The roles of putative neurotransmitters and neuromodulators in annelids and related invertebrates

Prot.lressin Neurohioh~,lyVol. 18, pp. 81 Io 120, 1982 Printed in Great Britain.All rights re~rved

THE AND

ROLES

OF

0301-0082/82/030081-40520.00/0 Copyright tD 1982 Pergamon Press Ltd

PUTATIVE

NEUROTRANSMITTERS

NEUROMODULATORS RELATED

IN

ANNELIDS

AND

INVERTEBRATES

C, R. GARDNER* and R. J. WALKERt *Roussel Laboratories. Kim.llisher Drire, Corin,qham, Sn'indon, Wiltshire, U.K. ~'Department o[ Neurophysiolo,q.v, Schtud of Biochemical and Ph.l'siolo~licalSciences, University of Southampton, Basset Crescent E, st, Southampton, U.K. (Received 4 February 1982) Contents Abbreviations 1. General Introduction 2. Acetylcholine 2.1. Earthworms and Leeches 2.1.1. Central nervous system 2.1.2. Somatic muscle 2.1.3. Visceral muscle 2.1.4. Luminescence 2.2. Other related invertebrates 3. Catecholamines 3.1. Earthworms 3.2. Leeches 3.3. Other related invertebrates 4. Other Phenylethylamines , 5. 5-Hydroxytryptamine 5.1. Annelid somatic muscle 5.2. Leech central neurones 5.3. Other related invertebrates 6. Amino acids 6.1. Earthworms 6.1.1. Gamma-aminobutyric acid 6.1.2. Excitatory amino acids 6.2. Leech central neurones 6.3. Other related invertebrates 7. Peptides 8. Other agents 8.1. Benzodiazepines 8.2. Convulsants, Barbiturates and Tetraethylammonium 9. General Conclusion References

81 81 82 82 82 83 84 85 85 87 87 92 93 95 98 98 100 103 104 104 104 105 106 107 108 109 109 ll0 Iii 112

Abbreviations ACh = acetylcholine; 5-HT -= 5-hydroxytryptamine; NA = noradrenaline; DA = doparnine; 5-HTP = 5hydroxytryptophan; GABA = gamma-aminobutyric acid; L-dopa = 3,4-dihydroxyphenylalanine; cAMP = cyclic adenosine monophosphate.

I. General Introduction T h e s a m e c o m p o u n d s a r e likely to act as t r a n s m i t t e r s t h r o u g h o u t t h e a n i m a l k i n g d o m t h o u g h it is p r o b a b l e t h a t t h e r e a r e specific differences b e t w e e n p h y l a . T h e a n n e l i d s a r e o n e o f the i n v e r t e b r a t e p h y l a which a r e i n c r e a s i n g l y used for p h y s i o l o g i c a l a n d p h a r m a c o l o g i c a l studies. In at least o n e respect they have been e m p l o y e d for m a n y y e a r s ; the b o d y wall m u s c l e o f t h e leech as a n a s s a y for a c e t y l c h o l i n e (Minz, 1932). T h e p h y l u m is d i v i d e d into t h r e e m a j o r classes, the Hirudinae o r leeches, the Olioochaeta w h i c h a r e largely t e r r e s t r i a l a n d f r e s h w a t e r w o r m s , a n d the Polychaeta w h i c h a r e m a i n l y m a r i n e w o r m s . O f these classes, t h e Hirudinae h a v e been m o s t extensively s t u d i e d o v e r t h e p a s t J...~.

Ix 2 3--^

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C, R. GARDNER AND R. J. WALKER

20 years following the first microelectrode recordings from leech neurones by Hagiwara and Morita (1962)and Eckert (1963). These initial studies used the large paired Retzius cells on the ventral surface of the segmental ganglia which were first described by Retzius (1891). However, since then many of the 350 or so neurones in the ganglia have been investigated and detailed maps prepared (Nicholls and Bayior, 1968; Stuart, 1970). It is becoming increasingly clear that it is most important to be able to work on identified neurones with known synaptic inputs and this is now possible using the leech ganglion preparation. The next step following cell identification is to try and establish functional roles for individual neurones and groups of neurones and to identify co-ordinating interneurones. This too has been achieved in the leech, for example, in terms of the control of the heart (Thompson and Stent, 1976b) and swimming (Poon, Friessen and Stent, 1978). Such detailed electrophysiological information is helpful for neuropharmacological studies and the leech central nervous system is ideal for such investigations. One of the major contributions to our understanding of the basic properties of nerve cells came from experiments on the squid giant fibre system (Hodgkin, 1964). However, both the polychaetes (Nicol, 1948) and the oligochaetes (Stough, 1926) contain giant fibre systems and these have also been studied in detail, particularly in the case of the oligochaetes. Thus the annelids provide ideal material for many aspects of the electrophysiology and pharmacology of the nervous system.

2. Acetylcholine (ACh) 2.1. EARTHWORMSAND LEECHES 2.1.1. Central Nerrous System

There have been few studies of the role of ACh in the CNS of annelids. A role for ACh in the ventral nerve cord of earthworms is suggested by the presence of acetylcholinesterase on the border between the neuronal layers and also scattered in the neuropile (Vigh-Teichmann and Goslar, 1969). However, the enzyme is not located on post-synaptic membranes but on neuroglial membranes (Dhainaut-Courtois and Dhainaut, 1974, in Nereidae; Teravainen, 1969, in Lumbricus) and it is not certain if its action is to hydrolyse released neurotransmitters. More direct studies are required to establish a physiological function for ACh and such studies have been hampered by connective tissue and muscle layers overlying the nerve cords of several species. The cholinergic system in the prostomium of Nereis has been investigated by Marsden et al. (1981) using choline acetyltransferase and acetylcholinesterase as histological markers. They present evidence for sensory epithelial cells with cholinergic axons which pass via the cephalic nerve into the brain and for motor cholinergic axons which pass via the same nerve to the prostomial muscle. Choline acetyltransferase positive fibres are also present in the second lobe of the corpora pedunculata. They conclude that there is a concentration of cholinergic activity in the anterior part of the brain. ACh is present in large quantities in leech segmental ganglia (Bacq and Coppee, 1937; Carayan-Gentil and Gautrelet, 1938; Schwab, 1949) and its synthetic enzyme, choline acetyl transferase is also present (Perkins and Cottrell, 1972) as is cholinesterase (Nistri et al., 1978). Isolated individual AE and L motoneurones contain both ACh and choline acetyl transferase (Sargent, 1977)whilst R cells, inhibitory motonet, rones to the dorsoventral flattener muscle and the T, P and N sensory cells did not. Thus the ACh present in the ganglia could be in cell bodies or in both nerve terminals and cell bodies. The presence of cholinesterase may suggest ACh-mediated neurotransmission in the CNS. ACh stimulates the spontaneous activity of the nerve cord of annelids (Umrath, 1952) notably the leech [Gaskell. 1914: Kostowski, 19651. This prompted studies of the action of ACh on identified neurones. The Retzius (R)cells of Hirmh~ were depolarised by ACh at relatively high concentrations (1-3 mml. This excitation was blocked by benzoquino-

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nium and decamethonium and reduced by atropine, hexamethonium and tubocurarine, the latter being the least effective (Kerkut and Walker, 1967; Gerasimov, 1968). Further studies have suggested that R cells possess both "nicotinic" excitatory receptors stimulated by nicotine (the ( - ) isomer is approx. 1000 times as potent as the ( + ) isomer), iobeline and dimethyl phenyl piperazinium and "muscarinic" inhibitory receptors stimulated by muscarine, furmethide and McN-A-343 etc., and blocked by atropine (Woodruff et al., 1971; Newton, 1972). ~-Bungarotoxin does not block the action of ACh on leech neurones (Magazanik, 1976). Recently, Sargent et al. (1977) have tested other identifiable cells in Hirudo and found that ACh depolarised P and N cells, hyperpolarised AE cells and evoked diphasic responses (depolarisation followed by hyperpolarisation) in T and L cells. This depolarisation only occurred at low concentrations of ACh and was abolished on repeated application, leaving the hyperpolarisation. The existence of such receptors does not constitute proof of a transmitter role for ACh but strongly suggests this. 2.1.2. Somatic muscle ACh has long been implicated as a neuromuscular transmitter in annelids (see review by Gerschenfeld, 1973; Gardner, 1976). Bacq and Coppee (1937) stimulated nerves to body wall musculature of Aphrodite, Arenicola, Hirudo, Lumbricus and Sipunculus and observed augmentation and prolongation of responses with eserine. Muscle responses to electrical stimulation in earthworms are similarly potentiated (Botsford, 1941). Body wall strips of several species of worms are contracted tonically by ACh and a range of nicotinic and muscarinic agonists and ACh contractions are potentiated by anticholinesterases (oligochaete Lumbricus, polychaetes Arenicola, Nereis, Abarenicola and Brachiomma Wu, 1939b; Bacq, 1947; Nicol, 1952; Florey and Florey, 1965; Andersson and Fange, 1976; Alvarez et al., 1969; Rozhkova et al., 1980; echiuroid worm Urechis, Lawry, 1966; and hirudinae Hirudo, Minz, 1932, 1955; Chang and Gaddum, 1933; Mclntosh and Perry, 1950). The ACh receptors in the body wall of earthworms, generally in longitudinal muscles, have been studied, but there are discrepancies between observations. Mennicke (1925) suggested that the receptors were muscarinic in nature, being blocked by atropine and not by curare. Responses were also blocked by morphine. However, subsequent studies have demonstrated blockade (Haluk and Baysal, 1972), partial blockade (Wu, 1939b; Andersson and Fange, 1967) or no blockade (Gardner and Cashin, 1975) with atropine in Lumbricus. The receptors may also not be strictly nicotinic in nature as tubocurarine and gallamine have not been consistently shown to block ACh responses (Ewer and Van den Berg, 1954, Microchaetus; Andersson and Fange, 1967; Gardner and Cashin, 1975) although blockade has been observed (Baysal, 1967; Haluk and Baysal, 1972). Tubocurarine potentiated responses to ACh in Sabellastarte (Alvarez et al., 1969). Electrophysiological studies have shown block of miniature excitatory junction potentials by tubocurarine but not atropine in Pheretima communissima (Ito et al., 1970) and block of junction potentials in Pheretima hawayana by curare (Chang, 1975). Potentiation of these potentials by the cholinesterase inhibitor, prostigmine, was observed in both these studies. Studies with agonists do not clarify the picture greatly. Wu (1939b) observed initial contraction with nicotine, then relaxation accompanied by block of ACh responses in body wall of Lumbricus. This might suggest a nicotinic receptor. Contractions were observed with succinylcholine and sebac0yldicholine (Rozhkova et al., 1980). Extension of the study of Gardner and Cashin (1975) indicated that carbachol and nicotine are more potent that ACh in contracting the cordless body wall of Lumbricus (equipotent molar ratios (EPMR) being 0.02 and 0.4 respectively, where ACh = 1) whereas acetyl-l~methylcholine and choline were less active than ACh (EPMRs = 2.6 and 5.6 respectively). However, no relaxation was observed with nicotine even after application of > 20 times effective doses. These data still favour a nicotinic nature for the receptors involved. Pipcrazine depolarises the muscle membrane of Pheretima hawayana but this effect is

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unlike the well known hyperpolarising action of this drug in the nematode worm Ascaris lumbricoides (Del Castillo et al., 1964) and is blocked by atropine (Chang and Bruno, 1970). Thus, it may not be possible to apply the receptor classification appropriate for mammals to earthworm ACh receptors. Substantial evidence exists to suggest that the nerve-longitudinal muscle junction of the leech is cholinergic. In the presence of anticholinesterases such as eserine or physostigmine which enhance and prolong contractions of leech dorsal muscle (Kahlson and Uvnas, 1935, 1938; Bhattacharya and Feldberg, 1958; Flacke and Yeoh, 1968a,b) low concentrations of ACh (10-s M) induce contractions of the muscle which can be antagonised by curare but not by atropine (Gaskell, 1914; Minz, 1932, 1955; Chang and Gaddum, 1933; Bacq and Coppee, 1937; Yeoh and Flacke, 1967; Higuti, 1967). This sensitivity led to the regular use of this preparation as a bioassay for ACh (Mclntosh and Perry, 1950). Nicotinic receptors sensitive to nicotine, carbachol, decamethonium and succinylcholine were demonstrated by Flacke and Yeoh (1968a,b) and the quantitative effects of d-tubocurarine on responses to ACh, nicotine and carbachoi were similar (Fiacke and Yeoh, 1968b). Rozhkova (1973) further confirmed the nicotinic nature of receptors in this preparation. Intracellular recordings from dorsal muscle fibres showed that miniature excitatory junction potentials were enhanced by neostigmine and blocked by d-tubocurarine but not atropine (Higuti, 1967 in Hirudo nipponia). Iontophoretic application of ACh evoked a depolarisation (leading to action potentials) which diminished on repeated application (Walker et al., 1968, in Hirudo medicinalis). The L cell may be a motoneurone releasing ACh onto these muscle fibres as the cell contains both ACh and choline acetyltransferase (Sargent, 1977) and stimulation of the cell evokes excitatory junction potentials which are blocked by tubocurarine (Kuffler, 1975). Furthermore, ACh-sensitive membrane regions coincided with, sites where local synaptic potentials could be recorded following stimulation of the L cells (Kuffler, 1978). Cholinergic agonists contract the mesodermal trunk muscles of Arenicola, Nereis and Capiteila larvae (Marsden and Lacelli, 1978). Acetylcholine and carbachol partially arrest the ciliary activity of Phyllodoce larvae. Dopamine and noradrenaline also appear to have some effect on the ciliary activity of these larvae. 2.1.3. Visceral muscle Several early pharmacological studies on Arenicola (Wells, 1937), Allolobophora and Lumbricus (Wu, 1939a) indicated that the annelid gut had an excitatory cholinergic control system, with receptors of a muscarinic nature. A more extensive study by Andersson and Fange (1967) showed ACh to be the most potent compound, followed by carbachol, and tetraethylammonium to have the weakest agonist action. There is a lot of conflicting evidence on the effect of cholinergic drugs on annelid hearts. The "hearts" of leeches are lateral pulsating contractile blood vessels or sinuses. The contractions are controlled by a pair of rhythmically bursting motoneurones in each segment (heart exciter, HE cells) (Thomson and Stent, 1976). In an early study Gaskell (1914) found that contractions of leech heart were inhibited by muscarine and accelerated by atropine. However, Prosser and Zimmerman (1943) found that low concentrations (10-9-10 -7 M) of ACh accelerated heartbeat in Arenicola and Lumbricus, while higher doses stopped the heart in systole. The action of ACh was blocked by atropine. Kiefer (1959) observed only an excitatory effect of ACh in Lumbricus. Acetylcholine, 0.01-0.1 mM, carbachol, 0.1-1 mM, and nicotine, 0.1-1 mM, but not muscarine, elicited overshooting depolarisations followed by prolonged depolarised plateaus in myoepithelial cells of the proventriculus of the polychaete, Syllis spon qiphila (Anderson and Mrose, 1976). The proventriculus is the portion of the alimentary canal located between the proboscis and intestine, d-Tubocurarine, 0.01-0.5 raM, reduces both direct electrical stimulation and the action of acetylcholine while atropine has no effect. Neostigmine, 0.1-1.0mM, prolonged the acetylcholine response. It is suggested that the excitatory input onto the proventriculus muscle may be cholinergic and in particular nicotinic.

