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Evolutionary aspects of transmitter molecules, their receptors and channels R. J. WALKER and L. HOLDEN-DYE Department of Physiology and Pharmacology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO9 3TU

Key words: transmitters, receptors, ion channels. INTRODUCTION

Multicellular organisms such as animals, fungi and plants evolved from unicellular or acellular eukaryote protists which in turn evolved from monera (bacteria) prokaryotes (Fig. 1). Prokaryotes do not possess DNA in chromosomes, do not have a nuclear membrane and their flagella lack the 9 + 2 microtubular organization of eukaryotes. In contrast, eukaryotes possess a nuclear membrane, cytoplasmic microtubules, a flagellum of the 9 + 2 structure and have mitochondria and plastids, together with DNA in chromosomes. It is possible that a permanent symbiotic relationship of a number of prokaryotes produced a eukaryote, i.e. a chimaera - the intracellular organelles of eukaryotes being individual prokaryote organisms. It is likely that animals evolved at least three times, i.e. Porifera (sponges), Coelenterates (cnidaria-jellyfish, Hydra and ctenophores-sea gooseberries) and the Platyhelminthes (flatworms) (Fig. 2, after Barnes, Calow & Olive, 1988). From these flatworm-like ancestors arose all the known phyla around 600 million years ago (Fig. 3). There are around 28 phyla, depending on the classification adopted, but most people agree on the major phyla, the subject of this review: Platyhelminthes, Nematodes, Arthropods, Molluscs, Annelida, Echinoderms and Chordates. This evolution began over a billion years ago, probably over 1*5 billions ago, but at this level what is half a billion years more or less ? All is relative in this world and for us it is difficult to appreciate even 100 years let alone 1 billion years. So in summary in terms of time let us suggest the following: prokaryotes (Monera) 3-5 billion; eukaryotes (Protista) - 1-4 billion; all known phyla - 0-6 billion; chemical transmitters1-0 billion. We can suggest that the chemicals and ion channels involved in transmission had largely evolved by 1 billion years ago since the platyhelminths possess all the normal transmitter molecules and probably also the ion channels.

Plantae 27500+

Several million sp.

Fungi 43000 Algae/protozoa 120000

Fig. 1. Evolution of fungi, plants and animals from Protista which in turn evolved from Monera.

Coelenterates

Phyla '28'

Protista (algae/protozoa) 120000

Fig. 2. Evolution of Porifera, Coelenterates and the remaining phyla from Protista.

Chelicerata 63000

Nematodes

15000 Platyhelminthes /^~\Nemertea" 25000 V. \S0'

Annelida. Echinodermata 15000^-1 6000

CLASSIFICATION OF TRANSMITTER MOLECULES

We should begin by defining a transmitter within the

Parasitology (1991) 102, S7-S29 Printed in Great Britain

Fig. 3. Evolution of the major phyla from a flatwormlike ancestor. The approximate number of species in each group is indicated.

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R. J. Walker and L. Holden-Dye Table 1. List of putative transmitters and modulators Choline esters: Catecholamines: Indoleamines: Imidazoleamine: Phenolamines: Purines: Excitatory amino acids: Inhibitory amino acids: Peptides:

acetylcholine dopamine, noradrenaline, adrenaline tryptamine, 5-hydroxytryptamine histamine octopamine adenosine, adenosine triphosphate L-glutamic acid, L-aspartic acid gamma-aminobutyric acid, glycine, taurine angiotensin, adrenocorticotropic hormone, arg-vasopressin, arg-vasotocin, ascaris heptapeptide, bombesin, carnosine, cholecystokinin, catch-relaxing peptide, endorphins, egglaying hormone (gastropod), enkephalins, FMRFamide family, gastrin, glucagon, head activating peptide, insulin, luteinizing hormone releasing hormone, motilin, neuromedins, neuropeptide Y, neurotensin, oxytocin, proctolin, pancreatic polypeptide, somatostatin, substance P, small cardiac peptide A and B, thyroid hormone releasing hormone, vasoactive intestinal peptide

context of this review. A transmitter is a chemical which is released from a neurone, often at the nerve terminal, which then diffuses across a relatively small gap, the synapse, usually 20—50 ran, and interacts with a specific part of the membrane of the follower cell, the receptor. This transmitter or ligand interaction is responsible for a change in the conformation of the follower cell membrane which generally results in a change in permeability of the membrane and a change in membrane potential. Substances can be released which diffuse across greater distances before acting on the effector cell and these can be termed local hormones. Chemicals may be released from neurones which modify synaptic transmission induced by a primary transmitter, these can be termed modulators. It is likely that at least certain neuropeptides released from nerve terminals may act in this way. It is convenient to classify transmitters and modulators into two groups, so-called classical transmitters and neuroactive peptides. Purines also act as transmitters or modulators at certain sites. A list of compounds which may have a transmitter/ modulator role are given in Table 1. T h e classical transmitters can be divided into a number of groups. One of the first compounds to be considered as a transmitter was acetylcholine (Fig. 4). This compound is synthesized from choline by the enzyme, choline acetyltransferase, and once released is rapidly degraded by acetylcholine esterase. Both enzymes act as important markers for the presence of acetylcholine activity, though choline acetyltransferase is the more selective marker for the identification of a cholinergic synapse. Uptake sites on nerve terminals for choline can also be used as markers for acetylcholine as a potential transmitter. Although there are many esters of choline, some of which occur naturally, acetylcholine is the only one which is a transmitter. Acetylcholine acts as a transmitter at many sites and there is evidence that the characteristics of the receptor differ from one site to another. Based on studies using agonists and

COO

coo

1 CH

CH2

2

1

CH

CH 2 NH 3 +

CH NH 3 +

COO'

COO"

L-glutamic acid

L-aspartic acid

NH2CH3COOH

NH2(CH2)3COOH

Glycine

GABA

CH3 . CO . O . CH2 . CH2 . N + (CH3)3 Acetylcholine

Fig. 4. Structural formulae of acetylcholine, L-glutamic acid, L-aspartic acid, y-aminobutyric acid (GABA) and glycine. antagonists, it has been shown that there are two basic acetylcholine receptor types, one where nicotine is an agonist, the 'nicotinic' site, and one where muscarine functions as an agonist, the 'muscarinic' site. These sites have subsequently been subdivided. Both nicotine and muscarine are agonists on many invertebrate tissues and these ligands are usually antagonized to varying degrees of selectivity by tubocurarine and atropine, respectively. Although acetylcholine activates both nicotinic and muscarinic receptors there is no evidence that these receptors are closely related (Venter et al. 1988). The two proteins differ in terms of size and structure, and antibodies against one receptor do not interact against the other. This lack of similarity is also true at the functional level where nicotinic receptors are linked to fast ion channels while muscarinic receptors are linked to second messengers. This classification forms a useful basis to which we shall return later. Another group of compounds which function as transmitters are certain amino acids. These can be divided into two groups, dicarboxylic amino acids

Evolutionary aspects of transmitter molecules OH . (OH). CH2 . NH 2

0 H

Octopamine

Dopamine

OH

OH OH-^^-CH.(OH).!

0 H

- \ 3 " C H (OH)CH2 . NH Adrenaline

Noradrenaline

OH

H2 . CH2—NH2 H C = C — C H 2 . CH2NH2 H

Serotonin

H Histamine

Fig. 5. Structural formulae of dopamine, octopamine, noradrenaline, adrenaline, serotonin (5-HT) and histamine. which are generally excitatory and monocarboxylic amino acids which are generally inhibitory for vertebrate neurones. Examples of the former are Lglutamate acid and L-aspartic acid while examples of the latter are gamma-aminobutyric acid (GABA) and glycine (Fig. 4). In addition to these amino acids, taurine and /?-alanine have also been proposed as putative inhibitory transmitters in vertebrates. Catecholamines form a group with three members which are transmitters, dopamine, noradrenaline and adrenaline (Fig. 5). Together with acetylcholine, adrenaline was one of the earliest compounds to be considered to have transmitter function. Although adrenaline has this role in certain vertebrate groups at the post-ganglionic sympathetic synapse, e.g. amphibia, noradrenaline is the transmitter released at this site in mammals. Adrenaline also has an important role as a hormone, released from the adrenal medulla. All three compounds have transmitter function in the vertebrate central nervous system. Interestingly, while dopamine is an important transmitter in invertebrates, noradrenaline functions very much as a trace amine, while there is little or no evidence for adrenaline as an invertebrate transmitter. There are three other amines which have roles as transmitters, i.e. 5-hydroxytryptamine (serotonin or enteramine), histamine and octopamine (Fig. 5). 5Hydroxytryptamine (5-HT) is a central transmitter in vertebrates and both a central and peripheral transmitter in invertebrates. In many examples it replaces noradrenaline as an excitatory transmitter for organs of invertebrates. Apart from 5-HT, tryptamine also occurs in the vertebrate central nervous system and is probably a transmitter, though such a role in invertebrates is far from certain. Histamine has a role as a central vertebrate trans-

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mitter and is present in many tissues in vertebrates. This amine is also present in invertebrates and is a transmitter in gastropod molluscs and in the visual system of arthropods. The final amine is octopamine which has important transmitter and neurohormonal roles in invertebrates, but a similar function in vertebrates has not yet been demonstrated. Using histochemical techniques, Grimmelikhuijzen (1986) failed to identify catecholamines or 5H T in Hydra neurones nor could acetylcholine be detected. So at least some coelenterates do not contain detectable amounts of classical transmitter molecules. There is now excellent evidence, especially from the work of Burnstock (1985), that purines such as adenosine and adenosine triphosphate act as transmitters, certainly in the periphery and also probably in the central nervous system. There is a vast literature on the presence and possible functions of neuroactive peptides in both vertebrates and invertebrates. There are now over 30 peptides which are present in and are likely to be released from neurones (Table 1). A number of these neuropeptide-like materials have even been identified in unicellular organisms including Clostridiwn, Rhodopseudomonas and Tetrahymena. These neuropeptides include adrenocorticotropic hormone, neurotensin, insulin, somatostatin, /?-endorphin, relaxin, arginine vasotocin and calcitonin (LeRoith et al. 1983, 1986). In many cases, these peptides coexist with classical transmitters and it is likely that virtually all, if not all, neurones contain at least two compounds which can be released following nerve stimulation. We shall return to this later in the review. In some cases, more than one peptide is present in a single neurone, e.g. a substance P-like material and an arg-vasopressin-like material in Helix neurones (Walker, Boyd & Osborne, 1987); small cardiac peptide (SCP) B and an FMRFamidelike peptide in Aplysia neurones (Lloyd et al. 1987); SCP-B and an FMRFamide-like peptide in Crustacea (Callaway, Masinovski & Graubard, 1987) and F L R F - and vasopressin-like immunoreactivity in locust neurones (Evans & Cournil, 1990). T h e evidence for two or more classical transmitters being present in a neurone is less common but does occur, e.g. dopamine and 5-HT are both present in the giant cerebral neurone of Lymnaea (Boer et al. 1984), as is a substance that shows immunoreactivity to a vasotocin antibody; 5-HT and GABA can both occur in leech Retzius neurones (Cline, 1986). PROPERTIES OF A SYNAPSE

