographics in the retina.

Neuronal localization of pancreatic polypeptide (PP) and vasoactive intestinal peptide (VIP) immunoreactivity in the earthworm (Lumbricus terrestris).

Scaling of the hydrostatic skeleton in the earthworm Lumbricus terrestris.

Phasic gain control of the transmission in cutaneous reflex pathways to motoneurones during 'fictive' locomotion.

Circalunidian clocks control tidal rhythms of locomotion in the American horseshoe crab, Limulus polyphemus.

The role of cutaneous afferents in the control of gamma-motoneurones during locomotion in the decerebrate cat.

7α-Hydroxypregnenolone, a key neuronal modulator of locomotion, stimulates upstream migration by means of the dopaminergic system in salmon.

Ceramide glycanase from the earthworm, Lumbricus terrestris.

Flies do the locomotion.

Neuronal control of the kidney: contribution to hypertension.

Can modular strategies simplify neural control of multidirectional human locomotion?

Optimal coordination and control of posture and locomotion.