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Isolated preparations of pharangeal muscle from Nereis virens exhibit rhythmic bursts of contractions accompanied by bursts of spike discharges (Alohan and Huddart, 1979). Caffeine, 2-4mM, and acetylcholine, I-3 mM, stimulated spontaneous activity and induced contractions in the muscle. At 10°C, adrenaline had no effect, but at 20°C it was slightly excitatory. Caffeine and acetylcholine stimulated the slow elilux of calcium from the muscle while caffeine had no effect on calcium influx but acetylcholine stimulated it. Thus, there is some indication that ACh is involved in control of activity of visceral and heart muscle in annelids, but no clear evidence of a well-defined mechanism. 2.1.4. Luminescence Light responses can be recorded from isolated elytra of Harmothoe iunulata following field or ganglionic stimulation. Acetylcholine, !.0 mM, can also produce a flash of light while nicotine, 10 mM, is inactive. These responses were unaffected by tubocurarine or flaxedii, 1.0 mM, while 10 mM atropine, homatropine or scopolamine reversibly blocked the light response (Nicolas et al., 1978). When the epithelial luminescent glands of Chaetopterus notopods are stimulated either via field or nerve stimulation, rapidly rising light emissions with relatively slow decays are elicited (Anctil, 1981). 1 mM procaine abolishes this response but recovery occurs on washing. Acetylcholine, 0.01-10 mM, induced luminescence from isolated notopods which persisted for up to 4mins. Pretreatment with erserine, 0.1-10mM, potentiated the response both to electrical stimulation and to acetylcholine. Eserine increased the sensitivity to acetylcholine by up to 1000 fold. Carbachol, 0.01-10 mM, also produced luminescence but only about one third of that observed with acetylcholine on the same notopod. Muscarine produced responses which were about 1.5 times greater than the response to acetyicholine at the same concentration. Both atropine and scopolamine reversibly blocked the response to both electrical stimulation or to acetyleholine, in the range 0.1-10 mM. Tubocurarine and lobeline were inactive as antagonists. This would suggest that the eholinergic system here is muscarinic but it would be of interest to test nicotine itself on the preparation. Dopamine, noradrenaline, adrenaline and 5-HT, I mM, failed to elicit luminescence when applied to the notopods. 2.2. OTHER RELATED INVERTEBRATES ACh and its receptors are quite widespread in worm and fluke phyla other than annelids. Some have already been mentioned, but several groups which have been recently studied provide interesting comparisons. Parasitic nematodes and platyhelminths have been shown to contain ACh, cholineacetyltransferase and acetylcholinesterase (see short review by Mansour, 1979). Pharmacological experiments with larger parasites have demonstrated sensitivity of muscles to ACh (Barker et al., 1966 in Schistosoma mansoni; Chance and Mansour, 1949, 1953 in Fasciola hepatica; Baldwin and Moyle, 1949 in Ascaris lumbricoides). Whilst neuromuscular preparations from Ascaris are contracted by ACh, in Fasciola carbachol, ACh and methylcholine relaxed the preparations. The cholinesterase inhibitors physostigmine and prostigmine showed the same response and also sensitize the preparations to ACh (Chance and Mansour, 1949, 1953). The receptors for ACh in trematodes may be different from those in mammalian systems as neither atropine nor d-tubocurarine affected neuromuscular activity. In contrast, the body wall muscles of the nematode, Ascaris lumbricoides are contracted by ACh and nicotinic agonists (especially dimethylphenyl piperazinium) whilst muscarinic agonists were much less effective (Baldwin and Moyle, 1949; Norton and de Beer, 1957; Natoff, 1969). A recent detailed study by Rozhkova et al. (1980) in Ascaris suum has shown nicotinic receptors similar to those in leech dorsal muscle. Muscarinic agonists methylfurmethide and D,2-methyl-4 dimethyl amino methyl-l,3-dioxolan (F-2268) showed only weak activity whilst arecoline was ineffective. Similarly atropine was only a weak blocker of ACh whilst tetraethyl ammonium, hexamethonium and

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tubocurarine were more effective, tubocurarine clearly the most potent. A range of nicotinic agonists were effective but the receptors differed from those on striated muscles of higher vertebrates in having relatively low sensitivity to bisquaternary nicotinomimetics and relatively high sensitivity to tetramethylammonium. In the same study cholinesterase was identified histochemically in nervous and muscle structures and its inhibition by eserine or phosphacol potentiated the muscle contractile responses to ACh, propionylcholine and butyrylcholine 2-6 fold. The action of cholinergic compounds has been investigated on the longitudinal or lateral body wall musculature of the echiuroid, Urechis unicinctus (Muneoka et al., 1981). Acetylcholine, 10-4-10 -3 M, induced contractions in the body wall muscle. Carbachol was about 100 times more potent as an excitant than acetylcholine while methacholine, bethanechol and choline were all less potent than acetyicholine. Contractions induced by direct electrical stimulation of the body wall musculature were enhanced when the muscle was incubated with 10-5 M eserine while probanthine at the same concentration greatly reduced the contractions, d-Tubocurarine also depressed the twitch contractions but was less potent than probanthine while atropine, hexamethonium and gallamine were inactive. Eserine also potentiates the action of acetylcholine. Acetylcholine only induces contractions when applied to the inner surface of the muscle. High magnesium Ringer only slightly depressed the response to acetylcholine. It is concluded that acetylcholine acts directly on the muscle fibres and is likely to be the excitatory transmitter. Evidence for a possible cholinergic mechanism in planaria has been investigated by Erzen and Brzin (1979). These authors report the presence of acetylcholine, cholineacetyltransferase and cholinesterase, there being a high level of cholinesterase activity but only low levels of cholineacetyltransferase and non-specific esterases. Compounds which are selective inhibitors of cholinesterase activity in mammals do not show such selectivity in planaria suggesting that the planarian enzyme has different properties from that in the vertebrates. It is suggested that there is probably a cholinergic system present in the nervous system of turbellaria and so acetyicholine is likely to act as a transmitter. A small phylum of considerable interest are the velvet worms (onychophora). They occupy an intermediate phylogenetic position between the annelids and arthropods and may have been related to a common primitive ancestor of both annelids and arthropods. The modern representatives (Peripatus) are segmented with a thin cuticle and a fairly primitive nervous system with twin nerve cords, broadly similar to annelids. Unlike annelids, onychophora move using segmental limbs with their own intrinsic musculature. Their respiratory, vascular, digestive and excretory systems bear a close resemblance to arthropods. Recent interest in ancestry of modern annelids and arthropods has shown that Epiperipatus does not show bismuth Golgi complex staining, a characteristic arthropod feature (Locke and Huie, 1977). Evidence suggests that ACh is an excitatory neuromuscular transmitter in Opisthopatus costesi (Florey and Florey, 1965) and Peripatopsis moseleyi (Ewer and van den Berg, 1954; Gardner and Robson, 1978)Acetylcholinesterase is present in the muscle of Opisthopatus and all three studies showed potentiation of ACh-induced contractions with anticholinesterases. Florey and Florey (1965) also showed potentiation of responses to motor nerve stimulation. Acetyl-p-methylcholine only caused a contraction after treatment with anticholinesterase in Opisthopatus (Florey and Fiorey, 1965) but no clear classification of cholinergic receptors seems possible in this phylum as neither tubocurarine nor atropine blocked responses to ACh in Peripatopsis and atropine contracted the muscle at higher concentrations (2.5 x 10 -5 g ml- ~, Ewer and van den Berg, 1954). ACh is present in the central nervous system of Opisthopatus (3.0-7.2 pg g - i , Florey and Florey, 1965). It would seem that the neurotransmission in the body wall of onychophora shows greater similarity to that in annelids than in arthropods. ACh reduces the force of contraction of the onychophoran heart without affecting the rhythm, which is in contrast to the excitatory action of ACh on many arthropod hearts (Florey and Florey, 1965). Another small phylum which may represent remnants of an ancient animal group is priapulida (van der Land, 1970). The body wall of the worm-like marine invertebrate

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Prialpus caudatus is contracted by ACh, tetramine, nicotine, carbachol and tetramethylammonium but not pilocarpine, and atropine blocks all these effects (Fange, 1969; Mattison et al., 1974) Hyoscine and benzoquinominum also block responses to ACh but gallamine did not, and tubocurarine, decamethonium and succinylcholine increased ACh responses, similar to the action of the cholinesterase inhibitor eserine (Mattison et al., 1974). Both the intestine and rectum of Prialpus are also contracted by ACh and carbachol (Mattison et al., 1974). Whilst the presence of ACh and cholinesterase are common in annelids and related invertebrates the characteristics of the ACh receptors are not always similar and may not be directly comparable to mammalian ACh receptors. 3. Catecholamines

3.1. EARTHWORMS With the development of quantitative methods for assay of catecholamines their presence was shown in the tissues of worms (Lumbricus and Nephtys, Ostlund, 1954; yon Euler, 1961; Clark, 1966). Several have found noradrenaline (NA)(0.8-1.6/~g/g)and dopamine (DA) (1.8-3.0/~g/g) to be present in the ventral nerve cord using this method (Rude, 1969a; Ehinger and Myhrberg, 1971; Gardner and Cashin 1975) or other procedures such as Azan staining (Koritsanzky, 1974, in cerebral g~glia of AIIoiobophora calionosa) or microdansylation (Robertson and Osborne, 1979, in Lumbricus terrestris). Higher levels of DA than NA suggest that DA is a neurotransmitter in its own right and not just a precursor of NA. Little adrenaline has been detected in the nerve cords of worms (Myhrberg and Rosengren, 1967). 3H-NA was taken up into small cells in the prostomial epidermis and the infracerebral organ of Nereis but there was no significant uptake into cerebral ganglia (Marsden et ai., 1981). There was no accumulation of 3H-DA into either cells or tracts of the cerebral ganglia. Histochemical studies using formaldehyde-induced fluorescence have located primary catecholamines in epidermal sensory cells in several annelids (mainly DA) and in one pair of cell bodies in each segmental ganglion in Lumbricus (possibly NA) (Bianchi, 1962, 1964, 1967, in Octolasium complanatum; Ehinger and Myhrberg, 1971; Myhrberg, 1967; Rude, 1966; Kerkut et ai., 1967; Teichmann and Aros, 1966; Clark, 1966, in Nephtys caeca; Plotnikova and Govyrin, 1966 (also in Ailolobophora) and Gardner and 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. Microspectrofluorimetric and fluorescence microscopic analysis of cells in the brain of Nereis has shown in the order of 30 cells showing cyan (blue-green) indicating eatecholamines (White and Marsden, 1978). The distribution of the cyan fluorophore in prostomial epidermal cells along the cirral and at the base of the antennal nerves suggests that catecholamines may be involved with sensory pathways which would support earlier observations. The regeneration of the monoaminergic system in the cerebral ganglion of Allolobophora has also been investigated using fluorescence microscopy and microspectrofluorimetric techniques (Koritsanszky and Hartwig, 1974). In control ganglia these authors observed NA-, DA- and 5-HT/5-hydroxytryptophan-containing neurones. However, four weeks after the removal of the cerebral ganglia, only DA- and 5-HT/5-HTPcontaining cell bodies reappeared. After 6 weeks, regeneration was complete and the intrinsic and afferent aminergic pathways of the cerebral ganglion were re-established. NA and DA may have a role in the sensory modulation of locomotion, by an action within the nerve cord, as they enhance rhythmic contractions in isolated sections of earthworm with nerve cord intact, but have no similar action on cordless sections (Bieger and Hornykiewicz, 1972a; Gardner and Cashin, 1975) (Fig. 1). If nerve cords are incubated with 14C-DA the tissue takes it up. It can then be released as t'*C-DA, t4C-NA and monoamine metabolites by electrical stimulation and this release is calcium dependent (Fig. 2). This further suggests the presence of catecholamine

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C . R . GARDNER AND R. J. WALKER

DA~ I0 ugl,,,I

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FIG. 1. The effect of dopamine (DA) on a weakly spontaneous section of earthworm with intact nerve cord. 10/~g/ml DA was without effect (upper trace) and only a washing artefact can be seen. 15 and 25 #g/ml evoked regular rhythmic contractions. This figure is reproduced with permission from Gardner and Cashin, 1975.

percentage of control 300

200

100

o time 0

10

20

30

40

mins

FIG. 2. Effect of electrical stimulation (25 V, 10 Hz, 10rain) on release of radioactivity from earthworm nerve cords preincubated with 90/~M 14C-DA for 75 min. After 30 rain washout with fluid replacement at 10 rain. intervals, stimulation was applied via bipolar platinum electrodes in contact with the cords with 0. I msec. (~---Q), I msec. (O---O) and 2 msec. ( × - - × ) pulse width (time 20 rain. on graph). Although 0.1 msec pulse width stimuh~tion had little effect stimulation with wider pulses increased emux of radioactivity. Each curve is the mean of four experiments taking the second sample prior to stimulation as 100%.