A typical synapse is shown diagrammatically in Fig. 6. A synapse acts as a barrier to synaptic transmission from one axon to another. There must be a mechanism to activate transmission across this barrier. There are two possible mechanisms, one

R. J. Walker and L. Holden-Dye

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many cases, in addition there is an uptake system for the transmitter itself. Physiologically, it must be shown that on pre-synaptic stimulation the chemical Release is released and acts on a specific component of the post-synaptic membrane, the receptor. This stimuReceptor lated release should be calcium-dependent and so it can be shown that in the absence of calcium no release occurs. Pre-synaptic stimulation will produce a postsynaptic response which can be recorded. The putative transmitter must be applied to the postVesicle synaptic membrane and shown to have the same or for storage very similar action to the response following presynaptic stimulation. These responses to the endoFig. 6. The basic organization of a synapse. genous and exogenous compound should have the same mechanism of action, e.g. the same ion conductance changes. If this is the case then the two electrical and the other chemical. Electrical synapses responses will have the same reversal potentials, i.e. have a very small gap between the pre- and postsynaptic membranes (around 1-2 nm) and are the membrane potential at which there is no apparent response. Changing the gradient for the ion(s) termed 'tight' junctions. They occur in the central involved will change the reversal potential for both and peripheral nervous systems in both vertebrates events to the same degree. Alternatively, the reand invertebrates. For example, in the crayfish there sponses may involve second messengers, such as is an electrical synapse between the lateral giant fibre cAMP, and potentiators or blockers of this system and the giant motor fibre (Furshpan & Potter, 1959) will induce changes in both responses. The exoand between the Retzius cells of the leech (Eckert, genous compound may be applied directly to the 1963); in the mammalian brain, in the mesencephalic preparation, ionophoretically or by pressure innucleus of the Vth cranial nerve (Baker & Llinas, jection. Finally, pharmacological tools can be used in 1971), in Deiter's nucleus (Korn, Sotelo & Crepel, the elucidation of the transmitter e.g. an antagonist 1973), and in the inferior olive (Llinas, Barker & which should block or reduce the stimulated reSotelo, 1974). sponse and the exogenously applied response. These In the fish brain there are electrical synapses onto antagonists should be specific or at least reasonably the giant Mauthner cells (Retzlafe, 1957). Synapses selective for the post-synaptic receptor. Compounds normally rectify, i.e. they only transmit information which also block ion channels can be of value. Once one way, from the pre- to the post-synaptic memthe identity of the transmitter is fairly clear then brane. Clearly, where a chemical is involved, in other blocking agents can be used which block transmission and only present in the pre-synaptic synthesis, storage, release, degradation or uptake of membrane then transmission can only be unidirecthe transmitter and the effect on the post-synaptic tional. There is always a delay at the synapse and this response following stimulation noted. Following this delay at a chemical synapse is 0-7-1-0 ms. At approach a chemical can be identified with a high electrical synapses the delay is shorter, being around degree of certainty. One of the more difficult criteria 100 fis. Transmission is rarely 1:1, usually a series of to fulfil is demonstration of release from central pre-synaptic potentials are required to release sufsynapses but this can be overcome in a number of ficient chemical to induce an action potential in the ways. For example, the use of labelled precursors or follower cell. Before a chemical can be considered to be the transmitter, use of slice preparations, use of pushpull cannulae and cups or the use of voltammetric transmitter at a particular synapse, a number of techniques. In summary, the requirements can be criteria have to be fufilled. These can be considered abbreviated to: presence, synthesis, release, inacunder a series of headings, e.g. anatomical, biotivation, identity of action and specific receptors chemical, physiological and pharmacological (Shep(Bradford, 1986). These are summarized in Table 2. herd, 1988). Firstly, in terms of anatomy, the chemical has to be shown to be present in the presynaptic terminal, usually contained within discrete VOLTAGE-GATED CHANNELS AND LIGANDorganelles, termed vesicles. Secondly, in terms of GATED ION CHANNELS biochemistry, enzymes must be present in the presynaptic terminal to synthesize the chemical from its Voltage-gated ion channels are channels whose precursors. This may involve a single step, e.g. in the permeability changes as the membrane potential changes, e.g., when the membrane potential is case of acetylcholine or may involve a series of steps, depolarized there is a large and rapid increase in e.g. for the synthesis of adrenaline. A pre-synaptic permeability to sodium ions. Voltage-gated sodium, uptake system must exist for the precursor(s) and, in Nerve terminal

Post-synaptic membrane

Evolutionary aspects of transmitter molecules

Sll

Table 2. Criteria to be satisfied before a compound can be considered a transmitter at a synapse (1) Presence in pre-synaptic terminal, usually within vesicles. (2) Systems for uptake of precursor molecule(s) and enzymes for synthesis of chemical. (3) Calcium-dependent release of chemical following activation of presynaptic cell. (4) Identity of action of endogenously and exogenously applied chemical via a specific part of the post-synaptic membrane, the receptor. (5) Selective antagonists to block responses to endogenous and exogenous application of chemical via the same mechanism. (6) Systems for the inactivation of the chemical following ligand—receptor interaction.

Table 3. A summary of voltage-gated and ligandgated ion channels Voltage-gated channel

Permeant ions

Sodium Potassium Calcium I-h or I-f Ligand-gated channel receptor Nicotinic Glutamate A T P P-2 subtype 5-HT-3 GABA-A Glycine

Na + K+ Ca2+ Na + , K + Small Small Small Small

Chelicerata Crustacea

Uniramia

cations cations cations cations

cici-

potassium and calcium channels are selective for their particular ion (Table 3). Another channel which is similar in properties is the I-h or I-f channel which is permeable to several small cations (Hille, 1989). Some chloride channels are also influenced by voltage changes but it is likely that these are not closely related to the cation channels. These voltagegated channels occur widely and are present in all the major phyla examined (Fig. 7). It is likely that they occur in all cells of eukaryotes (Hille, 1984). In excitable tissues such as nerve and muscle and in glands they are responsible for rhythmic, pacemaker activity, normally associated with a high permeability to sodium, i.e. a sodium leak current. They are also responsible for propagated action potentials and secretory events. Certain toxins have been employed in elucidating the properties of these channels, e.g. tetrodotoxin (TTX) and saxitoxin (STX) for sodium, w-conotoxin for L-type calcium channels and charybdotoxin for the calcium activated potassium channel. There are many substances which block calcium channels including inorganic ions such as cadmium, cobalt, manganese, nickel and lanthanum and organic compounds such as verapamil and the dihydropyridines, e.g. nifedipine. Calcium channels are normally closed at resting membrane potentials and consist of a number of subtypes including I-C which is responsible for the

Fig. 7. Occurrence of sodium and calcium channels in major groups.

pre-synaptic release of vesicular transmitters and Ltype, T-type and N-type. There is also a very slow IB calcium current which is associated with the slow bursting of some pacemaker cells and a number of potassium channels including, I-K, I-DR (delayed rectifier), I-C (calcium-dependent channel), I-A (fast transient)1, I-AR (anomolous rectifier) and I-M (M current). I-K activates very slowly with depolarization and is important in maintaining the resting potential. Based on cDNA clone analysis three sodium channels which are approximately 90 % homologous have been isolated from rat brain (Joho et al. 1990; Maue et al. 1990). These are summarized in Table 4. The voltage-gated channels have considerable homology in their amino acid sequence and consist of a single continuous polypeptide of about 2000 amino acids. The sequence includes four internal repeats which may aggregate as a square to form a pore. In addition to the main subunit there may also be a number of small subunits. It is likely that sodium, calcium and potassium channels are similar in these respects though the initial work was performed on the Electrophones sodium channel (Noda et al. 1984). LIGAND-GATED CHANNELS

There are a number of ligand-gated ion channels

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Table 4. Summary of cation channel types and some of their properties Sodium Type I Sodium Type II Sodium Type III Calcium Type I-C

Inactivating; tetrodotoxin-sensitive Slow-inactivating Very slow inactivation Unitary conductance 20-40 pS; block with Co, Mn, Cd, Ni, La Long-lasting, i.e., unitary; non-activating conductance Calcium Type L 25 pS Calcium Type T Small unitary conductance 9 pS; transient i.e., rapid inactivation Unitary conductance 13 pS; slow inactivation Calcium Type N Very slow current Calcium I-B Potassium I-K Very slow activation; unitary conductance 20 pS; blocked with tetraethylammonium (TEA), 4aminopyridine (4-AP), Ba, Co Potassium I-DR Very slowly inactivates; unitary conductance 10 pS; (Delayed rectifier) blocked with TEA, 4-AP, Ba, Co Potassium I-C Unitary conductance 10-20 pS; blocked with apamin, (Calcium-dependent) charybdotoxin Unitary conductance 10 pS; rapidly activated and Potassium I-A inactivated; blocked with 4-AP Turns off when depolarized and on when Potassium I-AR (Anomalous rectifier) hyperpolarized Does not inactivate Potassium I-M

including nicotinic acetylcholine, excitatory amino acid (primarily L-glutamate), GABA-A, glycine, purine P-2 (ATP), and 5-HT-3 ion channel (Table 3). These channels are responsible for very rapid events associated with inhibition or excitation usually at the synapse. The events are usually associated with the opening of channels, i.e. an increase in conductance and decrease in resistance but they can operate in the reverse, i.e. induce a decrease in conductance and increase in resistance. The channel and the ligand-binding site or receptor are components of the same transmembrane macromolecular protein complex, each receptor being associated with its own population of ion channels. It had been postulated that ion channels might be linked to more than one receptor in gastropod neurones (Swann & Carpenter, 1975) though subsequent work also using gastropod neurones suggested that each receptor had its own population of ion channels (Chad & Kerkut, 1981). However, a receptor can be linked to more than one type of ion channel on the post-synaptic membrane. This brings us to the concept that on many post-synaptic membranes there may be a potential to activate more than one type of ion channel and hence potentially different responses. For example, with gastropod neurones, acetylcholine can either excite or inhibit depending on the concentration applied (Wachtel & Kandel, 1971). For example, when the cholinergic interneurone L10 of the abdominal ganglion of Aplysia is stimulated at a slow rate, each action potential induces an epsp in the follower neurone L-7. If the rate of stimulation of L-10 is increased then the epsp fades and is replaced by an ipsp. Acetylcholine itself produces a

biphasic event, a brief depolarization followed by a sustained hyperpolarization on L-7. These events can be explained by postulating that the presence of two acetylcholine receptors on L-7, one containing a sodium channel within its macromolecule and one containing a chloride channel within its macromolecular configuration. The excitatory response is sodium mediated while that for inhibition is chloride mediated. Biphasic responses are common with many invertebrate neurones and can probably be associated with all or certainly most ligands. We now come to the question of the number of receptor subtypes for a ligand. Agonist and antagonist pharmacological studies suggest that most ligands can activate more than one type of receptor subtype and many attempts have been made to classify receptor and ion channel subtypes. Using molecular biological techniques for the elucidation of receptor and ion channel subunit structure has revealed a number of ligand-gated ion channels whose subunits have considerable polypeptide identity. This gene superfamily includes GABA —A, nicotinic acetylcholine and glycine receptors (Olsen & Tobin, 1990). It is also clear that within the subunits which make up a receptor there are a number of subunit subtypes, each being specific for a particular tissue or brain region (Malherbe et al. 1990). In terms of evolution we can speculate that from a given set of subunits for a particular receptor, that is a, /?, y, S, have developed oclt a 2 , a 3 , a 4 , fix, /?2, /?3, /?4, etc. These can account for all the slight variations in receptor subtypes which are continually being reported from pharmacological studies. A considerable amount of information is now known

Evolutionary aspects of transmitter molecules P

Synaptic gap

S13

Neurotoxin/ ligand binding

Open channel entrance

Membrane

Gated ion channel Cytoplasm

Fig. 8. An acetylcholine channel complex (from Smith, 1989). for the GABA-A, glycine and nicotinic acetylcholine receptor complex (Noda et al. 1983; Boulter et al. 1986; Schofield et al. 1987; Greeningloh et al. 1987). The first complex to be analysed was the nicotinic acetylcholine receptor-ion channel from the electric organ of the electric fish (Numa et al. 1983). This is composed of five subunits, two alpha, and one of beta, gamma and delta with molecular weights of 40, 49, 57 and 65 kDa, respectively. This complex is therefore a multimeric protein with a total molecular weight of around 268 kDa. These five subunits can form a circular configuration with a central pore through which the ions may pass (Toyoshima & Unwin, 1988). A diagram of an acetylcholine receptor-channel complex is shown in Fig. 8. This pentameric structure for muscle nicotinic receptors appears to be basic since it is present in all vertebrates so far investigated, though the number of different subunits may differ. It is not yet clear whether the mammalian brain nicotinic receptor has three alpha and two beta subunits or is tetrameric. A number of different nicotinic receptors have been identified in the mammalian brain (Luetje, Patrick & Seguela, 1990). Both a and /? subunits contribute to the formation of the nicotinic gated ion channel though the contribution of these subunits to the functional receptor is not clear. These two subunits can combine in at least six different ways to make six different neuronal nicotinic acetylcholine receptors. Early studies on the purification of the GABA-A receptor using the affinity label flunitrazepam identified two subunits, alpha (48-8 kDa) and beta (51-4 kDa). The molecular weight of the receptor indicated that it was likely to have the composition a-2, fi-2. However, more recent studies on expression of cDNA for the alpha and beta subunits have led to a modification of this initial proposal. The GABA receptor complex has subunits for, or binding sites for, at least four chemical groups, i.e., GABA and its analogues such as muscimol, benzodiazepines, picrotoxin and barbiturates where, although all subunits bind GABA and benzodiazepines with different affinities, benzodiazepines

show preference in labelling the a subunit while muscimol shows a preference for the /? subunit. At least this is the situation in vertebrates, while the situation in invertebrates varies between the phyla. For example, in insects there is evidence for benzodiazepine modulation of GABA (Lees et al. 1987) while this does not appear to be the case in nematodes (Holden-Dye et al. 1989). Barbiturates also fail to modulate the GABA response on nematode muscle and picrotoxin is relatively inactive as an antagonist though the GABA inhibitory response on this muscle is chloride-mediated. However, the situation in the vertebrates appears more complex than initially thought and there is evidence for additional receptor subunits, i.e. gamma, delta and epsilon and even subtypes of these subunits (Schofield, 1989). It is perhaps germaine to ask just what all this really means ? Reconstitution experiments using alpha and beta subunits failed to produce receptors which mimicked in vivo events. However, the incorporation of a gamma-2 subunit with the alpha and beta subunits produced a functional receptor which was modulated by benzodiazepines as predicted from pharmacological studies. Recently, attempts have been made to isolate GABA receptor subunits from the snail Lymnaea and a subunit which the authors have identified as a /? subunit has been isolated (Harvey et al. 1990). It will be interesting to determine whether or not this subunit recognizes benzodiazepines. The glycine receptor, like GABA-A, has a channel which is selective for anions and physiologically this means chloride. However, this complex does not recognize picrotoxin, instead strychnine acts as an antagonist. The glycine receptor-channel complex has 3 subunits, 48, 58 and 93 kDa. The composition of the glycine binding component complex is probably two to three 48 kDa subunits and one or two 58 kDa subunits. It is probable that the 48 kDa subunits form the anion channel. The 93 kDa subunit is located on the inner membrane surface where it can interact with the cytoskeleton. Strychnine binds to the 48 kDa subunit. Evidence suggests there are similarities between nicotinic acetylcholine, GABA-A and glycine receptor channel complexes and that they are members of an evolutionary superfamily. One of the excitatory amino acid receptor family has also been cloned (Hollman et al. 1989). The subunit sequence lacks the two cysteine residues on the extracellular region which have been shown to be present for all the other ligand-gated ion channel subunits that have previously been sequenced. Whether or not this is true for all the ligand-gated channels of the glutamate receptor family, including the N M D A receptor, remains to be seen. Amino acid homology between the nicotinic, glycine and GABA-A receptors is shown in Table 5. The suggestion of a receptor superfamily with reference to nicotinic acetylPAR

s

R. J. Walker and L. Holden-Dye

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Table 5. A summary of the percentage amino acid homology in the nicotinic acetylcholine receptor, GABA-A receptor and glycine receptor subunits (The sequences are those of bovine GABA-A, rat 48 kDa glycine and bovine muscle acetylcholine. The first figure is percentage identical residues and the figure in parentheses is percentage identical plus conservative subunits (after Smith 1989).)

Acetylcholine-a GABA-Aa GABA-A^

GABA-Aa

GABA-A/?

Glycine

19(38) 100 35 (57)

15(32) 35 (57) 100

15(37) 34 (56) 39 (59)

choline receptor and GABA-A, has been investigated by Benson (1988) using a range of antagonists against responses to acetylcholine and GABA of insect central neurones. For example, strychnine, bicuculline and picrotoxin all block insect nicotinic acetylcholine receptors, suggesting some similarity between subunits of the nicotinic acetylcholine complex and that of GABA-A and glycine. However, it should be noted that while picrotoxin blocks insect GABA receptors, bicuculline fails to block the somal receptor response to GABA in the experiments of Benson. The nicotinic acetylcholine receptor of nematode muscle is also blocked by strychnine, picrotoxin and bicuculline (Holden-Dye & Walker, 1990) though the GABA receptor on this muscle is far less sensitive to strychnine and picrotoxin with bicuculline being inactive. The actual site of action of these antagonists for the Ascaris muscle nicotinic receptor (i.e. is the block competitive in nature and at the neurotransmitter binding site or is it noncompetitive and at the channel site ?) has yet to be determined. However, it is known that non-competitive receptor antagonists (i.e. compounds that block the ion channel opened by receptor activation) can recognize more than one type of ligand-gated channel. A good example of this is the ability of MK801, a non-competitive antagonist of the N M D A receptor, to block vertebrate nicotonic-gated channels (Ramoa et al. 1990). Interestingly, this compound does not block nicotinic receptors in Ascaris (Holden-Dye & Walker, unpublished observations). Other compounds are known to act at both nicotinic and excitatory amino acid channels, including the dissociative anaesthetics PCP and ketamine, and the toxin from the digger wasp philanthotoxin (Rozental et al. 1989). This pharmacological similarity may reflect structural homology between the channels but we shall have to await the success of attempts to clone more members of the glutamate receptor family, in particular the N M D A receptor, to see whether or not sequence homology exists between the nicotinic receptor and the glutamate receptor family. There are also pharmacological similarities between the anion

Fig. 9. The pharmacological profiles of mammalian GABA-A, glycine and nicotinic receptors together with nematode GABA and nicotinic receptors and the insect nicotinic receptor in terms of antagonism by bicuculline, picrotoxin, strychnine and non-competitive inhibitors (NCI) such as quinacrine and tetraphenylphosphonium (Schwartz & Mindlin, 1988). Table 6. The range of ligands which following receptor activation are linked to G-proteins (After Hille, 1989.) Ligand

Receptor

Acetylcholine L-Glutamic acid GABA Purines 5-HT Catecholamines Neuroactive peptides Light Gustatory Olfactory

Muscarinic Quisqualate (not all) GABA-B P-l Adenosine A-l, A-2 5-HT-l, 5-HT-2 All types Many examples Rhodopsins Some Some

channel gated by GABA and the nicotinic-cation channel. Agents such as chlorpromazine and trimethylphenylphosphonium, which are known to have binding sites within the nicotinic cation channel (Hucho, Oberthur & Lottspeich, 1986; Giraudat et al. 1987) also block GABA-A receptor-mediated chloride flux in mammalian brain (Schwartz & Mindlin, 1988; see Fig. 9). These data are summarized in Fig. 9. Not all transmitter membrane receptors are directly associated with a channel. There are a number which when activated stimulate a G protein system which is membrane bound (Table 6). These are often associated with slower events in the postsynaptic cell and these receptors may not be concentrated at the synapse. The G protein is located