89

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I

FiG. 3. Extracellular recordings from groups of elements within a ganglion of the ventral nerve cord of Lumhricus via tungsten microelectrodes using standard amplification techniques. The nerve cord was still attached to a ventral strip of body wall which was pinned fiat in the recording chamber. The upper traces show responses to light touch (at marks) of the body wall in the same or nearby segments. The central trace shows the rhythmic spontaneous bursts of action potentials recorded. The frequency and duration of bursts were enhanced by application of a drop of 20 tag/ml DA in Ringer (Gardner and Cashin, 1975) to the nerve cord (lower trace).

releasing neural elements in the ventral nerve cord, probably nerve endings, consistent with their proposed role as sensory neurones. Some preliminary electrophysiological observations showed that spontaneously active groups of cells in the ventral nerve cord of Lumbricus, which responded to tactile stimuli to the body wall, were activated by application of DA to the nerve cord (Fig. 3). However, bursts of action potentials often correlated with rhythmic body wall contractions and it is not certain whether these action potentials were causitive to or a result of these contractions. Whilst touch, stretch (Collier, 1939) or illumination of such preparations after dark adaptation (Fig. 4) may elicit similar responses it has been suggested that the monoaminergic sensory cells are concerned with reception of photic or tactile stimuli as these stimuli increase their release from nervous tissue (Bieger and Hornykiewicz, t972b). The biosynthesis and degradation of the catecholamines show some similarities to mammalian systems. Isolated nerve cords of Lumbricus, when incubated with t4C-DA, store it and convert a portion to 14C-NA which is also retained in the nerve cord (Table 1) suggesting the presence of a dopamine-~-hydroxylase enzyme. Catecholamines may be catabolised by monoamine oxidases as inhibitors of these enzymes increase the concentrations in the nerve cord (Myhrberg, 1967; Gardner and Cashin, 1975). 5-HT concentrations in the nerve cord of Lumbricus were increased more markedly by monoamine oxidase inhibition than catecholamine levels. This may indicate an alternative means of catabolism of the catecholamines, perhaps by a catechol-O-methyl transferase (Fig. 5). After pretreatment with monoamine oxidase inhibitors and pyrogallol to inhibit any catechol-O-methyi transferases, catecholamines were effective at approximately 70 fold lower doses in modulating rhythmic contractions of earthworm sections with intact nerve

FIG. 4. Effect of white light (horizontal bars, 40 watt tungsten filament 10cm from preparation) on the spontaneous activity of a 3 hr dark adapted section of Lumbricus. The section was taken from just posterior to the cliteUum and was suspended in vitro (Gardner and Cashin, 1975). Time bar--5 rain.

90

C.R. GARDNER AND R. J. WALKER TABLE I. AMOUNTS OF

'4C-DA AND t4C-NA IN

MONOAMINI,.' RELrASE EXPERIMENTS FROM

SAMPLES FROM STIMULATED

LUMBRICUS NERVE CORDS, CORDS

WERE INCUBATED WITH 90 pM 14C-DA FOR 75 MIN AND AFTER 30 MIN WASHOUT EXPERIMENTS WERE PI'RFORMED. POOLED EFFLUX SAMPLES WERE ANALYSED BY THE COLUMN SEPARATION METHOD OF ANSELL A ND BEESON (1968) AND ADDITI()NALLY WITH TWD DIMENSIONAL CHROMATOGRAPHY (*FLEMING AND CLARK, 1970). NOTE THE INCREASED EFFLUX OF MONOAMINE METAB-

OLITES DURING ELECTRICAL STIMULATION (25V, 10Hz, 2MSEC. 10MIN.) WHICH MAY INDICATE AN INCREASED TURNOVER OF CATECHOLAMINES DURING

STIMULATION. ADDITIONALLY NOTE THE CONVERSION DF DA TO NA WITHIN THE CORD DURING THE EXPERIMENT

14C.DA Basal Efflux Basal Efflux* Stimulated Efflux Within Nerve Cord after 3 hr washout

% Radioactivity identifiied as: At eluent front (including cateeholamine 4C-NA metabolites)

62 58 27

7 7 8

20

63

24 35 65 - - (17% at origin)

cord (Bieger and Hornykiewicz, 1972a, b; Gardner and Cashin, 1975). The former authors found that DA caused a strong increase in contractions whilst NA was inhibitory, whilst the latter authors observed predominately increases in contractions with both NA and DA with inhibition in about 20% of applications of either catecholamine. These differences could stem from different regional effects of the enzyme inhibitors in the different tissues of the sections of worm. The storage mechanisms for monoamines are similar to those in mammals in that monoamines depleted by reserpine and catecholamines, but not by 5-HT, were depleted by 6-hydroxydopamine (Gardner and Cashin, 1975) (Fig. 5). Furthermore, the releasing agent, amphetamine, enhanced rhythmic contractions of body wall preparations of Lumbricus as long as the nerve cord was present. The DA receptors in the nerve cord of Lumbricus are similar to a class of DA receptors in mammals in that they are stimulated by apomorphine and piribedil (Saka and Tsuji, 1934; Bieger and Hornykiewicz, 1972a, b; Gardner and Cashin, 1975). The NA response may be via receptors with c( characteristics as isoprenaline in a similar dose range f ItlorelCelt©* i¢or*

I conltol

1

5 ~ pro* iZid

J

L, 1

5 I

reserpine

i 0"5

1 60HDA

.5 ~

¢onc n x

lJO-4(jrrt/fttl

FIG. 5. Subjective scores of blue-green fluorescence (open cohtmns) and yellow fluorescence (closed columns) within ventral nerve cords of Lumbricus, under the influence of some drugs. Mean scores from at least 20 transverse sections + S.E.M. are shown and statisticifl significance, *p < 0.01. **p < 0.(101 is indicated. This ligure is reproduced witl~ permission from Gardner and Cashin. 1975.

NEUROTRANSMITTERS AND NEUROMODULATORS IN ANNELIDS

ACh

S

ACh

91

S

120

o

80

U

.40 Ld

N

0

LtJ

~

O ~ ; M NOR--~NAL,NE O'~2~T~ ACh

120.

~: 8 0 -

40" 0

S

.im

ACh

S

E

FIG. 6. Effects of N A (n -- 5), G A B A (n = 9) and 5-HT (n = 4) on respon~ of body wall of Luraln,icus to fieldstimulation (S) and to ACh. Responses are the mean percentage of the initial response, -4-S.E.M. Test agents were added to the bath 3-4 rain. prior to A C h in order to allow their effectson fieldstimulated responses to develop. Responses to stimulation at the end of this period and following A C h responses in the presence of the candidate neurotransmitters are indicated by horizontal bars. This figure is reproduced with permission from Gardner, 19gla.

(0.05--0.2 mM) was relatively inactive. However, in order to determine whether these catecholamines act on separate receptors and the comparative receptor characteristics, further more detailed studies are required, perhaps of responses of single neurones within the nerve cord. Relaxation of cordless body wall of the earthworm by DA has been observed whilst NA caused a contraction (Andersson and Fange, 1967). However, more recent observations have shown that both NA and DA inhibit responses of cordless body wall of Lumbricus to field stimulation (Gardner and Cashin, 1975; Gardner, 1979). These responses are reduced by tetrodotoxin and do not appear to be due to activation of muscle fibres directly (Gardner, 1981a). ACh-induced contractions (tetrodotoxin-insensitive) were only inhibited at higher concentrations of the catecholamines (Bieger and Hornykiewicz, 1972a; Gardner 1981a; Fig. 6). These observations suggest prejunetional actions at lower concentrations and also post-junctional actions at higher concentrations, both effects being inhibitory to neuromuscular transmission. The action of DA was mimicked by apomorphine and piribedil. The receptors mediating the action of NA could be designated ~ in that NA and adrenaline are effective whilst isoprenaline is relatively ineffective (Gardner and Cashin, 1975; Gardner, 1979; Gardner, 1981a). Further characterisation of these receptors would require studies with pharmacological antagonists. This action of catecholamines in Lumbricus is inconsistent with a lack of effect of NA in electrophysiological studies of neuromuscular transmission in Pheretima hawayana (Chang, 1975). It is also apparently inconsistent with the effects of catecholamines on neuromuscular transmission in Pheretima communissima (Kuriyama et al., 1974). Spontaneous release of both excitatory (probably ACh) and inhibitory (proposed to be GABA) transmitters was enhanced via ~ receptors whilst the postjunctional membrane was hyperpolarised via /~ receptors. In the apparent absence of inhibitory transmission in Lurnbricus terrestris (see Section 6 on inhibitory amino acids) catecholamines inhibit excitatory transmission, predominantly by a prejunctional action. Both pre- and the weaker post-junctional actions are probably mediated via a receptors. Enhancement of '3

92

C.R. GARDNER AND R. J. WALKER

the release of an inhibitory transmitter or hyperpolarisation of the post-junctional membrane would only explain the post-junctional inhibitory action, involving inhibition of ACh-induced contractions. It remains possible that the 0t receptor-mediated effects observed under resting muscle membrane conditions in Pheretima communissima are not functionally significant at the higher action potential-induced level of transmitter release. However, it seems less easy to consider a facilitation of transmission under resting conditions and an inhibition of the same transmission during induction of postjunctional action potentials. Perhaps these different actions represent different roles for catecholamines in the different earthworm species. It is apparent that the central action of catecholamines in inducing rhythmic contractions of body wall sections of Lumbricus predominates over their inhibitory actions on neuromuscular transmission at similar concentrations. This does not necessarily imply that the two effects are mediated via different neuromuscular systems but it raises this possibility. The neuromuscular transmission, of Lumbricus is heterogenous (Roberts, 1962; Gunther, 1972; see review by Mill, 1975, pp. 252-264) and it is possible that the electrical stimulations employed in these studies may only activate components of it. 3.2. LEECHES

The action of catecholamine and related amines has been investigated on Leech Retzius cells (Sunderland, 1978; Sunderland et al., 1980; 1982), DA was the most potent catecholamine, being about 3 times more potent than NA on Hirudo Retzius cells and about 8 times more potent than NA on Haemopis cells (Table 2). There was also a slight difference in potency between DA and 5-HT on these two species, DA being only half the potency of 5-HT on Haemopis cells but about one quarter of the potency on Hirudo cells. DA has a clear hyperpolarising action on Retzius cells in contrast to the findings with NA and adrenaline. In the case of these two latter catecholamines the response could be either hyperpolarisation, depolarisation or a biphasic response, depolarisation followed by hyperpolarisation. For example, out of 69 Haemopis preparations, 26 were hyperpolarised, 18 depolarised and 10 showed a biphasic response to NA; the remaining 15 were unaffected. In contrast with DA, out of 123 Hirudo preparations, 122 were inhibited and TABLE 2. TO COMPARE THE RELATIVE POTENCIES OF A RANGE OF DOPAMINE ANALOGUESAND RELATED COMPOUNDS ON TIlE RETZIUS CELLS OF THE LEECHES HIRUDO AND HAEMOPIS. THE RELATIVE POTENCIES WERE CALCULATED AS THE EQUIPOTENTMOLAR RATIOS(EPMRs) AND REFERRED TO 5-HT AS THE STANDARDWHICH IS TAKEN AS 1. WHERE VALUES ARE GREATER THAN I THEN THE COMPOUND UNDER TEST IS LE~ POTENT THAN 5-HT

No. of

Compound

5-Hydroxytryptamine Dopamine Tyramine 3-Methoxytyramine Morphine Ephedrine A.D.T.N.

6-Hydroxydopamine Octopamine Phenylethylamine Noradrenaline Amphetamine Methoxamine Isoprenaline Pseudoephedrine Phenylephrine Metaraminol lsoetharine Adrenaline Salbutamol

H irudo EPMR

No. of

H aemo pis EPMR

Observations

Range

Mean

Observations

Range

I0

-2.14-10.69 5.28 2.0-19.92 4.20-12.59 8.20 1.26-22.78 10.61 4.28-46.63 10.29-15.43 2.54-31.75 0.22-40.33 8.72-40.87 5.91-98.20 0.67-107.04 14.38-149.30 96.53 102.10 4.06-608.20

1.00 4.28 5.28 6.31 6.75 8.20 9.25 10.61 12.76 13.72 13.76 19.63 24.79 26.72 40.58 49.92 96.53 102.10 145.90