Evolutionary aspects of transmitter molecules

on the inner surface of the cell membrane and can activate a membrane-bound enzyme which can produce second messengers. The second messenger can activate a kinase protein which in turn can phosphorylate a target protein. These targets may be ion channels but can be enzymes, components of the cytoskeleton or pumps and transporters. Due to this cascade of events the action of the agonist is amplified but takes tens of milliseconds to exert its effect. The response may persist after the agonist is removed. Thus, many physiological responses involving ion channels are regulated through second messenger systems. This topic is discussed in detail in an excellent review by Hille (1989). EVOLUTION OF ION CHANNELS

This topic has been studied in detail by Hille (1984, 1988, 1989). He suggested that mechanosensitivestretch-receptor-activated channels were present in prokaryotes over 1500 million years ago. These were preserved in early stem eukaryotes and at an early stage in the evolution of eukaryotes the voltage-gated cation channel evolved. From this channel evolved firstly a potassium and a calcium channel, probably 12—1400 million years ago. During the evolution of the Protista these further divided into subtypes of potassium and calcium channel. Early in the evolution of the animal phyla, say 8—900 million years ago, the sodium channel evolved from a calcium subtype channel. Electrophysiological evidence suggests that voltage-gated potassium and calcium channels are present in protozoa and algae while voltage-gated sodium channels and ligand-gated channels are generally considered to be absent. There is evidence, however, that a sodium-dependent, TTX-insensitive action potential is present in the protistan heliozoan Actinocoryne (FebreChevalier, 1989). Sodium can also modulate calcium events in Tetrahymena and T T X can alter the behaviour of the animal (Brown, 1989). Sodium-free saline has little effect on Tetrahymena behaviour while in saline with less than 0-1 mM calcium the animals die. Sodium action potentials and ligandgated channels are certainly present in coelenterates (Anderson & Schwab, 1982) and in all of the more advanced phyla that have been studied. For example, in platyhelminths, planarians possess neurones with typical TTX-sensitive sodium channels and sodium channels which are TTX-insensitive (Keenan & Koopowitz, 1984). A comparison of the amino acid sequences in the channel proteins show that the four homologous domains of sodium channels have greater identity in their sequences with the corresponding domains of calcium channels than to each other, i.e. 6 1 % compared to 50%. Their sequence identity is less with respect to the one potassium domain, i.e. 2 7 % channel. From this information Hille (1989) concluded that a single

S15

voltage-sensitive channel structure evolved into potassium channels and, by two gene duplications, to calcium channels. Sodium channels then arose from the calcium channels. T h e development of potassium and calcium channels allowed early eukaryotes to use intracellular free calcium as a signalling system. Such a system also needs the evolution of calmodulin and calmodulin-sensitive proteins to complete the response. OVERVIEW OF THE PHYSIOLOGICAL ROLES FOR TRANSMITTERS

In this section of the review we shall only consider examples where there is clear evidence for a transmitter to act at a particular site. The earlier literature has been referred to by Leake & Walker (1980). Acetylcholine has important physiological functions in all the phyla so far studied. In the annelids and in particular the leeches where its function has been investigated in detail, acetylcholine is an excitatory motor transmitter for the body wall musculature (Sargent, 1977). These motoneurones can also synapse centrally and so acetylcholine can also act as an interneuronal central transmitter. Likewise acetylcholine is the major excitatory motor transmitter in the nematodes (Johnson & Stretton, 1985) and chordates. In the chordates it also has an interneuronal role. In the arthropods and molluscs acetylcholine can act as a motor transmitter (Marder, 1976; Liebeswar et al. 1975) though it is not the major motor transmitter in either phylum. In contrast it is an important interneuronal transmitter in both phyla (Kandel et al. 1967; Blankenship, Wachtel & Kandel, 1971 ; Wang-Bennett, Sovan & Glantz, 1988). Finally in the arthropods acetylcholine is the major sensory transmitter (Barker et al. 1972; Hildebrand, Townsel & Kravitz, 1974). GABA has an important role as an inhibitory motor transmitter in annelids, nematodes and arthropods (Usherwood & Grundfest, 1964; Takeuchi & Takeuchi, 1966; Cline, 1986; Cline, Nusbaum & Kristan, 1985; Johnson & Stretton, 1987). There is little evidence for GABA as a motor transmitter in either molluscs or chordates. GABA is an interneuronal transmitter in all phyla that have been studied (Walker & Holden-Dye, 1989), and is the major interneuronal inhibitory transmitter in chordates (Roberts, 1986; Enna & Karbon, 1986). Glycine is another important inhibitory interneuronal transmitter in chordates (Werman, Davidoff & Aprison, 1968) but so far there is relatively little evidence for it in invertebrate phyla. However, there is one important report from the gastropod molluscs (Sawada et al. 19846). These authors have identified glycine in a group of neurones, R-3/14 (which also contain a peptide; Knock et al. 1989), where it is released onto and modifies contractility of the 2-2

R. jf. Walker and L. Holden-Dye anterior aorta of Aplysia. Since it has been identified here as a transmitter it is likely to have a similar role in other phyla and so should not be ignored. There is good evidence for L-glutamate as an excitatory motor transmitter in both arthropods and molluscs (Takeuchi & Takeuchi, 1964; Usherwood & Cull-Candy, 1975; Shinozaki, 1980; Sawada et al. 1984a). There is evidence for this amino acid as a sensory transmitter in chordates and an interneuronal transmitter in arthropods, molluscs and chordates (Judge, Kerkut & Walker, 1977; Fagg & Foster, 1983; Sombati & Hoyle, 1984; Nistri, 1985). In terms of the evolution of the functions of a group of compounds, the catecholamines and octopamine comprise an interesting example. The major catecholamine in invertebrate phyla is dopamine. This amine is a motor transmitter in arthropods and molluscs (House, 1973; Tritt & Bryne, 1982; Walker, 1986; Marder, Hooper & Eisen, 1987). In addition, it functions as an interneuronal transmitter in annelids, arthropods, molluscs and chordates (Berry & Cottrell, 1975; Klemm, 1985; Grace & Bunney, 1985). The evidence for noradrenaline in the invertebrate phyla is far less convincing than for dopamine; however, there is evidence either for noradrenalinecontaining neurones or enzymes for its synthesis in molluscs (Osborne, 1984). In addition, there is evidence for the presence of noradrenaline in Limulus, scorpions, crayfish, insects and ticks (Klemm & Axelsson, 1973; Stone, Binnington & Neish, 1978; O'Connor, Watson & Wyse, 1982; Elofsson et al. 1982; Roberts et al. 1983; Bakary, Fuzeau-Braesch & Papin, 1988). There are relatively high levels of noradrenaline in echinoderms (Juorio & Robertson, 1977). Noradrenaline plays an important role in chordates, both in the autonomic nervous system and in the central nervous system (Rogawski, 1985). Although adrenaline has been identified in many invertebrate phyla there is no good evidence for it as a transmitter or modulator at an identified synpase. This contrasts with the position in chordates where adrenaline substitutes for noradrenaline in the sympathetic nervous system of some groups and where it also plays a role in the brain. In addition, adrenaline acts as a hormone, being the major amine released from the adrenal medulla. Octopamine has been identified as playing an important modulatory role in neuromuscular and sensory physiology and as having a motor and an interneuronal transmitter function in arthropods and molluscs (Evans et al. 1976; Evans & O'Shea, 1978; Christensen et al. 1983; Morton & Evans, 1984; Flamm & Harris-Warrick, 1986; Ramirez & Orchard, 1990). Octopamine also has a hormonal role in arthropods particularly for flight and stress in insects (Evans, 1985). 5-HT functions as a motor transmitter in annelids,

S16

particularly in leeches, where it is an inhibitory transmitter for body wall musculature (Sawada & Coggeshall, 1976). 5-HT is widely distributed in arthropods where it functions as a motor, interneuronal and sensory transmitter. There are many 5HT-immunoreactive neurones in the insect brain in, for example, the optic lobe where it probably modulates the overall activity of other optic lobe interneurones (Nassel, Meyer & Klemm, 1985; Nassel, 1988). 5-HT has been identified in certain insect sensory neurones and so may act as a sensory transmitter in this group (Lutz & Tyrer, 1988). There are also 5-HT-containing sensory neurones in crustaceans (Beltz et al. 1984) which also contain acetylcholine (Katz & Harris-Warrick, 1990). 5-HT modulates salivary gland secretion in certain insects (Maddrell & Phillis, 1975) and other peripheral sites (Nassel, 1988). 5-HT neurones have been identified in gastropod molluscs and these cells make both peripheral and central connections indicating both a motor and interneuronal role for this amine (Cottrell & Macon, 1974; Mayeri et al. 1974; Liebeswar et al. 1975). 5-HT is involved in many aspects of the behaviour of molluscs including feeding, nociception and general arousal patterns (Kupfermann & Weiss, 1981). 5-HT in chordates is primarily associated with a central role as an interneuronal transmitter (Vander Maelen, 1985). Histamine is another amine which has evolved transmitter functions. There is good evidence for it to have an interneuronal role in vertebrates (Haas, 1985; Airaksinen & Panula, 1988) and in two invertebrate phyla, arthropods and molluscs. There are histamine-containing neurones in gastropods and these make synaptic connections with follower neurones, again indicating an interneuronal function (Turner & Cottrell, 1977; Osborne, 1984; McCaman & Weinreich, 1985). In arthropods, there is evidence for histamine as a sensory transmitter released by photoreceptors onto follower neurones in both Crustacea (Callaway & Stuart, 1989) and insects (Hardie, 1987, 1989). It is probable that histamine also serves as an interneuronal transmitter in this phylum. Finally, mention should be made of purines as transmitters or modulators since there is good evidence for such a role in vertebrates (Stone, 1989). In invertebrates, the evidence is less clear though these compounds are likely to have important roles and future experiments will prove this (Sathanan & Burnstock, 1976; Carr et al. 1986; McCaman, 1986; Cox & Walker, 1988; Barraco & Stefano, 1990). NEUROACTIVE PEPTIDES AND COLOCALIZATION

Neuroactive peptides have been the focus of considerable attention over the past 15 years (see Table 1). In invertebrates two neuroactive peptides were