I0 I0 6 5 6

-2.14--4.28 1.40-I 1.65 2.0-3.48 1.47-6.23

2 3 7 6 6 3 5 17 I 6 6 I 6

2.28-22.78 0.78-9.86 3.05-106.90 10.29-36.00 3.79-34.38 3.67-11.00 11.36-326.96

7 I 6 5 1 3 1 5 3 6 3 2 6 3 7 I I 6 3

inactivc

3

Mean I 2.35 4.16 2.75 3.76 12.53 4.32 57.88 18.58 17.65 7.44 108.08

inactive 233.74 1.99-398.12 2.57-27.00 296.10 14.54-608.21

inactive

233.74 104.35 22.62 296.10 141.66

NEUROTRANSMITTERSAND NEUROMODULATORSIN ANNELIDS

93

only one failed to respond while out of 93 Haemopis preparations, 88 were inhibited by DA and 5 failed to respond. Repeated applications of NA desensitised the inhibitory response. Adrenaline gave the same variety of responses as NA but was less potent (Table 2). It is possible that the excitatory actions of NA and adrenaline were indirect; however, they persisted in high magnesium Ringer which suggests that they are direct and are activating an additional receptor system on which DA is inactive. Phenylethylamine gave an overwhelmingly inhibitory response on Retzius cells, only one Haemopis cell was excited and as can be seen from Table 2, its potency was similar to that of NA. Tyramine was found to only inhibit Retzius cells and was only slightly less potent than DA, particularly on Hirudo cells. 3-methoxytyramine was also a potent DA agonist and in the case of Haemopis was only slightly less potent than DA, again it only showed inhibition. 6-Hydroxydopamine and ADTN (I,2,3,4-tetrahydro-6,7-dihydroxy-2-napthylamine) were also potent DA agonists, devoid of any excitatory component in their response. Morphine and apomorphine were also inhibitory, morphine being particularly potent on Haemopis cells (Table 2). Amphetamine also showed only inhibitory actions and was more potent on Haemopis than on Hirudo Retzius cells. Octopamine also gave a variety of responses but the dominant action was inhibitory. On Hirudo cells, octopamine was about 3 times less potent than DA, while on Haemopis cells it was over 25 times less potent than DA. Phenylephrine gave similar responses to NA and adrenaline, that is, inhibition and/or excitation. It was considerably less potent than dopamine in both species. The major action of methoxamine was also inhibitory and this compound was about 6 times less potent than DA on Hirudo cells but about 50 times less potent on Haemopis cells. Metaraminol also showed both excitatory and inhibitory actions on leech Retzius cells and was approximately 25 and 10 times respectively less potent than DA on Hirudo and Haemopis cells, lsoprenaline was inactive on all 17 Haemopis preparations on which it was tested but inhibited 43 of the 45 Hirudo preparations. This compound was the only one which had such a clear difference between the two species. On Hirudo cells it was about 6 times less potent than DA. Salbutamol was inactive on all the cells tested in both species. A number of other compounds were also tested on Retzius cells but not in sufficient numbers for a definite conclusion in terms of their major action or for a clear potency value. These compounds included a-methylnoradrenaline, isoetharine, ephedrine, N-methylephedrine and pseudoephedrine. These compounds all inhibited cell activity. In terms of the actions of catecholamines and related amines on Retzius cells of Hirudo and Haemopis there are certain differences in the mean potencies, that is, the differences in potency between DA and many of the other compounds is far less in the case of Hirudo than with Haemopis. In which case it could be suggested that the DA receptor for Hirudo is less specific than that of Haemopis in terms of its potency ratio with 5-HT. The possible nature of the excitatory component of the NA and adrenaline responses is also of interest but further work is required on this before it can be concluded as to whether there is a specific excitatory catecholamine receptor on leech Retzius cells or not. 3.3. OTHER RELATED INVERTEBRATES

catecholamines are present in a wide range of invertebrates (Kerkut, 1973). Catecholamines can be synthesised and metabolised in protozoan cells (Janakidevi et al., 1966) and the catecholamines may help to regulate carbohydrate metabolism within the cell (Kerkut, 1973). Catecholamines are also present in coelenterates (Dahl et al., 1963) and echinoderms (Cottrell, 1967; also see Kerkut, 1973; Leake and Walker, 1980; chapter 7, pp. 144-148). Similarly, other worms also contain catecholamines. The occurrence of catecholamines in three species of planarians was investigated by Welsh and King (1970). In extracts of Ouoesia they found levels of DA from 0.25-1.5/~g/g while the values for NA ranged from 0.12-1.5/~g/g wet weight. Procotyla and Phagocata yielded only detectable amounts of DA. This study confirms a fluorescence study where cells containing 5-HT or a catechol-

94

C.R. GARDNER AND R. J. WALKER

amine were identified in three planarian species (Welsh and Williams, 1970). A histochemical and physiological study on the presence and action of dopamine on planarians was undertaken by Carolei et al. (1975) using Duyesia ,qonocephala. Pretreatment with L-dopa greatly enhanced the fluorescence observed in planarian sections while benserazide only weakly increased the fluorescence. Reserpine pretreatment markedly reduced the fluorescence while piribedil, apomorphine, haloperidol and clonidine had no effect. Changes in the motor activity of Duyesia following drug pretreatment was noted with L-dopa, benserazide, D-amphetamine, apomorphine, peribedil, haloperidol and reserpine while clonidine had no effect. L-Dopa induced hyperkinesias which was described as an abnormal screw-like movement. A similar effect was observed with D-amphetamine, apomorphine and piribedil while benserazide decreased motility. Haloperidol produced a gradual reduction of movements until the animals became totally immobile while with reserpine pretreatment planarians became motionless. Treatment with 2-Br-~t-ergocryptamine produced a typical curling in one plane which was similar to that observed following treatment with atropine. Dihydroergometine produced an increase in motor activity with hyperkinesias, similar to the activity produced with DA agonists. The authors compare the motor activity observed in planaria, a three dimensional screw-like movement, with the stereotype behaviour observed in mammals following activation of the DA system. In a later paper, Palladini et al. (1980) observed that manganese also induced screw-like movements similar to those produced when the DA system was activated. This screw-like movement induced by DA agonists is associated with an increase in cAMP levels in Dugesia (Venturini et al., 1981). Likewise DA antagonists reduce motor activity and cAMP levels. Pretreatment with morphine also reduces both motor activity and cAMP levels, while naloxone induces strong hyperkinesias and an increase in cAMP levels. The naloxone effect can be inhibited with reserpine or haloperidol pretreatment. In the nematode Caenorhabditis elegans, DA is present in eight mechanosensory cephalic and deirid cells in hermaphrodites, males having six further DA-containing cells caudally (Sulston et al., 1975). Reserpine depleted axonal but not somatic DA. However, muscle tone of Ascaris lumbricoides was not affected by NA or related amines (Ash and Tucker, 1966) and the physiological role of these catecholamines remains to be elucidated. NA and DA are present in trematodes (Schistosoma mansoni and Fasciola hepatica, Bennett et al., 1969; Chou et al., 1972; Gianutsos and Bennett, 1977). DA is also present in Paragoniraus westermani and P. ohirai but absent in the cestode Hymenolepis diminuta (Chou et al., 1972). Green fluorescence was observed in central ganglia and linking commissures, nerve cords and fibre networks between them (Bennett and Bueding, 1971). Little is known of the physiological roles of the catecholamines in trematodes but they may have no role in control of motility, as increase in DA levels together with marked decrease in NA due to treatment of S. mansoni with disulfuran did not affect motility (Bennett and Gianutsos, 1978). However, both NA and DA have been reported to decrease motor activity of Fasciola hepatica via adrenoreceptors rather than DA receptors (Tkach, 1968). Another physiological role for one or more of these catecholamines is suggested by increases in length of S. mansoni when incubated with NA or DA. Pharmacological studies suggested that this response was mediated via DA receptors (Tomosky et al. (i 974). Catecholamines inhibit twitch contraction of body wall strips of the echiuroid, Urechis unicinctus (Muneoka and Kamura, 1982). The order of potency for the catecholamines was NA > adrenaline ~ isoprenaline. The threshold concentration for the NA inhibition was 10 -9 M. DA, phenylethanolamine and histamine had little or no effect on this muscle at 10 -6 M. NA inhibited the contractions induced by both acetylcholine and L-proline. The inhibitory action of NA was blocked by phentolamine and phenoxybenzamine but not by propranolol or alprenolol. In the presence of phentolamine, tetanic contraction of the muscle strip was enhanced. The resting potential of these muscle fibres is around - 4 8 mV and this resting potential is hyperpolarised to - 5 5 mV following the appli-

NEUROTRANSMITTERS AND NELJROM()I)UI.ATORSIN ANNI'LIDS

95

cation of 10 -6 M NA. 5-HT has no effect on the membrane potential but does enhance twitch contraction and relaxation. Bio-assay estimations suggest that the body wall contains 5-HT while the ventral nerve cord contains NA. Octopamine also enhances the contraction of this muscle but has no effect on the rate of relaxation. It is suggested that NA and also possibly 5-HT may have a role in modulating muscle con~traction in

Urechis. Catecholamines may be present in onychophora as blue-green fluorescence has been observed in sensory nerves of peripheral sense organs, in tracts in the nerve cord and in scattered neurones within the cortex of the cord in Perip, topsis sedqwicki and P. moseleyi (Gardner et al., 1978). Neither adrenaline, NA nor DA had an effect on longitudinal body wall sections of Peripatopsis moseleyi (Ewer and van den Berg, 1954; Gardner and Robson, 1978) and NA had no effect on similar preparations from Opisthopatus costesi (Florey and Florey, 1965) or on contractions of isolated hearts from this species (Florey and Florey, 1965). However, if preparations of ventral body wall with nerve cords intact are prepared in P. moseleyi both NA and DA evoke clear responses. Increases in muscle tone were observed with superimposed rhythmic contractions, particularly with DA (Fig. 7) (Gardner and Robson, 1978). lproniazid evoked similar responses suggesting the presence of a monoamine oxidase. These responses have similarities with responses to catecholamines in the equivalent preparations from the annelid Lumbricus terrestris (Gardner and Cashin, 1975) and are consistent with a role for the catecholamines within the nerve cord, perhaps as neurotransmitters in sensory neurones. Adrenaline and NA evoked only occasional contractions of the longitudinal body wall muscle of Priapulus caudatus, were ineffective on the intestine, but strongly contracted the rectum (Mattison et al., 1974). A study of agonists on the rectum response showed similar effects of adrenaline, NA, DA, isoprenaline and phenylephrine. Thus, in terms of agonist responses, the receptors involved in this response do not fit into any category of the mammalian adrenoreceptor classification.

4. Other Phenylethylamines

The discovery of octopamine-mediated neurotransmission in some invertebrates (e.g. lobster, locust and firefly, Harmer, 1980; Leake and Walker, 1980, pp. 144-174; Talamo, 1980) led to extensive studies of the presence of this and related phenylethylamines in i

t ACh 10

NA

t

ACh 20

80

t

t

5HT 50

~

.

~

FIo. 7. Responses of ventral preparations of Peripatopsis (with intact nerve cords) to ACh and monoamines. ACh induced a smooth, rapidly rising, dose-related contraction. NA caused a slower increase in tone with some rhythmic activity. DA evoked more rhythmic activity and less increase in tone. 5-HT initially increased then decreased these activities. Drug concentrations are in/~g/ml. Time mark--5 min.

96

C.R. GARDNER AND R. J. WALKER

vertebrates and other invertebrates and their potential physiological roles (Harmer, 1980; Talamo, 1980). Octopamine is present in a wide range of invertebrates (Robertson and Juorio, 1976; Juorio and Robertson, 1977; Evans, 1978a) and this includes annelids. Octopamine would appear to be the major catecholamine-like substance in Lumbricus terrestris, there being 7.4/zg/g present in cerebral ganglia, 8.1 #g/g in subpharyngeal ganglia and 5.3/ag/g in the ventral nerve cord (Robertson, 1975). Robertson and Osborne (1979) further confirmed the predominance of octopamine using radiochemical enzymic assays and cited preliminary data indicating that it was almost entirely p-octopamine. An adenyl cyclase enzyme sensitive to octopamine is present in the nervous tissue of Lumbricus and it is specific to octopamine when considering phenylethylamines in that DA and NA did not activate the enzyme, in fact there was some inhibition, and phentolamine did not block the effect of octopamine (Robertson and Osborne, 1979). Glutamate, GABA, glycine and adenosine similarly did not activate the adenyl cyclase. 5-HT, however, did stimulate adenyl cyclase activity in Lumbricus nervous tissue and it remains to be determined whether the enzymes are different. The addition of GTP to the assay system enhanced the magnitude of the octopaminestimulated activity (Robertson and Osborne, 1979) consistent with reports in invertebrates and vertebrates that guanyl nucleotides may play an important role in regulation of ~Ldeayl cyclase activity in nervous tissue (Clement-Cormier et al., 1975; Hegstrand et al., 1976; Harmer and Horn, 1977; Sulakhe, 1977). However, no definite physiological or biochemical role for octopamine in Lumbricus had so far been identified. The previous evidence suggests that it may function as a r~e:~o~transmitter in the central nervous system. Gardner (1979)observed a contraction re.sl:tO,~e to ~octopamine in the isolated body wall of Lurnbricus terrestris different from responses to catecholamines. A study with a series of related phenylethylamines has indicated that this response requires the presence of p-hydroxy groups but tends to be reduced or converted to catecholamine responses by c~-methyl or m-hydroxy or m-methoxy substitution (Gardner, 1979; Table 3). Synephrine was more potent in Lumbricus as it is in stimulating l.uminescence in Photuris (Carlson, 1969) and adenyl cyclase in the haemolymph of the lobster (Batelle and Kravitz, 1978). Two c~2 receptor agonists, clonidine and xylazine, according to mammalian classifications, did not ha~e catecholamine like effects in Lumbricus, despite the fact that they may act on cct receptors in higher concentrations. However, they both showed responses similar to octopamine and synephrine (Table 3). The levels of octopamine in the leech have been assayed using a radiochemical-enzymatic method (Webb & Orchard, 1980). Levels of 12-40 and 7-19 ng/mg tissue protein respectively were detected in the ganglia and in the non-ganglion nervous tissue. Octopamine was detected in the blood at a concentration of 1.96 x 10 -6 i . Pretreatment of leeches with iproniazid or p-chlorophenylalanine resulted in a rise in octopamine levels in the nerve cord while pretreatment with fusaric acid or reserpine resulted in a fall in octopamine levels. In a second paper Webb and Orchard (1981) report the synthesis, release and re-uptake of octopamine in the ventral nerve cord. Incubation with either labelled tyrosine or tyramine produced labelled octopamine. In addition trace amounts of dopamine were also synthesised. There is evidence for both a sodium sensitive and a sodium insensitive component in the uptake system for octopamine. The release of labelled octopamine from the nerve cord was calcium dependent and could be induced using high potassium Ringer. The production of hydroxymandelic acid was reduced in the presence of iproniazid which suggests the presence of a monoamine oxidase in the nerve cord. It is suggested that octopamine can be synthesised in the leech nerve cord from tyrosine and tyramine while a major metabolite is probably hydroxymandelic acid. These papers represent an important contribution to a possible role for octopamine in the leech. Octopamine also has an inhibitory action on leech Retzius ceil activity but there is no evidence as to its site of action on these cells (Sunderland, 1978). However, on other

]NEUROTRANSMITTERSAND NEUROMODULATOR$IN ANNELIDS

97

TAItLI" 3. EFFI':('TS OF MONOAMINES AND RELATI!I) SUIISTANCES ON THF BODY WALL OF" LUMBRI('('.~ DURING FIt!LI) STIMULATION. + + + STRONG EFFECT AT < O . I raM, + + STRONG EFFr('T AT 0. l - 0 . 5 raM, + CONSISTI~NT EFFI!('T AT > 0 . 5 nlM, -t- WFAK EFFI'('T AT > 0 . S m M

R,

DL-Isoprenaline DL-Adrenaline L-Noradrenaline Dopamine p-Tyramine DL-p-Octopamine DL-p-Synephrine Normetanephrine Metanephrine Metaraminol Phenylephrine /~-OH-Phenylethylamine Ephedrine DL-Amphetamine p-Chloroamphetamine 5-HT CIonidine Xylazine Benzylpiperazine

CH-- CH-- NH

..