Evolutionary aspects of transmitter molecules

identified in the mid-seventies, proctolin in the cockroach (Brown & Starratt, 1975) and FMRFamide in the lamellibranch mollusc, Macrocallista, (Price & Greenberg, 1977). Proctolin is a pentapeptide with the sequence Arg-Tyr-Leu-Pro-Thr, and is co-localized with L-glutamate in certain insect motoneurones (O'Shea & Bishop, 1982) and in the crayfish (Bishop et al. 1987). In each case proctolin release modifies muscle tension. Proctolin is also colocalized with acetylcholine.with 5-HT and with dopamine in crayfish neurones (Siwicki, Bettz & Kravitz, 1987) and so is likely to have important roles in the modulation of the function of many transmitters. Subsequent to the discovery of FMRFamide this peptide has been identified as a member of a family of peptides (Price, 1986; Price et al. 1987; Ebberink et al. 1987; Greenberg et al. 1988). These include FLRFamide, SPFLRFamide, and four peptides of the general formula XDPFLRFamide where X is one of the following: pGlu, Gly, Ser, Asn. This family also occurs in other groups. For example, in insects a number of FMRFamide-like peptides have been identified (Robb & Evans, 1990) including ProAsp- Val-Asp-His-Val -Phe- Leu- Arg-Phe-amide (Robb, Packman & Evans, 1989) and DPKQDFMRFamide in Drosophila (Nambu et al. 1988). FMRFamide-like immunoreactivity has also been identified in Crustacea and two octapeptides identified in the lobster, Ser-Asp-Arg-Asn-Phe-Leu-ArgPhe-amide and Thr-Asn-Arg-Asn-Phe-Leu-ArgPhe-amide, named FL1-3 and FL1-4 respectively (Trimmer, Kobierski & Kravitz, 1987), in the leech (Li & Calabrese, 1987), in sea anemones (Grimmelikhuijzen & Graff, 1986) and in vertebrates (Dockray et al. 1983: Dockray, 1985; Chen, Tsai & Shen, 1989). The FMRFamide family of peptides present in coelenterates is 4—7 amino acids in length and has a carboxyl terminal which is either -LRGamide or -GRFamide (Grimmelikhuijzen & Graff, 1985; Grimmelikhuijzen et al. 1988, 1990). This provides further evidence that this family of peptides appeared early in animal evolution. In the leech there is a SCP-like peptide which reacts to antibodies against SCP-B and FMRFamide (Evans & Calabrese, 1989). Evidence would suggest that these peptides can act as neurohormones, neuromodulators and as neurotransmitters and have important implications in terms of the evolution of transmitters (Penzlin, 1989). More recently, other interesting neuroactive peptides have been isolated from molluscs including the catch-relaxing peptide, CARP, Ala-Met-Pro-MetLeu-Arg-Leu-amide (Hirata et al. 1987), Ala-ProGly-Trp-amide (Kuroki et al. 1990) which is related to the crustacean red-pigment concentrating hormone (RPCH), Gly-Ala-Pro-Met-Phe-Val-amine and Gly-Ser-Pro-Met-Phe-Val-amide which both inhibit phasic contractions of muscles (Fujisawa et al. 1989). CARP has central as well as peripheral

S17 actions; it has excitatory and inhibitory actions on central neurones of gastropods (Mat Jais et al. 1990) and so, like FMRFamide, is likely to have important functions in modifying behaviour. It is likely that in each phylum endogenous neuroactive peptides will be identified and in turn these will be shown to occur elsewhere. For example, in Ascaris there is a heptapeptide, Lys-Asn-GluPhe-Ue-Arg-Phe-amide which modifies neuronal activity (Cowden, Stretton & Davis, 1989) and also inhibits gastropod central neurones (Pedder & Walker - unpublished observation). In the echinoderm Asterias, two neuropeptides have been identified, Gly-Phe-Asn-Ser-Ala-Leu-Met-Phe-amide and Ser-Gly-Pro-Tyr-Ser-Phe-Asn-Ser-Gly-LeuThr-Phe-amide. In the coelenterates, Bodenmuller & Schaller (1981) have isolated an 11-amino acid sequence which is required for head growth in Hydra, hence its name, the Head Activator Peptide. Its sequence is, < pGlu-Pro-Pro-Gly-Gly-Ser-LysVal-Ile-Leu-Phe. This peptide triggers cells to divide and stimulates multipolar cells to become neurones. It is synthesized in neurones and stored in granules. This amino acid sequence is conserved through evolution since it is present in the human hypothalamus at a concentration equivalent to that contained in 100 million Hydra. Besides FMRFamide, two cardioactive peptides, SCP-A and SCP-B have been identified in molluscs (Lloyd, 1982). Neuroactive peptides associated with reproduction in Aplysia and other gastropods have been identified including the Egg Laying Hormone (ELH) and two atrial gland peptides (Chiu et al. 1979; Schlesinger, Babirak & Blankenship, 1981). These peptides have actions on many neurones in the brain and clearly have important modulatory effects on central activity (Brownell, 1983). Finally, in this section we shall consider some examples of co-localization of classical transmitters and neuroactive peptides with particular reference to vertebrate-like peptides that have been identified in invertebrate phyla, primarily using immunocytochemical methods. Co-localization occurs in vertebrates (Table 7) where a classical transmitter can occur in the same neurone as a number of different peptides. The main vertebrate peptides which have been tentatively identified in invertebrates are shown in Table 8. From these the evidence for arg-vasotocin being present in the nervous system of Aplysia is good (Moore et al. 1981) where it alters the activity of central neurones. This peptide also suppresses the amplitude of the gill-withdrawal reflex induced by tactile stimulation of the siphon (Thornhill et al. 1981). Arg-vasopressin-like material is present in Helix brain and Arg-vasotocin alters the firing pattern of identified neurones (Boyd, Osborne & Walker, 1987). Arg-vasopressin-like material is co-localized in neurones which also contain a substance P-like material (Walker et al.

R. J. Walker and L. Holden-Dye

S18

Table 7. Examples of co-localization of classical transmitters and neuroactive peptides in vertebrate neurones (After Osborne, 1983; O'Donohue et al. 1985.) Classical transmitter

Neuropeptide

Tissue

Dopamine

Enkephalin Cholecystokinin Somatostatin Enkephalin

Carotid body Brain Sympathetic ganglia Sympathetic ganglia Adrenal medulla Locus coeruleus Adrenal medulla Brain Brain Autonomic ganglia

Noradrenaline

5-HT Acetylcholine

GABA

Neurotensin Substance P Enkephalin Vasoactive intestinal peptide Enkephalin Neurotensin Somatostatin Substance P Somatostatin

Preganglionic nerves Cochlear nerve Preganglionic nerves Heart Ciliary ganglion Brain

Table 8. Vertebrate peptide-like material identified in molluscan tissues using immunocytochemistry Adrenocorticotropic hormone (ACTH-17-39; 1-24) Arginine vasotocin Bombesin Cholecystokinin (CCK) /?-Endorphin Met-Enkephalin Gastrin a-Melanocyte stimulating hormone (a-MSH) Oxytocin Pancreatic polypeptide (PP) Substance P Thyrotropin releasing hormone (TRH) Vasoactive intestinal peptide (VIP)

Glucagon

Calcitonin Leu-Enkephalin Insulin Neurotensin Somatostatin

Vasopressin

Table 9. Examples of co-localization of classical transmitters and neuroactive peptides in neurones. (In the case of the vertebrate peptides it is probably correct to describe them as immunoreactive-like material.) Molluscs

Arthropods

Annelids

5-HT and substance P in Helix 5-HT and cholecystokinin in Helix Egg Laying Hormone and a-bag cell peptide in Aplysia Glycine and a 12 amino acid peptide in Aplysia Acetylcholine and a peptide in Aplysia Acetylcholine and CCK/gastrin in Aplysia Acetylcholine and FMRFamide in Aplysia and Helix SCPs and FMRFamide in Aplysia Acetylcholine and SCP-B in Tritonia 5-HT and SCP-B in Tritonia SCP-B and FMRFamide in Tritonia 5-HT, dopamine and vasotocin in Lymnaea Dopamine and a-endorphin in Bombyx L-Glutamate and proctolin in cockroach/locust FMRFamide and BPP in cockroach L-Glutamate and proctolin in lobster Dopamine and proctolin in lobster Acetylcholine and proctolin in lobster Acetylcholine and 5-HT in crab Acetylcholine and FMRFamide in leech

Evolutionary aspects of transmitter molecules

1987). However, it is very unlikely that it is authentic substance P and much more likely to be a related tachykinin (Boyd, Osborne & Walker, 1984, 1985). This substance P-like immunoreactivity .also colocalizes with 5-HT in neurones which innervate the heart of Helix and so the tachykinin may have a modulatory role in heart activity (Boyd et al. 1984). A neurone, B-13, in Aplysia contains both acetylcholine and a cholecystokinin/gastrin-like material (Ono, 1989). In an elegant series of experiments she demonstrated that when the cholinergic synaptic component is blocked, there still remains a slow depolarizing component. This slow component is absent in cell B-4 which contains only acetylcholine but not the peptide. This suggests a clear transmitter role for the peptide at this site. Acetylcholine also colocalizes with FMRFamide in cell C-3 Helix (Bewick, Price & Cottrell, 1990).

S19

TRANSMITTERS IN PL AT YHELM I NTHS

immunoreactivity have been identified in planaria (Reuter, Karhi & Schot, 1984; Wikgren & Reuter, 1985). There is a large volume of literature concerning the role for 5-HT in trematodes, particularly in Fasciola and Schistosoma (Mansour, 1984; Webb, 1988). 5-HT is certainly present in this group (Fairweather et al. 1987) and trematodes can synthesize it from 5-hydroxytryptophan but not from tryptophan itself. 5-HT would appear to be involved in motor activity in trematodes and this or a related indole may well act as the motor excitatory transmitter. The actual nature of the endogenous ligand in trematodes is not established since careful analysis suggests that the compound in trematode tissue is not authentic 5-HT as it does not show the chemical characteristics of this amine (Andreini et al. 1970; Tomosky-Sykes et al. 1977). 5-HT stimulates glucose uptake and can increase glycogen metabolism. These actions may involve activation of adenylate cyclase since 5-HT stimulates the level of cAMP in trematode tissue. Dopamine and noradrenaline have been identified in Fasciola and Schistosoma (Gianutsos & Bennett, 1977) and in the light of the important roles of dopamine demonstrated in other invertebrates it is likely to be important in this group. Catecholamine-containing neurones have been demonstrated histochemically in Schistosoma (Machado, Machado & Pellegrino, 1972; Orido, 1989) and this is probably dopamine. A well-developed cholinergic system has also been demonstrated in trematodes (Niewiadomska & Moczon, 1982) and it may function as an inhibitory transmitter for bodywall musculature (McKay et al. 1989). Using immunocytochemical methods a number of peptides have been tentatively identified in Fasciola, including FMRFamide, pancreatic polypeptide, peptide tyrosine and substance P (Magee et al. 1989). These peptides may be involved in release of eggs and movement of ova through the ootype.