R~

/ R , " Rz R.~

R,

OH OH OH OH OH OH OH OH OH H H H H H CI

OH OH OH OH OH OH OH H H H H OH H OH OCH 3 OH OCH ~ OH OH OH OH OH H OH H OH H H H H

H H H H H H H H H CH 3 H H CH~ CH3 CH3

R4

R5 CH(CH3)2 CH.~ H H H H CH~ H CH 3 H CH~ H H H H

Reduced response + + + + + + + + + + + 0 0 0 0 :t0 0 + 0 0 + 0 +

Increased tone 0 0 0 0 + + + + + + + + + + + 0 0 0 0 + + + + +

Increased response and spontaneity

0 0 0 + 0 + + 0 0 0 0 + 0 + + ++ + ++ + 0 0

invertebrate systems there would appear to be evidence for specific octopamine receptors (Atkinson et al., 1977; Batta et al., 1979; Battelle and Kravitz 1978; Evans, 1978b, 1980; Harmer and Horn, 1977; Nathanson, 1979; Robertson and Carlson, 1976; Roberts and Walker, 1981). In a recent paper Evans (1981) has attempted to classify the different types of octopamine receptor responsible for the action of octopamine on the locust extensortibiae muscle. The findings from this paper provide an excellent basis for the study of octopamine receptors in other systems including the annelids. In the locust muscle preparation there would appear to be three types of receptor: octopamine-1 receptors, which mediate the slowing of the myogenic rhythm of these muscles; octopamine-2A receptors, which mediate the increase in amplitude of slow motoneurone twitch tension; octopamine-2B receptors, which mediate the increase in relaxation rate of twitch tension induced by firing either the fast or slow motoneurones. Chlorpromazine and yohimbine are much better blockers of octopamine-I receptors than of octopamine-2 receptors while metoclopramide is much better as a blocker of the octopamine-2 receptors. Clonidine is a better agonist on the octopamine-I receptors while naphazoline is better as an octopamine-2 agonist. Octopamine-2A can be separated from octopamine-2B receptors in that metoclopramide, mianserin and cyproheptadine block octopamine-2A in preference to 2B receptors. Naphazoline is a better agonist against octopamine-2A than is tolazoline while tolazoline is a better agonist then clonidine for the octopamine-2B receptors. On another invertebrate preparation, the central neurones of Limulus, octopamine is also inhibitory (Roberts and Walker, 1981). These again appear to be specific octopamine receptors and are blocked by both phentolamine and cyproheptadine, both of which block the insect muscle octopamine receptors described by Evans (1981) and so do not help in comparing the Limulus central receptors with those of insect muscle. In terms of agonists, clonidine is approximately equipotent with naphazoline which would suggest that these octopamine receptors are not like the octopamine-I receptors of insect muscle. Xylazine is either less potent than clonidine or inactive on Limulus octopamine receptors but Evans did not use this compound in his study. Once again it does not seem possible to transfer such mammalian receptor classifications to invertebrates. It would be interesting to investigate the actions of clonidine and xylazine on octopamine responses in mammals. The potential physiological roles of J.P.~. 18~2/3--,

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C . R . GARDNER AND R. J. WALKER

octopamine and/or synephrine or other phenylethylamines in annelids remain to be elucidated.

5. 5-Hydroxytryptamine (5-HT) 5.1. ANNELIDSOMATIC MUSCLE A role for 5-HT at annelid neuromuscular junctions has been suggested, but not simply as the major motor neurotransmitter. In the leech, nerve terminals containing dense core vesicles innervate body wall muscles en passage and synapse onto clear vesicle (probably cholinergic) terminals which also innervate the muscle (Yaksta-Sauerland and Coggeshall, 1973; Coleman, 1975). The dense core vesicles contain a chromaffin substance and strongly take up 3H-5-HT but not 3H-NA. Autoradiographic studies in Nereidae have similarly shown uptake of 3H-5-HT into neuromuscular nerve terminals (Dhainaut-Co'urtois and Dhainaut, 1974). The uptake of tritiated 5-HT, NA, 5-hydroxytryptophan (5-HTP), DOPA and DA by the cerebral ganglia and prostomial nervous system of Nereis has been investigated using light autoradiography (Marsden et al., 1981). Tritiated 5-HT was strongly taken up by the antennal, palpal, tegumentary and nuchal nerves and a number of ganglionic nuclei in the midbrain and neurosecretory neuropile and into the infracerebral organ. These results suggest the presence of a 5-HT motor system from cerebral cells sending axon processes to prostomial muscle and epidermis via cerebral nerves. But surprisingly there appeared to be no uptake of labelled 5-HTP. Whilst Mill and Knapp (1970)identified only one morphological type of nerve terminal at neuromuscular junctions in the longitudinal muscle of the earthworm, other studies have shown two (Smallwood, 1926; Nishihara, 1967; Rosenbluth, 1972). The last author showed in Lumbricidae one type with clear vesicles resembling the cholinergic neuromuscular junctions of vertebrate skeletal muscle and a second type with granular vesicles. These granular vesicles may contain a monoamine such as NA or 5-HT. As is true in many invertebrates (see Gerschenfeld, 1973) there are large amounts of 5-HT in annelid nerve cords (e.g. 7-10/~g/g in L u m b r i c u s - - W e l s h and Moorhead, 1960; Myhrberg, 1967; Rude, 1969a, b; Gardner and Cashin, 1975). The use of fluorescence microscopy has allowed the demonstration of 5-HT-containing cell bodies in the ganglia of the earthworms Lumbricus and Octolasium (Bianchi, 1962, 1964; Myhrberg, 1967; Kerkut et al., 1967; Rude, 1969a; Gardner and Cashin, 1975) and of the leech Hirudo (Ehinger et al., 1968; Kuzmina, 1968; Marsden and Kerkut, 1969, Rude, 1969a). Using micro thin-layer chromatographic analysis, Jost et al. (1981) have shown the presence of 5-HT in the central nervous system of Nereis. Three weeks following the injection of p-chlorophenylalanine, the levels of 5-HT were greatly reduced. There was no conclusive evidence for the occurrence of any other indoleamines in the Nereis brain although two types of yellow fluorescence in brain cells had previously been observed (White and Marsden, 1978). Some fibres of the 5-HT-containing neurones in Lumbricus travel to the periphery into muscle layers (Rude, 1969b; Myhrberg, 1967; Gardner, 1975) and it is possible that these may innervate muscles of the body wall. Of the seven 5-HT-containing cells in the segmental ganglia of Hirudo only the two giant Retzius cells send branches to the periphery (Sunderland et al., 1974; Smith et al., 1975; Lent and Frazer, 1977; Mason and Leake, 1978). Stimulation of the Retzius cell evoked slow reduction of tone of the resting longitudinal muscle and increased the rate of the slow phase of relaxation following isometric contraction of the muscle. This response was mimicked by 5-HT (Mason et al., 1979). Furthermore, stimulation of peripheral nerves to the muscle evokes a junction potential with an excitatory component which is blocked by d-tubocurarine and a chloride dependent inhibitory component which is blocked by the 5-HT antagonists BOL-148 and cyproheptadine (Sawada and Coggeshall, 1976).

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The possible actions of the 5-HT containing Retzius cells on leech body wall musculature has been investigated by Leake et al. (1981) and Leake and Sunderland (1982). Stimulation of the Retzius cells or application of 5-HT, 3 x 10 -~ M, both induce a delayed but prolonged decrease in the basal tone of the muscle. The tone recovers after a delay of 10-30 min. Both stimulation of the Retzius cells and direct application of 5-HT increase the relaxation rate following contractions of the body wall musculature either by direct electrical stimulation or activation of motoneurones. Out of a range of potential antagonists, only cyproheptadine, 10 -'~ M, and atropine, 5 x 10 -6 M, reversibly blocked the decrease in basal tension induced by Retzius cell activation. Two of the compounds tested, methysergide and ergometrine, both at 10-5 M, had direct relaxing effects on the muscle but in addition methysergide did antagonise the action of the Retzius cells. None of the antagonists tested appeared to block the action of 5-HT on the muscle. The relaxing action of 5-HT on the muscle cannot be mimicked by DA which suggests that the receptor on the muscle which is activated by 5-HT is different from the central 5-HT/DA Retzius cell receptor (Leake et al., 1980). Accumulated evidence confirms the presence of 5-HT within Retzius cells (Rude et al., 1969; Marsden and Kerkut, 1970; Coggeshall, 1972; Osborne et al., 1972, McCaman et ai., 1973; Coleman, 1975; McAdoo and Coggeshall, 1976; Lent et al., 1979). In addition to 5-HT, 5-HTP and tryptophan have been shown to be present in the leech Retzius cells (Osborne et ai., 1972). Also Retzius cells can take up tryptophan, 5-HTP and 5-HT (Hildebrand et al., 1971; Coleman, 1975) and can synthesise 5-HT (Hildebrand et al., 1971), probably via an aromatic amino acid decarboxylase enzyme (Coggeshall et al., 1972) and a tryptophan hydroxylase enzyme (Osborne et ai., 1972). Inactivation of 5-HT may not involve a monoamine oxidase as there is little evidence of the presence of such an enzyme in leech tissues (Yaksta and Coggeshall, 1973; Corte and Nistri, 1974). 5-HT has an inhibitory action on isolated leech muscle, inducing relaxation (Mason et al., 1979; Poloni, 1955) and antagonising ACh-induced contractions (Schain, 1961). Furthermore, it hyperpolarises muscle fibres and depresses spontaneous excitatory junction potentials (Walker et al., 1968; Sawada and CoggeshaU, 1976). Thus there is considerable evidence for 5-HT as an inhibitory neuromuscular transmitter in the leech. It is also possible that Retzius cells control mucus-releasing cells in the skin of the leech via release of 5-HT (Lent, 1973; Coleman, 1975). However, there are few dense core vesicle-containing nerve endings on mucus cells and further investigation is required of this potential role of 5-HT. The neuromuscular actions of 5-HT do not appear to be similar in all annelids and related worms. For example, 5-HT relaxes dorsal longitudinal muscle strips of sabellid worms but did not inhibit contractions induced by ACh (Aivarez et al., 1969). Another annelid which has been studied in some detail is the oligochaete Lumbricus terrestris. Initial observations of Andersson and Fange (1967) showed that 5-HT contracted the isolated body wall but inhibited visceral muscle. In sections of body wall with intact nerve cord, 5-HT consistently enhanced tension-induced rhythmic contractions (Bieger and Hornykiewicz, 1972a; Gardner and Cashin, 1975). Quiescent sections of cordless body wall were induced to contract rhythmically by 5-HT but not by other monoamines (Gardner and Cashin, 1975) and lower concentrations of 5-HT markedly enhanced the contractile responses of these preparations to field stimulation (Gardner, 1979; 1981a). 5-HT depresses the tonic more than the phasic contractions of body wall of Pheretima communnissima evoked by direct electrical stimulation. This was speculated to be due to an action on the motility of calcium within the muscle fibres (Tashiro and Yamamoto, 1971). This represents a postjunctional action of 5-HT but the effects of 5-HT in Lumbricus may not be due to a postsynaptic action. Tetrodotoxin inhibits body wall responses to field stimulation and prevents 5-HT from inducing spontaneity (Gardner, 1981a). In addition, ACh-induced contractions, which are assumed to be due to a postjunctional action, are not enhanced by 5-HT although simultaneously recorded responses to field stimulation were markedly increased (Fig. 7). The 5-HT receptors involved in these responses have similar characteristics to adenyl

100

C.R. GARDNER AND R. J. WALKER TABLE 4. EFFI!CT OF A SERII'SOF NliUROACTIVI! AGENTS ()N ISOLATH) FIELD STIMULATED BOl)Y WALL ()F LtMBRICU$. THI! RANKINGS INDI(2ATI"THE DEGRrF "IX)WHICH RliSPONSI" SIZI" WAS INCREASED AND SI~)NTANEITY INI)U('I!D. + + + STRONG EFFECT AT <0,1 raM. + + STRONG EFH!('T AT 0.1-0.5 mM, + CONSISTENT EFFI~('T AT >0.5 mM, __+ WI!AK EFFECT AT >0.5mM. DFLAYS IN EXCESS OF 5MIN TO ONSET OF RESPONSI.S ARE SHOWN IN PARENTHESES 5-Hydroxytryptamine 5-Methoxytryptamine 7-Methyltryptamine Tryptamine Bufotenin 5-Hydroxytryptophan Tryptophan

+ + + + + + 0

+ + + + +

p-Chloramphetamine Fenfluramine Amphetamine

+ + + (3- I 1 min) + + (5-25 min) + (8-24 min)