This phylum comprises three groups which will be discussed in this section: planarians, trematodes and cestodes. In planarians there is evidence for acetylcholine in certain neurones (Koopowitz & Keenan, 1982). Around 40 neurones in Notoplana appear to be dopaminergic (Koopowitz & Keenan, 1982) while the occurrence of other catecholamines is less clear. There is evidence for dopamine receptors in planaria where this amine alters motor activity (Venturini et al. 1989). 5-HT stimulates DNA and RNA synthesis and cell division in this group and can also influence regeneration (Webb, 1988). Welsh and Moorhead in their classical study found 5-HT in planaria (Welsh & Moorhead, 1960). In terms of neuroactive peptides, a somatostatin-like immunoreactivity has been identified in Dugesia (Schilt, Richoux & Dubois, 1981) and a met-enkephalin-like material, also in Dugesia (Venturini et al. 1983), together possibly with endorphins. Substance P and FMRFamide-like

Unlike trematodes, cestodes are capable of synthesizing 5-HT from both tryptophan and 5-hydroxytryptophan (Ribeiro & Webb, 1983a) and 5 - H T is present in this group, for example, in Hymenolepis (Lee, Bueding & Schiller, 1978). There is a highaffinity uptake mechanism for 5-HT and a highaffinity binding site (Webb, 1985). Endogenous release of 5-HT from tissue slices of Hymenolepis has been demonstrated and this release is calciumdependent (Gordon & Webb, 1989). This amine has been identified in neurones and branches from the commissural neurites divide to form terminal varicosities on the longitudinal muscles (Webb & Mizukawa, 1985). When applied to the muscle, 5H T does not always induce contractions, suggesting it may not act as the excitatory transmitter (Webb, 1988). It may act as a modulator of muscle activity possibly through a cyclase and myosin kinase system. 5-HT also increases glycogenolysis, glycolysis and

Table 9 summarizes some of the examples of colocalization so far identified to invertebrate phyla. It is difficult at this stage to observe any clear pattern in terms of co-localization of particular classical transmitters and neuroactive peptides. It is likely that as additional examples are identified then certain patterns may emerge in terms of either classical transmitters associated with particular peptides or possibly there may be a functional relationship, for example, certain peptides associated more with a motor or interneuronal cell type. Clearly, acetylcholine co-localizes with many different peptides and a peptide (e.g. enkephalin or proctolin) can be co-localized with many different classical transmitters. This has been discussed by O'Donohue et al. (1985). All that can be concluded is that probably most, if not all, neurones contain more than one material that can be released following presynaptic terminal activation.

R. Jf. Walker and L. Holden-Dye phosphofructokinase. Octopamine is also present in cestodes (Ribeiro & Webb, 19836) while dopamine and noradrenaline appear to be absent (Chou, Bennett & Bueding, 1972). There is evidence for a possible transmitter role for L-glutamate in Hymenolepis. This amino acid contracts the body wall musculature and there is a high-affinity uptake system (Webb, 1985, 1986). There is also a highaffinity binding site for glutamate and glutamate is released from tissue slices of Hymenolepis (Webb, 1988). Glutamate-like immunoreactivity is present in the longitudinal nerve cord of Hymenolepis and there is a glutamate-linked adenylate cyclase present (Webb & Eklove, 1989; Eklove & Webb, 1990a, b). Glutamate and aspartate both excite longitudinal nerve cord activity (Keenan & Koopowitz, 1982). All this evidence suggests an excitatory transmitter role in this group for L-glutamic acid. Immunocytochemical techniques revealed the presence of FMRFamide-like, vasotocin-like, leu-enkephalin-like and neurotensin-like immunoreactivity in the nervous system of Diphyllobothrium (Gustafsson et al. 1985) and 5-HT in neurones and ganglionic commissures of the brain of cestodes (Gustafsson et al. 1985, 1986; Fairweather et al. 1988). TRANSMITTERS IN NEMATODES

The normal range of transmitter molecules have been identified in nematodes including catecholamines, 5-HT, acetylcholine and GABA. Earlier work has been reviewed by Willett (1980). Dopamine neurones have been identified in Caenorhabditis elegans (Sulston, Dew & Brenner, 1975) and enzymes for its synthesis, i.e. tyrosine hydroxylase and aromatic amino acid decarboxylases are present. There is also evidence for the presence of dopaminebeta-hydroxylase and phenylethanolamine-iVmethyltransferase (Kisiel, Duebert & Zuckermann, 1976) which would suggest the presence of noradrenaline and adrenaline. In addition, a catecholamine-sensitive adenylate cyclase has been demonstrated (Willett et al. 1979) though its characteristics are more that of a beta adrenergic receptor rather than a dopaminergic system. The tissue levels of dopamine are much higher than the levels for either noradrenaline or adrenaline although the catecholamine-activated cyclase is activated to a greater extent by noradrenaline and adrenaline than by dopamine. Selective high-affinity uptake systems have been demonstrated for both noradrenaline and for 5-HT. The presence of enzymes for the inactivation of catecholamines has also been investigated and while catechol-O-methyltransferase is present, monoamine oxidase appears to be absent (Wright & Awan, 1978). It has been suggested that catecholamines may play an important role in the control of body wall musculature since catecholamine neurones occur in the ventral cord (Goh &

S20

Davey, 1976). As mentioned earlier there is excellent evidence for both acetylcholine and GABA as neuromuscular transmitters in nematodes, mediating excitation and inhibition respectively (Johnson & Stretton, 1985, 1987). Recent studies have revealed information regarding the acetylcholine and GABA pharmacological profiles of the receptors in Ascaris (Holden-Dye et al. 1989; Colquhoun, Holden-Dye & Walker, manuscript in preparation). The GABA receptors resemble GABA-A receptors in that they are linked to chloride ion channels and are activated by muscimol, dihydromuscimol being a potent agonist. This receptor differs from the vertebrate GABA-A in that it has an extremely low sensitivity to picrotoxin while bicuculline is inactive. It also lacks a TBPS and benzodiazepine-sensitive binding site. In terms of GABA agonists, apart from sulphurcontaining compounds, the agonist profile correlates very well with the mammalian GABA-A site. The acetylcholine receptor resembles a vertebrate nicotinic ganglion site. DMPP and analogues are potent agonists while mecamylamine and benzoquinonium are potent antagonists though it must be noted that benzoquinonium is a neuromuscular blocker. Tubocurarine and a-bungarotoxin have relatively low potencies as antagonists while dihydro-/?-erythroidine is inactive. Neostigmine potentiates the action of acetylcholine while physostigmine depresses the response, suggesting that physostigmine is able to bind to the part of the receptor which recognizes acetylcholine and so acts as an antagonist. Immunocytochemical studies have shown the presence of 5HT, GABA, ACTH and FMRFamide-like material in the nervous tissue of the nematode, Goodeyus ulmi (Leach, Trudgill & Gahan, 1987). No immunoreactivity was found to either VIP or somatostatin14. Recently, a detailed study on the immunocytochemical localization of neuropeptides in Ascaris nervous system has revealed positive immunoreactivity to 12 neuropeptides (Sithigorngul, Stretton & Cowden, 1990). These were luteinizing hormone releasing hormone (LHRH), Aplysia peptide L - l l , Aplysia peptide 12-B, small cardiac peptide B (SCP-B), neuropeptide Y, FMRFamide, gastrin-17, cholecystokinin octapeptide (CCK-8), amelanocyte stimulating hormone (a-MSH), calcitonin gene related peptide (CGRP), corticotropin releasing factor (CRF) and vasoactive intestinal peptide (VIP). In addition, a number of these peptide-like immunoreactive materials co-localized in the same neurone (e.g. Aplysia peptide 12-B, neuropeptide Y, FMRFamide, cholecystokinin, CGRP, CRF; FMRFamide, cholecystokinin; SCPB, neuropeptide Y, FMRFamide, cholecystokinin; SCP-B, neuropeptide Y, FMRFamide; Aplysia peptide 12-B, neuropeptide Y, FMRFamide). It is of interest that an FAlRFamide-like material was present in a number of different cell types. Both 5HT and octopamine have been found in Caenor-

S21

Evolutionary aspects of transmitter molecules

Table 10. Summary of the occurrence and physiological roles for classical transmitters in platyhelminths, nematodes, arthropods, molluscs and annelids ( + , Indicates present in phylum; sensory, intern (interneuronal) and motor indicate these roles for the transmitters in each phylum; ? indicates limited evidence only. The lack of clear evidence for platyhelminths shows more work is required in this phylum.)

Acetylcholine

Platyhelminths

Nematodes

Arthropods

Molluscus

Annelids

+ motor ?

+ motor

+ sensory intern motor + intern motor

+ motor intern

+ motor intern

+ motor intern sensory + motor intern + motor intern + motor intern 4- intern + motor intern

+ motor intern

GABA L-Glutamate Dopamine Octopamine Histamine 5-HT

+ motor

+ + +

+ sensory

+ motor?

+ intern motor + intern motor + intern motor + sensory +sensory intern motor

habditis elegans, 5-HT being detected histochemically in two pharyngeal neurones while octopamine was detected radio-enzymatically and is probably restricted to a cell (Horvitz et al. 1982). Octopamine and 5-HT produce distinct but opposite behavioural responses, suggesting they may act physiologically as antagonists. The evidence for 5H T in nematodes is controversial (Willett, 1980) and Willett made the interesting observation that the only nematodes where 5-HT has been demonstrated are those occurring in the vertebrate intestine. It is possible that the 5-HT may be derived from the host. However, from an evolutionary viewpoint it seems very unlikely that this phylum would have failed to evolve mechanisms to synthesize 5-HT and to employ it as a transmitter or modulator since it is so important in other phyla.