Quipazine TM PP MK 212

+ (10-30 min) + ( I2-26 rain) 0

cyclase-linked receptors in mammals (Hamon et al., 1980) and the liver fluke (Northup and Mansour, 1978)in that they show ranked responses to substituted tryptamines, with bufotenin as a partial agonist, but are insensitive to piperazine-containing 5-HT agonists such as MK 212, quipazine and m-trifluormethyl phenylpiperazine (Gardner, 1981a, Table 4). A 5-HT-stimulated adenyl cyclase is present in the nervous tissue of L. terrestris (Robertson and Osborne, 1979) leaving it possible that the neuromuscular response may be mediated via an adenyl cyclase-linked receptor. The 5-HT mechanisms in Lumbricus have some similarities to mammalian neurotransmission. Inhibition of monoamine oxidase increases 5°HT levels in nervous tissue (Gardner and Cashin, 1975; Fig. 5)and increased 5-HT but decreased 5-hydroxyindole acetic acid, a major metabolite of 5-HT in mammals, in the first five segments of Lumbric,s (Izquierdo et al., 1979). Monoamine oxidase also sensitised earthworm sections to 5-HT (Bieger and Hornykiewicz, 1972a; Gardner and Cashin, 1975). 5-HTP appears to be a precursor of 5-HT in the body wall and the stores of 5-HT are depleted by reserpine but not 6-hydroxydopamine (in the nerve cord) and can be released by p-chloramphetamine and fenfluramine (in the body wall) (Gardner and Cashin, 1975; Gardner, 1981b). There appears to be an uptake mechanism in the isolated body wall preparation as chlorimipramine and fluoxetine potentiate 5-HT and have a similar effect to 5-HT at higher concentrations (Gardner, 1981b). This evidence suggests that there may be 5-HT-containing motoneurones within the nerve cord which send processes to the body wall muscle, perhaps terminating in axoaxonic synapses with the major excitatory neurotransmission. Such axo-axonic connections have, however, not been identified in electron microscope studies (Nishihara, 1967; Mill and Knapp, 1970; Rosenbluth, 1972). In addition, the rhythmic nature of contractions induced by higher concentrations of 5-HT in the isolated body wall may require further explanation. 5.2. LEECHCENTRAL NEURONES 5-HT was first shown to have an inhibitory action on leech Retzius neurones by Kerkut and Walker (1967). The ionic mechanism associated with this inhibition has been subsequently investigated by Walker and Smith (1973) and found to be predominantly a chloride event. There was little or no evidence for an involvement of other ions. If it is assumed that this 5-HT inhibition is a chloride event then a value for internal chloride for these cells and a value for the equilibrium potential for chloride can be obtained. It was found that internal chloride was 8.4 mM which gave a chloride equilibrium potential of - 7 0 mV. Tryptamine, cyproheptadine and mianserin were tested as possible 5-HT antagonists. Tryptamine was found to be an agonist, being some 120 times less potent than 5-HT while the other two compounds were found to block 5-HT but this block was rather inconsistent and required high concentrations. Both compounds also had direct actions on the cells. Biphasic effects were also observed, a brief excitatory phase preceding the inhibition. Strychnine also hyperpolarises Retzius cells and an attempt was made to try and decide if 5-HT and strychnine were acting on the same or different receptors.

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I01

Mianserin was found to reversibly antagonise the action of 5-HT but had no effect on that to strychnine. There was no evidence for cross desensitisation between the two compounds and it was concluded that each was acting on a separate receptor. T w o structure activity studies on the Retzius cell 5-HT receptor have been performed (Smith and Walker, 1974; 19751. The equipotent molar ratios of a range of tryptamine analogues were obtained, comparing their activity with 5-HT. All the compounds tested were less potent than 5-HT and the study suggests that for potent 5-HT like activity, the hydroxyl group is required in the 5 position; a two carbon side chain with a terminal nitrogen but an indene nucleus is almost as active as an indole. The substitution of methyl, fluoro, chloro, methoxy or acetyl groups onto the 5-HT molecules progressively reduces the potency. 5-Methoxylation or terminal N methylation of tryptamine increased the potency of tryptamine and these compounds tended to have a different slope for their dose response curves to that of 5-HT. It is suggested that they may act on a different receptor. In addition, tryptamine methylated analogues often depolarised the Retzius cells prior to hyperpolarising them. In the second paper a large number of compounds were examined for possible antagonism against 5-HT. Morphine, atropine and dibenamine were the best antagonists of 5-HT but dibenamine also had a direct excitatory effect on the neurones. Methysergide was a weak antagonist. A large number of other potential 5-HT antagonists failed to have any effect on the 5-HT response though some of them had direct depolarising actions on the neurones. A possible role for 5-HT as an inhibitory transmitter in the leech central nervous system has been proposed (Smith et hi., 1975b). Chloride dependent inhibitory postsynaptic potentials (ipsps) can be recorded from Retzius cells and these ipsps can be reversibly blocked by morphine. Pretreatment of the ganglion with imipramine potentiates the duration of the 5-HT inhibition, suggesting the presence of an uptake system for 5-HT. Fluorescence microscopy studies reveal the presence of six 5-HT containing neurones and these cells specifically take up labelled 5-HTP injected in vivo into leeches. Electron microscope studies demonstrated the presence of 100 nm dense core vesicles in the Retzius cells and similar dense core vesicles occur in the ganglion neuropile. 5-HT has previously been shown to be unequivocally present in the Retzius cells (Rude et al., 1969a). These observations all indicate the possibility that 5-HT may be a central transmitter in the annelids. It was also shown by Kerkut and Walker (1967) that the Retzius cells of leeches are hyperpolarised by DA and the mechanism for this inhibitory effect has been investigated and compared with that of 5-HT (Sunderland et al., 1979). As with 5-HT, it was found that the DA hyperpolarisation was a chloride conductance increase event. It was not possible to reverse the DA response by hyperpolarising the membrane potential. Values for internal chloride and chloride equilibrium potential were calculated and found to be very similar to those values obtained from similar experiments using 5-HT (Walker and Smith, 1973). It was concluded that DA and 5-HT activated the same type of chloride ionophore in the membrane of the Retzius cell. A structure-activity study has been performed on the DA response of both Hirudo and Haemopis Retzius cells and certain differences have been observed (Leake et al., 1980; Sunderland, 1978). The results are summarised in Table 2. In both species the potency values obtained were compared to DA but DA itself was compared to 5-HT. Compared to 5-HT, DA was slightly more potent on Haemopis Retzius cells than on Hirudo. Tyramine was of the same order of potency as DA in both species, suggesting that this is perhaps not a typical DA receptor. However, ADTN (l,2,3,4-tetrahydro-6,7-dihydroxy2-naphthylamine) is reasonably active in both species. NA and adrenaline are considerably less potent than DA while isoprenaline is inactive on Haemopis Retzius cells but is only 6--7 times less potent than DA on Hirudo Retzius cells. Salbutamol was inactive on all the cells examined. 6-hydroxydopamine was about half as potent as DA in both species. Octopamine was about 3 times less potent than DA on Hirudo neurones but about 25 times less potent than DA on Haemopis neurones which might also indicate a difference between the two neurones. Two general comments can be made from the table

102

C.R. GARDNI'R AND R. J. WALKER

which must be taken into consideration in drawing any strong inferences from the EPMR values. Firstly in some cases there is a very wide range in potencies for individual experiments, one very different value could markedly change the mean value and this is particularly important when very few determinations have been made. Which brings one to the second point, that is, many more determinations are required for many of the compounds and the table can only provide a possible indication of the relative potencies of the different compounds and for differences between the two species. Since it is clear that both 5-HT and DA act via. chloride ionophores on leech Retzius cells, an attempt was made to show that each amine is acting on its own specific receptor (Sunderland et al., 1980). In this study DA was again found to be less potent than 5-HT in the two species but by a greater margin, 4.1 times less potent in the case of Haemopis and 20 times in the case of Hirudo. Dose response curves for the two amines on both species were compared. In the case of Haemopis both amines produced the same maximum hyperpolarisation and the slopes of the two curves are similar, DA is displaced to the right, indicating that it is less potent. In the case of Hirudo, the maximum dopamine response was less than the 5-HT maximum response although the slope of the two curves is similar, the DA curve being displaced to the right. Cross desensitisation occurs with both amines in both species. A comparison was made of the time courses of the responses of the two amines with respect to doses which produced a half maximum response and those which produced a maximum response. The time course of the response to DA and 5oHT in Haemopis and to 5-HT in Hirudo were similar, the time to half maximum being 3--4sec. and that to maximum being 10--15 sec. In Hirudo, the DA response was much slower, the time to reach half maximum hyperpolarisation being 10 sec and to maximum hyperpolarisation being 35--40 sec. Nine compounds were used to try and distinguish the sites of action of DA and 5-HT. Atropine, morphine and strychnine were the most consistent antagonists but they blocked both amines equally. Fluphenazine, metociopramide and ergometrine were less consistent as antagonists but again blocked both amines equally. Methysergide, phentolamine and propranolol were unable to antagonise the actions of either amine. In addition atropine, morphine, fluphenazine and strychnine had direct inhibitory actions while phentolamine had a direct excitatory action. The direct

C9

C~

FIG. 8. Diagram for a joint 5-HT/DA receptor on the leech Retzius cell. A--shows the positions of the active sites of the receptor. Sites 3 and 4 are hydroxyl binding sites, site I is an i~romatic ring binding site and site 2 is a terminal nitrogen binding site. The remaining site represents a third hydroxyl binding site which may be present in H,*emopis but not Hirudo. B--indicates the way in which 5-HT could interact with the receptor. C - indicates the way in which DA could interact with the receptor. D--indicates the way in which 7HT could interact with the receptor (after Sunderland 1978).

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103

inhibitory effect of morphine was antagonised by atropine and fluphenazine while phentolamine and propranolol were ineffective. The evidence from these experiments would suggest rather surprisingly that DA and 5-HT are acting via the same receptor. A model for this joint 5-HT/DA receptor has been proposed (Leake et al., 1980; Sunderland 1978) and is shown in Fig. 8. Figure 8A represents the receptor site where sites 3 and 4 are hydroxyl binding sites, site I is an aromatic ring binding site and site 2 is a terminal nitrogen binding site. The remaining site which is not numbered represents a third hydroxyl binding site which may exist in Haemopis but is not present in Hirudo. In Fig. 8B, the way in which the 5-HT molecule could interact with this receptor is shown while in Fig. 8C, the way in which DA could interact is shown; in Fig. 8D, the possible interaction between 7HT and the receptor is shown. 5.3. OTHER RELATED INVERTEBRATES

5-HT, like ACh and primary catecholamines, appeared early in the course of evolution. It is present in planarians (Welsh and Williams, 1970), onychophorans (Gardner et al., 1978), nematodes (Anya, 1973) and trematodes (Bennett et al., 1969). In planarians 5-HT occurred in cerebral ganglia, ventral nerve cord, the ventral part of the peripheral plexus and in the pharynx. Some 5-HT-containing cells sent processes to the ventral epidermis and may thus be sensory or cilioregulatory. The latter role may be supported by a decrease in locomotion of Dugesia tigrina by reserpine. Depletion of 5-HT by this agent would result in a decreased rate of beating of cilia. The action of melatonin, 5-HT and tryptamine have been tested in terms of their ability to contract pigment cells on the planarian, Dugesia lugubris (Csaba et al., 1980). All three compounds contracted the pigment cells, tryptamine having a slightly greater effect than the other two compounds. The onychophorans Peripatopisis moseleyi and P. sedgwicki show yellow fluorescent (presumed 5-HT-containing) cells and some yellow fluorescent fibre tracts in the ventral nerve cords. Some 5-HT containing fibres were also observed among the body wall muscles (Gardner et al., 1978). 5-HT appears to inhibit isolated body wall muscles, but evokes a mixed rhythmic excitation then inhibition if the nerve cords are intact (Gardner and Robson, 1978, Fig. 7). This suggests an excitatory effect of 5-HT on muscle activity via the central nervous system but a direct relaxation of the body wall muscles. The body wall of another more ancestral phylum, priapulida, does not respond to 5-HT, but the intestine is weakly inhibited and the rectum strongly contracted (Mattisson et al., 1974). The distribution of 5-HT in the rectal ganglion, pharyngeal muscles and contractile portions of the body wall, together with a highly selective uptake process into pharyngeal nerves and two cells in each lateral ganglion in nematodes (Goh and Davey, 1976) suggests that 5-HT may be a neurotransmitter in the nematodes (Anva, 1973). Of particular interest is the role of 5-HT in parasitic flatworms. Although amino acids, particularly lysine (Tomosky-Sykes et al., 1977) may give false positive yellow fluorescence, studies in starved Fasciola hepatica (Mansour, 1979) and in Schistosoma mansoni have indicated the presence of 5-HT (Bennett et al., 1969). In the latter species, 5-HT occurred in the head region near the commissure, in nerve trunks and in storage granules throughout the parenchyma (Bennett and Bueding~ 1971). Intact liver flukes are capable of synthesising 5-HT from 5-HTP via a decarboxylase enzyme but not from tryptophan. It was speculated that 5-HTP or 5-HT itself may be provided to the fluke from the hosts' blood (see review by Mansour, 1979). A high affinity uptake mechanism has been demonstrated for 5-HT in Schistosoma mansoni (Bennett and Bueding, 1973) and similar uptake in schistosomuls is blocked by fluoxetine (Catto and Ottensen, 1978). Fluoxetine, however, has other pharmacological actions in S. mansoni, probably unrelated to the 5-HT uptake mechanism (Pax et al., 1979). 5-HT, related indoleamines and lysergic acid diethylamide (LSD) increased motility of several parasitic flatworms via an action not involving central ganglia (in Fasciola hepatica). The stimulant effects of 5-HT and LSD were blocked by 2-bromo LSD (BOL) in

104

C.R. GARDNER AND R. J. WALKER

F. hepatica and by depletion of 5-HT with reserpine (Mansour, I979; Tomosky et al., 1974). Methysergide and dihydroergotamine may be partial agonists in S. mansoni (see Mansour, 1979). 5-HT and related indoleamines stimulate a GTP-dependent adenyl cyclase in F. hepatica; LSD is only a weak agonist alone but antagonises 5-HT (Northup and Mansour, 1978). BOL is an antagonist. Phosphodiesterase is present and participates in cyclic AMP regulation in the organism. Phosphodiesterase inhibitors increase fluke motility similar to 5-HT but this action may not entirely be due to inhibition of the phosphodiesterase enzyme (Mansour and Mansour, 1977, 1979). This data strongly suggests a neurotransmitter role for 5-HT at some sites, perhaps both central and neuromuscular, in the parasitic flatworms.