TRANSMITTERS IN ARTHROPODS

Probably all the classical transmitters have been identified in arthropods, particularly in insects and crustaceans and aspects of this work have already been discussed. However, the three classical transmitters which have been studied in detail in this phylum are acetylcholine, GABA and L-glutamic acid (see Table 10). Acetylcholine is the most important sensory transmitter in arthropods and also plays a significant role in the central nervous system (Sattelle, 1985). There are three types of acetylcholine receptors, nicotinic, muscarinic and mixed nicotinic/muscarinic (Breer, 1981; Lummis & Sattelle, 1985). There is good evidence for both abungarotoxin-sensitive and insensitive nicotinic receptors. At the post-synaptic membrane of sensory synapses, the action of acetylcholine is nicotinic and a-bungarotoxin-sensitive. The receptor proteins for the nicotinic a-bungarotoxin-sensitive acetylcholine receptor have been purified and incorporated into lipid bilayers where they form acetylcholineactivated cation channels (Hanke & Breer, 1987). A

+

+ intern

+ + motor intern

regulatory polypeptide of this acetylcholine receptor has also been purified and found to inhibit 125I abungarotoxin binding to pre-treated neuronal membranes (Benke, Stahner & Breer, 1989). Muscarinic receptors are located pre-synaptically on afferent inputs which are involved in lipid mobilization. Octopamine can also modify behaviour in insects when injected into the central nervous system, and has recently been shown to modulate the activity of insect wing stretch receptors (Ramirez & Orchard, 1990). 5-HT shares a number of the roles of octopamine as a neurohormone and modulator of neuromuscular transmission. Using immunocytochemical techniques 5-HT has been shown to be widely distributed in insects and has an important role as an interneuronal transmitter (Nassel, 1988), as in vertebrates. Salivary gland and Malpighian tubule secretion are also under the control of 5-HT (Berridge & Patel, 1968; Maddrell & Phillips, 1975) and it has a role in heart regulation (Collins & Miller, 1977). Finally, as previously mentioned, the arthropods are one of two phyla where histamine has a transmitter function. This is as a sensory transmitter, elucidated from the work of Hardie (1988, 1989) and Simmons & Hardie (1988). Enkephalin-like immunoreactivity in retinular cells of the lobster eyestalk (Mancillas et al. 1981), suggests a role in the primary photoreceptors. Substance P was found in the ' neurosecretory' cells which are synaptically linked to the retinular cells. SUMMARY

Classical transmitters are present in all phyla that have been studied; however, our detailed understanding of the process of neurotransmission in these phyla is patchy and has centred on those neurotransmitter receptor mechanisms which are amenable to study with the tools available at the time, for example, high-affinity ligands, tissues with high density of receptor protein, suitable electrophysio-

R. J, Walker and L. Holden-Dye logical recording systems. Studies also clearly show that many neurones exhibit co-localization of classical transmitters and neuropeptides. However, the physiological implications of this co-localization have yet to be elucidated in the vast majority of examples. The application of molecular biological techniques to the study of neurotransmitter receptors (to date mainly in vertebrates) is contributing to our understanding of the evolution of these proteins. Striking similarities in the structure of ligand-gated receptors have been revealed. Thus, although ligand-gated receptors differ markedly in terms of the endogenous ligands they recognize and the ion channels that they gate, the structural similarities suggest a strong evolutionary relationship. Pharmacological differences also exist between receptors that recognize the same neurotransmitter but in different phyla, and this may also be exploited to further the understanding of structure-function relationships for receptors. Thus, for instance, some invertebrate GABA receptors are similar to mammalian GABAA receptors but lack a modulatory site operated by benzodiazepines. Knowledge of the structure and subunit composition of these receptors and comparison with those that have already been elucidated for the mammalian nervous system might indicate the functional importance of certain amino acid residues or receptor subunits. These differences could also be exploited in the development of new agents to control agrochemical pests and parasites of medical importance. The study of the pharmacology of receptor proteins for neurotransmitters in invertebrates, together with the application of biochemical and molecular biological techniques to elucidate the structure of these molecules, is now gathering momentum. For certain receptors, e.g. the nicotinic receptor, we can expect to have fundamental information on the function of this receptor at the molecular level in both invertebrates and vertebrates in the near future. REFERENCES

S22

& LLINAS, R. (1971). Electrotonic coupling between neurones in the rat mesencephalic nucleus. Journal of Physiology 212, 45-63.

BAKER, R.

BARKER, D. L., HERBERT, E., HILDEBRAND, J. G. & KRAVITZ,

E. A. (1972). Acetylcholine and lobster sensory neurones. Journal of Physiology (London) 226, 205-29. BARNES, R. S. K., CALOW, P. & OLIVE, P. J. W. ( 1 9 8 8 ) .

The

Invertebrates: a New Synthesis. Oxford: Blackwell Scientific Publ. BARRACO, R. A. & STEFANO, G. B. (1990). Pharmacological evidence for the modulation of monoamine release by adenosine in the invertebrate nervous system. Journal of Neurochemistry 54, 2002-6. BELTZ, B., EISEN, J. S., FLAMM, R. E., HARRIS-WARRICK, R. M., HOOPER, s. L. & MARDER, E. (1984). Serotonergic innervation and modulation of the stomatogastric ganglion of three decapod crustaceans, (Panulirus interruptus, Homarus atnericanus and Cancer irroratus). Journal of experimental Biology 109, 35-54. BENKE, D., STAHNER, I. & BREER, H. ( 1 9 8 9 ) . M o n o c l o n a l

antibodies detecting regulatory polypeptides of the insect neuronal acetylcholine receptor. Journal of experimental Biology 147, 329-42. BENSON, j . A. (1988). Transmitter receptors on insect neuronal somata: GABAergic and cholinergic pharmacology. In Molecular Basis of Drug and Pesticide Action, pp. 193-206 (ed. Lunt, G. G.) Amsterdam: Elsevier Science Publishers, BV. BENSON, j . A. (1989). A novel GABA receptor in the heart of a primitive arthropod. Journal of experimental Biology 147, 421-38. BERRIDGE, M. j . & PATEL, N. G. (1968). Insect salivary glands: stimulation of fluid secretion by 5-HT and cAMP. Science 162, 462-3. BERRY, M. s. & COTTRELL, G. A. (1975). Excitatory, inhibitory and biphasic synaptic potentials mediated by an identified dopamine-containing neurone. Journal of Physiology (London) 244, 589-612. BEWICK, G. S., PRICE, D. A. & COTTRELL, G. A. ( 1 9 9 0 ) . T h e

fast response mediated by the C-3 motoneurone of Helix is not attributable to the contained FMRFamide. Journal of experimental Biology 148, 201-19. BISHOP, C. A., WINE, J. J., NAGY, F. & o'SHEA, M. R. ( 1 9 8 7 ) .

Physiological consequences of a peptide co-transmitter in a crayfish nerve-muscle preparation. Journal of Neuroscience 7, 1769-79. BLANKENSHIP, J. E., WACHTEL, H. & KANDEL, E. R. ( 1 9 7 1 ) .

histaminergic system in the guinea pig central nervous system: Journal of comparative Neurology 273, 163-86. ANDERSON, p. A. v. & SCHWAB, w. E. (1982). Recent advances and model systems in coelenterate neurobiology. Progress in Neurobiology 19, 213-36.

Ionic mechanisms of excitatory, inhibitory and dual synaptic actions mediated by an identified interneurone in abdominal ganglion of Aplysia. Journal of Neurophysiology 34, 76-92. BODENMULLER, H. & SCHALLER, H. c. (1981). Conserved amino acid sequence of a neuropeptide, the head activator, from coelenterates to humans. Nature, London 293, 579-80.

ANDREINI, G. L., BERETTA, C , FAUSTINI, R. & GALLINA, G.

BOER, H. H . , SCHOT, L. P. C , STEINBUSCH, H. W. M.,

AIRAKSINEN, M. S. & PANULA, P. (1988). T h e

(1970). Spectrophotometric and chromatographic characterization of a butanol extract from Fasciola hepatica. Experientia 26, 166-7. BAKARY, Z. EL, FUZEAU-BRAESCH, S. & PAPIN, C. ( 1 9 8 8 ) .

Detection of biogenic amines and nychthermeral variations in the scorpion Leiurus quinquestriatus. Comparative Biochemistry and Physiology 90C, 173-7.

c. & REICHELT, D. (1984). Co-existence of immunoreactivity to anti-dopamine, anti-serotonin and anti-vasotocin in the cerebral giant neuron of the pond snail, Lymnaea stagnalis. Cell and Tissue Research 238, 411-12.

MONTAGNE,

BOULTER, J . , EVANS, K., GOLDMAN, D., MARTIN, G., TRECO, D., HEINEMANN,

s. &

PATRICK,

j . (1986). Isolation of a

Evolutionary aspects of transmitter molecules cDNA clone coding for a possible neural nicotinic acetylcholine receptor a-subunit. Nature, London 319, 368-74. BOYD, P. J., OSBORNE, N . N . & WALKER, R. J. ( 1 9 8 4 ) . T h e

pharmacological actions of 5-hydroxytryptamine, FMRFamide and Substance P and their possible occurrence in the heart of the snail Helix aspersa. Neurochemistry International 6, 633-40. BOYD, P. J., OSBORNE, N . N . & WALKER, R. J. ( 1 9 8 5 ) .

Localization of Substance P-like material in the central and peripheral nervous system of the snail, Helix aspersa. Histochemistry 84, 97-103. BOYD, P. ] . , OSBORNE, N . N . & WALKER, R. J. ( 1 9 8 7 ) .

Localization of Arg-Vasopressin-like material in central neurones and mechanism of action of ArgVasotocin on identified neurones of the snail, Helix aspersa. Neuropharmacology 26, 1633-47. BRADFORD, H. F. (1986). Chemical Neurobiology. New York: Freeman. BREER, H. (1981). Characterization of synaptosomes from the central nervous system of insect. Neurochemistry International 3, 155-63. BROWN, B. E. & STARRATT, A. N. (1975). Isolation of proctolin, a myotropic peptide, from Periplaneta americana. Journal of Insect Physiology 21, 1879-81. BROWN, i. (1989). Studies on the physico-chemical basis of behaviour in Tetrahymena. Ph.D thesis. University of Southampton, England. BROWNELL, p. H. (1983). Neuroendocrine mechanisms of visceromotor behaviour in Aplysia. In Molluscan Neuro-Endocrinology (ed. Lever, J. & Boer, H. H.) pp. 256-263; Amsterdam: North Holland. BURNSTOCK, G. (1985). Purinergic mechanisms broaden their sphere of influence. Trends in Neuroscience 8, 5-6. CALLAWAY, J. C , MASINOVSKI, B. & GRAUBARD, K. ( 1 9 8 7 ) .