6. Amino .Acids 6.1. EARTHWORMS

6.1.1. Gamma-aminobutyric acid (GA BA) Qualitative evidence was found for the presence of GABA in the nervous tissues of Nereis (Dhainaut and Dhainaut, 1974; Dhainaut et al., 1969) but paper chromatographic methods demonstrated little GABA in the nervous system of Eisenia, although GABAtransaminase, which catabolises GABA, was found in a similar concentration to that in mouse brain (Pasantes et al., 1962). It remains possible, however, 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). This latter view is supported by a recent study using a microdansylation technique. Glutamate, glycine, aspartate, alanine and taurine were the predominant amino acids but there was relatively little GABA in the nervous tissue of Lumbricus (Robertson and Osborne, 1979). Using a micro thin-layer chromatographic analysis, a number of amino acids including proline, glutamate, aspartate and taurine were found to be present in the nervous tissue of Nereis (Jost et al., 1981). GABA was only found in sexually mature worms; the reason for this is not clear. The amino acid composition of the leech nerve cord and Retzius cells has been investigated in detail (Osborne et al., 1972). Both the Retzius cells and nerve cord had approximately the same amount of the following amino acids: lysine, ornithine, isoleucine, leucine, phenylalanine, methionine, GABA and arginine. There was more histidine, serine, proline, valine, threonine, glutamine, asparagine, alanine and glycine in the cord compared to the Retzius cells while the converse was true for aspartate and glutamate. Investigations of the role of GABA on neuromuscular transmission in worms have yielded some conflicting results. Nerve stimulation induces inhibitory as well as excitatory junction potentials in Pheretima comraunissima (Hidaka et al., 1969; Ito et al., 1969). Spontaneous inhibitory and excitatory miniature end plate potentials could be recorded. GABA hyperpolarised these muscles and both it and the inhibitory potentials were blocked by picrotoxin (see reviews by Kuriyama et al., 1974; Tashiro and Kuriyama, 1977). However, no strong evidence for GABA-mediated neurotransmission was obtained from similar studies in Pheretima hawayana (although GABA was inhibitory at high concentrations) (Chang, 1975). Eiectrophysiological recordings have not been made in Lumbricus but contractions of the body wall induced by ACh or field stimulation could not be inhibited by GABA (Koidl, 1974; Gardner, 1981a) or muscimol, taurine and glycine (Gardner, 1981a). Similarly, GABA did not inhibit ACh-induced contractions of the body wall of Aherenicola pac!fica (FIorey and FIorey, 1965). Contractions of the body wall musculature of Nereis induced by cholinergic agonists are however, inhibited by GABA (Marsden--personal communication).

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Stimulation of sections of isolated body wall of Lumbricus has failed to provide evidence of an inhibitory mechanism to the longitudinal muscle although one may exist in the innervation of the circular muscle (Drewes and Pax, 1971). A chloride-mediated mechanism in the muscle membranes is suggested by enhancement of contractions of the body wall by picrotoxin (Vereschagin and Sytinskii, 1960). This accumulated evidence in Lumbricus does not lend support to the suggested presence of a functional GABA mediated inhibitory neuromuscular transmission. This raises the possibility that there are considerable differences in neuromuscular transmission in different species of earthworm. GABA, 0.05-10 mM, reversibly depressed the luminescence obtained following electrical stimulation of the epithelial luminescent glands of Chaetopterus notopods (Anctil, 1981). The action of acetylcholine on this preparation was also completely abolished by pre-incubation with GABA. Muscimol was approximately equipotent with GABA in depressing the luminescence but the time for recovery was .~ much longer. Picrotoxin, 0.1-1 raM, potentiated the luminescence elicited by el~trlcal stimulation and partially reversed the depressant action of GABA. Bicuculline had a similar effect but was slightly less potent. Both picrotoxin and bicuculline potentiated the acetylcholine response. This work presents some evidence for a possible role for GABA as an inhibitory transmitter in the control of luminescence in Chaetopterus. Early work of Vereschagin and Sytinskii (1960) showed that picrotoxin enhanced the bioelectricai activity of the earthworm nervous system, suggesting a chloride mediated, inhibitory mechanism. In further studies using isolated sections of Lumbricus terrestris with nerve cord intact Gardner observed a marked and consistent inhibitory effect of GABA on spontaneous rhythmic contractions at concentrations between l0 and 400/~M (Fig. 9). Muscimol was active at similar concentration but its inhibitory effect was much longer lasting, often resulting in the complete cessation of spontaneous activity despite washing the preparation. Taurine and baclofen showed weak and inconsistent responses at similar concentrations to GABA and glycine was inactive (Fig. 9). As GABA has little effect on neuromuscular transmission in this species (Gardner, 1981a) it is probable that this action is via GABA receptors within the central nervous system. Picrotoxin (10-500/~M) and bicuculline (5-500#M) increased the basal tone of preparations and the frequency of the spontaneous contractions. Responses to GABA were attenuated in the presence of either drug but such a change may be due to the increased basal level of activity. Thus, whilst GABA receptors may not be present at neuromuscular junctions of Lumhricus they may be present in the central nervous system. 6.1.2. Excitatory amino acids Glutamate enhanced neuromuscular transmission in Pheretima hawayana (Chang, 1975) but had little effect in Pheretima communissima (Kuriyama et al., 1974). Aspartate and glutamate evoked small increases in basal tone and responses to field stimulation in isolated sections of body wall of Lumbricus at concentrations greater than 0.5 mM

IIll

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I

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J

FIG. 9. Effect of GflBA (closed bars) and glycine (open bar) on spontaneous activity of a section of Lumbricus body wall with intact nerve cord. Drug concentrations are given in ~g/ml. Time marks--2 min.

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(Gardner, 1981a). However, kainic acid was approximately nine times more potent. Similar potency ratios have been observed in mammalian neurones (Biscoe et al., 1976; McLennan and Wheal, 1978) and leech Retzius cells (James et al., 1980). Reasonable reproducibility of kainate-induced contractions allowed investigation of some pharmacological antagonists. Glutamate diethyl ester and diamino pimelic acid (both 0.5-1 mM) had little effect alone and inconsistently reduced responses to kainate, a-amino adipic acid (0.5-1 mM) tended to reduce the responses to field stimulation and also those to kainate. Recovery of kainate control responses was occasionally not obtainable despite regular washing of the preparations. In preparations of Lumbricus with nerve cord intact high concentrations (in the region of I raM) of aspartate and glutamate increased basal tone and frequency of spontaneous contractions. Kainate induced this response at lower concentrations than those which contracted cordless preparations (5--60/~M). This may indicate an additional action of kainate within the central nervous system. Diamino pimelic acid and ~-amino adipic acid had no consistent effect on these preparations at 0.5 mM but evoked increases in basal tone in about half the applications at I mM. There was a tendency to potentiation of responses to kainate with both agents. However, after washing them from the bath, the subsequent responses to kainate were reduced in 309/0 of applications. Excitatory amino acid receptors may be heterogeneous in both mammalian and invertebrate systems (Johnston et al., 1974; Hall et al., 1978; Watkins, 1978; Nistri and Constanti, 1979) and the agents used here may be predominately antagonists of a site which is not activated by kainate. 3H-kainic acid binds specifically to sites in nervous tissue with both high and low affinities. These binding sites have a widespread phylogenetic distribution, being found in species as primitive as hydra and as advanced as man (London et al., 1980). Equal proportions of low and high affinity sites were found in Lumbricus terrestris. Since the seaweed Digenea simplex, of which kainate is the active component, is ascaricidal (Takemoto, 1978) it is also possible that such sites (or receptors) are present in nematodes. The full pharmacological characteristics of these excitatory amino acid receptors remains to be determined. The existence of pharmacological receptors does not necessarily imply a physiological role for an agent. The application of electro-physiological measurements to the effects of these amino acids would help to resolve some of these problems. 6.2. LEECH CENTRAL NEURONES

L-Glutamate can exhibit either an excitatory action or a biphasic action, inhibition followed by excitation, on leech Retzius neurones. The excitatory component of this response has been investigated in some detail but comparatively little has been done on the inhibitory component. The ionic mechanism for the excitatory action of glutamate has been studied by James and Walker (1979) and has been shown to be mainly due to an increase in conductance associated with sodium ions. However, there would appear to be a potassium component but this potassium component appears to be linked to sodium, Chloride does not appear to be involved in the response. However, preliminary studies on the inhibitory component of the L-glutamate response shows that it is a pure chloride event (Manan et al., unpublished) with a reversal potential similar to that found for the chloride equilibrium potential determined from earlier studies using 5-HT and DA which have been described above. The pharmacology of the chloride response is interesting since it is not blocked by picrotoxin in which in many preparations acts as a chloride ionophore blocker. Neither is it blocked by chlorisondamine which again blocks glutamate in other preparations. A structure-activity analysis has been made of the excitatory glutamate receptor (James and Walker. 1979; James et al.. 1980). A number of compounds, that is, kainate, quisqualate, I)L-cis-l-amino-l,3-dicarboxycyclopentane. m.-homocysteic acid and ibotehate were more potent than L-glutamate. 4-Fluoroglutamate. OL-~-methylglutamate, and

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7-methylglutamate were similar in potency to glutamate while DL-~-aminoadipate, L-aspartate and DL-cis-l-amino-l,3-dicarboxycyclohexane were around ten times less potent than L-glutamate. D-glutamate and D-asparate were 25 and 112 times respectively less potent than L-glutamate. L-cysteine sulphinic acid was 68 times less potent than L-glutamate while a large number of other glutamate-like compounds were either inactive or far more than 100 times less potent than L-glutamate. Among the compounds which are inactive is N-methyI-D-aspartate. This study suggested that glutamate is probably interacting with this receptor in an extended form. Of particular interest was the high potency of kainate and quisqualate, where both compounds were more than 100 times more potent than L-glutamate. The actions of kainate and kainate analogues was further investigated on leech Retzius cells (James et al., 1980) where it was found that dihydrokainate was approximately equipotent with kainate as an excitant of leech Retzius cells. Cross desensitisation was observed between kainate and glutamate. Esterification of either kainate or dihydrokainate rendered the compounds inactive as did the addition of a benzyloxycarbonyl group on the nitrogen of either compound. The finding concerning the potency of dihydrokainate is of particular interest since it was suggested that the double bond in the side chain of the kainate molecule was essential for kainatelike activity and certainly in a number of other preparations, dihydrokainate is either inactive or very much less active than kainate. This would suggest that perhaps kainate is acting on Retzius cells at a different receptor site than the one where it acts on some other tissues. A further analysis has been made of the actions of L-glutamate and kainate on leech Retzius cells in an attempt to try and decide whether the two compounds are acting on the same or on different receptors (Walker et al., 1982). ~-Ketokainate was found to have kainate-like activity on Retzius cells but to be about 10 times less active than kainate while the allo form of a-ketokainate was about 3 times less potent than a-ketokainate. Using desensitisation techniques, an attempt was made to try and determine the site of action of kainate. A standard iontophoretic dose of kainate was applied to the cells and then various compounds added directly to the bath containing the preparation and two parameters noted: firstly, the time that the bath applied compound caused an increase in conductance and secondly the time taken for the iontophoretic response to kainate to return to control values. Application of 1 #mole L-glutamate resulted in a conductance change for one minute but the kainate response did not recover for 30 minutes. Application of 20 nmoles carbachol resulted in a conductance change for 2-3 min but then the kainate response returned immediately after this. Application of 10nmols kainate, 2 nmoles quisqualate or 25 nmoles ibotenate caused a conductance increase for 5-8 min but the recovery time of the kainate iontophoretic response varied. In the case following bath application of kainate the recovery was immediate while after quisqualate or ibotenate, the recovery time was 60-90 min. Although these results do not allow a conclusive answer it is clear that there are interactions at the membrane level between L-glutamate and kainate on leech Retzius cells and that the two amino acids may be acting at the same site. Equally it would appear that carbachol is acting at a different site. Another interesting series of glutamate-like compounds has been tested on leech Retzius cells, the homoibotenate analogues and AMPA, RS-g-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (Walker et al., 1982). The potency ratios for homoibotenate, 4-methyl-homoibotenate. 4-bromohomoibotenate and AMPA are 0.37, 0.012, 0.007 and 0.008 respectively, all being more potent than L-glutamate. These potency ratios are approximately in the same order as those obtained for these compounds on mammalian central neurones which suggests that leech Retzius cell glutamate excitatory receptors may resemble those located on mammalian central neurones. 6.3. OTHER RELATED INVERTEBRATES

GABA produces a rapid reversible relaxation of the body wall musculature of Ascaris lumbricoides (Ash and Tucker, 1966) and electrophysiologicai studies have shown a

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hyperpolarising action of GABA (Jarman, 1964; del Castilio et al., 1964, 1967; Brading and Caldweil, 1971), which is similar to the response evoked by stimulating the inhibitory nerves to the muscle. The antihelminthic agent, piperazine, showed a similar response to GABA in evoking a chloride-mediated hyperpolarisation only at the junctional regions of the muscle. Another antihelminthic, the macrocyclic lactone, avermectin Bta, blocks the action of GABA in the lobster but does not do so in Ascaris (Fritz et al., 1979). The paralytic action of avermectin Bta on Ascaris was therefore proposed to be via another mechanism of action. Picrotoxin has been shown not to block GABA-induced relaxation of Ascaris muscle strips (unpublished observations cited in Fritz et al., 1979). However, a study employing microiontophoretic applications to the bag region of Ascaris suum muscle showed antagonism of GABA by bicuculline, picrotoxin and benzylpenicillin (Martin, 1980). Of interest is an observed lack of bicuculline sensitive 3H-GABA binding sites in a range of invertebrates, including planarians (Mann and Er~na, 1980). These authors suggested that this may be due to different biochemical and/or pharmacological characteristics of vertebrate and invertebrate GABA receptors. The actions of GABA and glycine have been investigated on electrically evoked activity recorded from the submuscular ventral longitudinal nerve cord of the polyclad flatworm, Notoplana (Keenan et al., 1979). Both compounds at concentrations of 1-10 ~UMdepressed activity and this effect was reversed on washing. In general the preparations were less sensitive to glycine than to GABA. The action of GABA and glycine were non-specifically blocked by picrotoxin, bicuculline methiodide and strychnine. Picrotoxin and strychnine have direct excitatory effects on the nervous system while in general bicuculline is devoid of direct actions. Pentylenetetrazol, 100/ZM, increases the amplitude of the evoked response and induces some spontaneous discharge in the nerve cord. This type of firing pattern is also sometimes observed with strychnine and bicuculline but not with picrotoxin. However, these authors could not implicate GABA or glycine as the neurotransmitter of a tonic inhibitory system from the brain to the nerve cord. A series of amino acids and adenosine were tested for their ability to contract the body wall musculature of the echiuroid, Urechis unicinctus (Muneoka et al., 1981). The most potent amino acid was L-proline with a threshold of 10-6 M. Equipotent molar ratios for the other amino acids were compared to proline, which was taken as unity. D-Proline, hydroxy-L-proline, sarcosine, glycine, L-alanine, fl-alanine and taurine had respectively the following E P M R values: 333, 117, 18, 167, 192, 33 and 117. A large number of amino acids and bases failed to elicit a contraction, including aspartate, glutamate, GABA, adenosine and ATP. Pretreatment of the muscle with eserine, 10-SM, enhanced the response to L-proline while probantine depressed the response. The contractions to amino acids were either abolished or greatly depressed by high magnesium Ringer. The amino acids only produced contractions when they were applied to the outer surface of the body wall strip. It is suggested that the amino acids act indirectly to induce twitch by stimulating the epidermal chemoreceptor organs which are assumed to be connected to the body wall musculature via a subepidermal nerve network.

7. Peptides With the introduction of radioimmunoassay and immunocytochemical techniques there is now considerable evidence for the occurrence of vertebrate neuro- and gut peptides in the invertebrates, including the annelids. Using immunocytochemical techniques Sundler et a/. (1977) demonstrated the presence of bovine pancreatic polypeptide (BPP)-like material and vasoactive intestinal peptide (VIP)-Iike material in the central nervous system of Lumbricus. Soma showing immunoreactivity to VIP were located in the suboesophageal ganglion. Immunoreactive fibres were present running in the cord through the first ten segments. Soma showing immunoreactivity to BPP were present in the cerebral ganglion but there were no VIP reactive cells in this ganglion. BPP soma

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were also present in the suboesophageal ganglion and in other ganglia of the cord. BPP reactive fibres occurred in ganglia below the tenth segment indicating a wider distribution than for VIP. Cell bodies showing reactivity to BPP and VIP were located in different parts of the ganglion. No cells showed reactivity for both peptides and no fibres showed reactivity outside the cord. The presence of peptide containing neurones in the nervous system of annelids and in particular, L,mbricus, has been discussed by Oksche (1978; 1982). in initial studies no evidence was found for either opiate binding activity or enkephalin activity in a number of invertebrates (Pert et al., 1974: Simantov et al., 1976). However, more recently evidence has been obtained for two classes of opiate receptors in the nervous tissue of a bivalve mollusc, Mytilus (Stefano et al., 1980; Kream et al., 1980). Additionally opiate binding has been identified in'the brain of Locusta and Helix (S.-Rozsa et al., 1981) and of Leucophaea maderae using either 3H-naloxone or 3H-D-AiaMet-enkephalinamide as iigand (Stefano and Scharrer, 1981). Evidence has also recently been obtained for enkephalin and ]~-endorphin in the central nervous system of L,~mbricus (Alumets et al., 1979). Many neurone cell bodies and fibres showed intense enkephalin immunoreactivity in the cerebral ganglion. A few immunoreactive neurone bodies and fibres were also present in the subpharyngeal ganglion and abdominal ganglion. The distribution of cells showing immunoreactivity for//-endorphin had a similar pattern of distribution but there were fewer of them and they tended to be larger. The cell bodies which contained enkephalin were distinct from those containing ]~-endorphin. This study demonstrates that there are neurones in Lumbricus which contain either enkephalin or 18-endorphin or closely related peptides. It is therefore possible that Lumbricus contains enkephalin receptors. Evidence has also been obtained for cells in the suboesophageal ganglia of the earthworm, Dendrobaena, to contain a-endorphin. No immunoreactivity was found to/~-endorphin, neurophysin or vasopressin in this species (Remy and Dubois, 1978). Neurosecretary cells have long been known in earthworms and initial theories suggested that terminals of such cells release peptide "hormones" into the vascular system as the hypophysis does in mammals (Aros and Vigh, 1961). However, recent studies have revealed synaptic terminals of these cells both centrally and in the periphery (Aros et al., 1977). Presumed peptide-containing terminals contained large granular vesicles (diameter more than 1500 ]~). Using immunoreactive techniques, Zipser (1980) has localised enkephalin in a specific neurone in the posterior two thirds of the segmental ganglia of the leech Haemopis. In the anterior ganglia, immunoreactive varicosities but not cell bodies, were located. This technique has been extended to distinguish identifiable neurones in the leech nervous system (Zipser and McKay, 1981). No physiological roles have yet been proposed for synaptic effects of peptides in annelids. Morphine suppresses responses of Lurnhricus body wall to field stimulation (Gardner, unpublished observation) but further studies are necessary to demonstrate sites of action and function of potential neuropeptide transmitters. This would seem to be a valuable future avenue of research.

8. Other Agents 8.1. BENZODIAZEPINES

Specific binding of benzodiazepines (so-called benzodiazepine receptors) has been observed in a wide range of vertebrates, but not in invertebrates, including the earthworm (Nielsen et al., 1978) and the leech (Corradetti et al., 1980). The actions of benzodiazepines in mammals have been associated with a potentiation of GABA-mediated neurotransmissions, the system being closely associated with a chloride ionophore (Costa et al., 1979). However, no such mechanism has so far been detected in anndids, or other invertebrates that have been investigated. Benzodiazepines do not appear to act via a GABA system (Corradetti et al., 1980--flurazeparn, chlorazepate), and they do not potentiate GABA on neurone R I5 of Aplysia (Tremblay and Grenon, 1980--chlordiazepoxide, flurazepam) or in lobster muscle (Nistri and Constanti, 1978--flurazepam).

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C.R. GARDNER AND R. J. WALKER

This group of agents does, however, have a variety of pharmacological actions in invertebrate preparations. Flurazepam and chlorazepate have an initial excitant, then a marked depressant, effect on leech behaviour (Corradetti et al., 1980) and flurazepam induces rhythmic contractions in longitudinal muscle strips of the earthworm, Lumbricus terrestris with no consistent effect on responses to field stimulation. G A B A has no effect in this preparation (Gardner, 1981b). The mechanisms of these effects remain to be elucidated, but block of sodium channels has been suggested for their reduction of glutamate responses on lobster muscles (Nistri and Constanti, 1978) and block of axonal conduction in crayfish and squid giant axons (Wang and James, 1979). A series of soluble benzodiazepines have a range of actions on central neurones of Aplysia. Reduction of overshoot and increases in the amplitude of the action potential occur in several neurones with oscillatory firing in some cases (Hoyer et al., 1976). ACh-induced depolarisations and chloride-mediated hyperpolarisations were reduced whilst potassium-dependent hyperpolarisations were largely unaffected (Hoyer, 1977). Pre- and post-synaptic actions on neurotransmission and non-synaptic neuronal membrane effects have been implicated in these responses (Tremblay and Grenon, 1980; Hoyer, 1977). Some benzodiazepines (e.g. Ro 11-3128, 3-methylclonazepam and clonazepam) exert, in addition to their central mammalian actions, a schistosomicidai effect (Pax et al., 1978; Stohler, 1978). Ro 11-3128 produces a sustained contracture of the musculature of schistosomes (Pax et al., 1978), an effect which is not mediated via the sites which bind 3H-benzodiazepines in mammalian brain (Mohler et al., 1981). The schistosomicidal effect appears to be mediated via low affinity benzodiazepine binding sites on the epidermis of schistosomes which can be labelled with 3H-Ro 11-3128 (Bennett, 1981). In view of these actions in invertebrates, the effects of benzodiazepines in mammals should, perhaps, be studied with wider pharmacological considerations. 8.2. CONVULSANTS. BARBITURATES AND TETRAETHYLAMMONIUM

During the past ten years Prichard and his colleagues, particularly Kieinhaus have studied the action of convulsants and barbiturates on leech Retzius cells and to a lesser extent on sensory neurones. Bemegride, 10 mM, was shown to induce paroxysmal bursts in leech Retzius cells (Prichard, 1972b). Shortly after the application of bemegride (36 sec-9.5 min) the firing rate of the cell increased and became less regular and paroxysms of rapid firing associated with variable depolarisations began to occur at irregular intervals. The action of bemegride was reversible, the cell activity returning to normal one minute following the removal of bemegride. The action of this compound on Retzius cells appears to be indirect since the appearance of paroxysms can be prevented by using either 20 mM magnesium Ringer or 0.1 mM atropine. Penicillin is also capable of inducing paroxysmal discharge in leech ganglia (Prichard and Glaser, 1972). These findings are summarised by Kleinhaus (1975). Pentylenetetrazol, 1-10 raM, produces a direct hyperpolarising effect on Retzius cells and this action is associated with an increase in chloride conductance. This effect persists in the presence of 20 mM magnesium. The hyperpolarising effect is preceded by an initial depolarisation which is blocked in high magnesium Ringer (Prichard, 1971a). Prichard (1971b) also found that strychnine, 0.1-1 mM, has a hyperpolarising action on Retzius cells which is mediated via an increase in chloride conductance. Phenobarbital, 10 mM, initially excites and then inhibits Retzius cell activity (Prichard, 1972a). The excitatory phase disappears in high magnesium Ringer but the inhibitory phase persists. The inhibitory phase is associated with an increase in permeability to potassium ions. This dual action of phenobarbital was further analysed (Prichard and Kleinhaus, 1974) and it was found that 0.1-10 mM produced large excitatory post synaptic potentials (epsp) while 1.0-10 mM produced hyperpolarisations. In contrast, two other barbiturates, pentobarbital and secobarbital depolarise Retzius cells and increase firing rate via an increase in resistance, i.e. a decrease in conductance (Kleinhaus and Prichard, 1977c). All the barbiturates tested increased the duration of the action poten-

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tials. Both actions of pentobarbital were more obvious at pH 6.8 than at 8.8. This increase in action potential duration was further analysed (Kleinhaus and Prichard, 1977a). They found that the sodium salts of phenobarbital, barbital, pentobarbital, secobarbital, methohexital and thiopental, 1.0-5.0 mM, all induced prolonged actions potentials. The prolongation of the action potentials was enhanced in low calcium but reduced in high calcium. These prolonged action potentials were unaffected by 50/~m tetrodotoxin or chloride free Ringer but were dependent on external sodium. It is suggested that the barbiturates block a voltage dependent inward calcium current which activates a potassium conductance necessary for normal repolarisation. Kleinhaus and Prichard (1979) extended this study to a comparison of the interaction of barbiturates and divalent cations on sensory cells in the leech. They found that barbiturates effect the action potential in pressure (P), noxious (N) and touch (T) cells in a manner similar to that found in Retzius cells. They suggest that the barbiturates act via a calcium-sensitive repolarisation process normally present to varying degrees in the different cell types. Externally applied tetraethylammonium (TEA), 10-25 mM, greatly prolongs the action potentials of leech Retzius cells and also at the higher concentrations increases their amplitude (Klcinhaus and Prichard, 1975). Both actions are easily reversed on washing. Intracellularly applied TEA depolarises the membrane potential in addition to its action on the action potentials. In the absence of TEA, Retzius cell action potentials are sodium-dependent but it would appear that the prolonged action potentials found in the presence of external TEA have a large calcium component. Kleinhaus and Prichard suggest that TEA induces a voltage sensitive, slowly activated calcium current to become a major factor in the inward current of the action potential, probably by blocking the outward potassium current which normally counteracts it. This study was extended to the sensory T, P and N cells (Kieinhaus and Prichard, 1977b). TEA was found to prolong the duration of the action potentials in the order Retzius > N > P > T cells. In the presence of TEA but in the absence of sodium, the N and P cells exhibited a prolonged action potential with a calcium dependent component similar to that previously described for the Retzius cells. Such an event was not observed in the T cell.

9. General Conclusion From the evidence discussed in this review it is likely that acetylcholine is a motor excitatory transmitter onto body wall muscle in the leeches and probably throughout the annelids. While a central transmitter role for this substance is less clear, if one makes a comparison with other phyla such as arthropods and molluscs, then it remains a strong possibility. One or more catecholamines are clearly present in the annelids and of these the most probable transmitter is dopamine while there is little or no evidence for adrenaline. Metabolic studies suggest that annelid nervous tissue is capable of synthesising noradrenaline and it is likely that this amine is an endogenous catecholamine in this phylum. However there is not yet clear evidence for a physiological role for catecholamines in the annelids. It is probable that both dopamine and the related amine octopamine do have central roles in at least the leeches. The evidence suggests a physiological role for 5-HT in the regulation of body wall muscle tone in the leech and in addition 5-HT probably has a role in the CNS as an inhibitory transmitter. 5-HT is also present in many of the other groups discussed in this review and is likely to have similar physiological functions in them. GABA is probably present in annelids but its physiological role is far from clear. Glutamate and related amino acids excite annelid muscle and leech central neurones but again no clear physiological role has been demonstrated. Though by analogy with other phyla it is highly probable that glutamate and GABA have at least central transmitter roles in the annelids. A number of vertebrate peptides or related material including enkephalin have been found in annelid nervous tissue and so these compounds must be considered seriously as candidiates for a physiological role in this phylum. As can be seen from the above comments the neuropharmacology of this

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g r o u p o f a n i m a l s is still in its i n f a n c y a n d t h e r e is c o n s i d e r a b l e s c o p e for r e s e a r c h at all levels.

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