S23 CHRISTENSEN, T. A., SHERMAN, T. G., MCCAMAN, R. E. & CARLSON, A. D. (1983). Presence of octapamine in

firefly photomotor neurons. Neuroscience 9, 183-9. H. T. (1986). Evidence for GABA as a neurotransmitter in the leech. Journal of Neuroscience 6, 2848-56.

CLINE,

CLINE, H., NUSBAUM, M. P. & KR1STAN, W. B. ( 1 9 8 5 ) .

Identified GABAergic inhibitory motor neurones in the leech nervous system take up GABA. Brain Research 348, 359-62. COLLINS, c. & MILLER, T. A. (1977). Studies on the action of biogenic amines on cockroach heart. Journal of experimental Biology 67', 1—15.

& MACON, j . B. (1974). Synaptic connections of two symmetrically placed giant serotonin-containing neurones. Journal of Physiology

COTTRELL, G. A.

236, 435-64. COWDEN, C , STRETTON, A. O. W. & DAVIS, R. E. ( 1 9 8 9 ) .

AF1, a sequenced bioactive neuropeptide isolated from the nematode, Ascaris summ. Neuron 2, 1465-73. cox, R. T. L. & WALKER, R. j . (1988). An analysis of the

inhibitory responses of dopamine and octopamine on Helix central neurones. Comparative Biochemistry and Physiology 88C, 121-30. DOCKRAY, G. J., REEVE, J. R., SHIRLEY, J., GAYTON, R. J. & BARNARD, c. s. (1983). A novel active pentapeptide from chicken brain identified by antibodies to FMRFamide. Nature, London 305, 328-30. EBBERINK, R. H. M., PRICE, D. A., VAN LOENHOUT, H . , DOBLE, K. E., RIEHM, J. P . , GERAERTS, W. P . M. &

GREENBERG, M. j . (1987). The brain of Lymnaea contains a family of FMRFamide-like peptides. Peptides 8, 515-22. ECKERT, R. (1963). Electrical interaction of paired ganglion cells in the leech. Journal of General Physiology 46, 575-87.

Co-localization of SCP-B-like and FMRFamide-like immunoreactivities in crustacean nervous system. Brain Research 405, 295-304. CALLAWAY, j . c. & STUART, A. E. (1989). Biochemical and physiological evidence that histamine is the transmitter of barnacle photoreceptors. Visual Neurosciences 3, 311-25.

EKLOVE, H. & WEBB, R. A. (1990a) Glutamate-like

CARR, W. E. S., GLEESON, R. A., ACHE, B. W. & MILSTEAD, M.

ELOFSSON, R., LAXMYR, L., ROSENGREN, E. & HANSSON, C.

L. (1986). Olfactory receptors of the spiny lobster: ATPase-sensitive cells with similarities to P-2-type purinoceptors of vertebrates. Journal of Comparative Physiology 158, 331-8. CHAD, j . E. & KERKUT, G. A. (1981). Summation of

membrane current conductance responses as evidence for independent ionophore populations. Comparative Biochemistry and Physiology 69C, 61-5. CHEN, S-T., TSAI, M. s. & SHEN, c. L. (1989). Distribution

of FMRFamide-like immunoreactivity in the central nervous system of the Formosan monkey (Macaca cyclopsis). Peptides 10, 825-34. CHIU, A. Y., HUNKAP1LLER, M. W., HELLER, E., STUART, D.

K., HOOD, L. E. & STRUMWASSER, F. (1979). Purification

and primary structure of the neuropeptide egg-laying hormone of Aplysia californica. Proceedings of the National Academy of Sciences, USA 76, 6656-60. CHOU, T. T., BENNETT, J. & BUEDING, E. ( 1 9 7 2 ) .

Occurrence and concentration of biogenic amines in trematodes. Journal of Parasitology 58, 1098-102.

immunoreactivity in the cestode Hymenolepis diminuta. Canadian Journal of Zoology (in the Press). EKLOVE, H. & WEBB, R. A. (19906). The effect of Lglutamate and related agents on adenylate cyclase in the cestode Hymenolepis diminuta. Canadian Journal of Physiology and Pharmacology (in the Press). (1982). Identification and quantitative measurements of biogenic amines and DOPA in the central nervous system and haemolymph of the crayfish, Pacifastacus leniusculus (Crustacea). Comparative Biochemistry and Physiology 71C, 19 5-201. ENNA, s. j . & KARBON, E. w. (1986). GABA receptors: An overview. In Benzodiazepine/GABA Receptors and Chloride Channels - Structural and Functional Properties, pp. 41-56, (ed. Olsen, R. W. & Venter, J. C.) New York: Alan R. Liss Inc. EVANS, P. D. (1985). Octopamine. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (ed. Kerkut, G. A. & Gilbert, L. I.), Vol. 11, pp. 499-530. Oxford: Pergamon Press. EVANS, P. D. & CALABRESE, R. L. (1989). Small cardioactive peptide-like immunoreactivity and its colocalization with FMRFamide-like immunoreactivity in the central nervous system of the leech, Hirudo medicinalis. Cell and Tissue Research 257, 187-99. EVANS, P. D. &COURNIL, I. (1990). Co-localization of FLRF-

R. J. Walker and L. Holden-Dye and vasopressin-like immunoreactivity in a single pair of sexually dimorphic neurones in the nervous system of the locust. Journal of Comparative Neurology 292, 331-48. EVANS, P. D., KRAV1TZ, E. A., TALAMO, B. R. & WALLACE, B.

G. (1976). The association of octopamine with specific neurones along lobster nerve trunks. Journal of Physiology 262, 51-70. EVANS, P. D. & O'SHEA, M. (1978). The identification of an

octopaminergic neurone and the modulation of a myogenic rhythm in the locust. Journal of experimental Biology 73, 235-60. FAGG, G. E. & FOSTER, A. c. (1983). Amino acid neurotransmitters and their pathways in the mammalian central nervous system. Neuroscience 9, 701-19. FAIRWEATHER, I., MACARTNEY, G. A., JOHNSTON, C. F . , HALTON, D. W. & BUCHANAN, K. D. ( 1 9 8 8 ) .

Immunocytochemical demonstration of 5hydroxytryptamine (serotonin) and vertebrate neuropeptides in the nervous system of excysted cysticercoid larvae of the rat tapeworm, Hymenolepis diminuta {Cestoda, Cyclophyllidea). Parasitology Research 74, 371-9. FAIRWEATHER, I., MAULE, A. G., MITCHELL, S. H . , JOHNSTON, C. F . & HALTON, D. W. ( 1 9 8 7 ) .

Immunocytochemical demonstration of 5hydroxytryptamine (serotonin) in the nervous system of the liver fluke, Fasciola hepatica (Trematoda, Digenea). Parasitology Research 73, 255-8. FEBRE-CHEVALIER, c. (1989). Excitability and contractility in a primitive eukaryote, the helizoan Actinocoryne contractilis. In Evolution of the First Nervous Systems. St. Andrews, Scotland. (Unpublished abstract.) FLAMM, R. E. & HARRIS-WARRICK, R. M. (1986). Aminergic modulation in lobster stomatogastric ganglion. II. Target neurons of dopamine, octopamine and serotonin within the pyloric circuite. Journal of Neurophysiology 55, 866-81. FUJISAWA, Y . , KUBOTA, I . , IKEDA, T. & MUNEOKA, Y.

(1989). Bioactive peptides isolated from the anterior byssus retractor muscle of the bivalve mollusc, Mytilus edulid. Peptide Chemistry 51-6. FURSHPAN, E. j . & POTTER, D. D. (1959). Transmission at the giant motor synapses of the crayfish. Journal of Physiology 145, 289-325. GIANUTSOS, G. & BENNETT, j . L. (1977). The regional distribution of dopamine and norepinephrine in Schistosoma mansoni and Fasciola hepatica. Comparative Biochemistry and Physiology 58C, 157—9. GIRAUDAT, J., DENNIS, M . , HEIDMANN, T., HAUMONT, P . T . ,

p. & CHANGEUX, j . p. (1987). Structure of the high affinity site for non competitive blockers of the acetylcholine receptor: [3H]-chlorpromazine labels homologous residues in the beta and delta chains. Biochemistry 26, 2410-18. GOH, s. L. & DAVEY, K. G. (1976). Localization and distribution of catecholaminergic structures in the nervous system of Phocanema decipiens (Nematoda). International Journal for Parasitology 6, 403-11. LEDERER,

GRACE, A. A. & BUNNEY, B. s. (1985). Dopamine. In

Neurotransmitter Actions in the Vertebrate Central Nervous System, pp. 285-319 (ed. Rogawski, M. A. & Barker, J. L.) New York: Plenum Press. GRATION, K. A. F . , CLARK, R. B. & USHERWOOD, P . N . R.

S24

-(1979). Three types of L-glutamate receptor on junctional membrane of locust fibres. Brain Research 171, 360-4. GREENBERG, M. J., PAYZA, K., NACHMAN, R. J., HOLMAN, G.

M. & RICE, D. A. (1988). Relationships between the FMRFamide-related peptides and other peptide families. Peptides 9 (Suppl. 1), 125-35. GREENINGLOH, G., RIENITZ, A., SCHMITT, B., METHFESSEL, C , ZENSEN, M., BEYREUTHER, K., GUNDELFINGER, E. D. & BETSZ, H. (1987). The strychnine binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors. Nature, London 328, 215-20. GRIMMELIKHUIJZEN, c. j . P. (1986). FMRFamide-like peptides in the primitive nervous systems of coelenterates and complex nervous systems of higher animals. In Handbook of Comparative Opioid and Related Neuropeptides Mechanism; pp. 103-115; (ed. Stephano, G.) Boca Raton, Florida: CRC Press. GRIMMELIKHUIJZEN, C. J. P. & GRAFF, D. (1985). A r g - P h e -

amide-like peptides in the primitive nervous systems of coelenterates. Peptides 6, (Suppl. 3), 477-83. GRIMMELIKHUIJZEN, c. j . p. & GRAFF, D. (1986). Isolation

of < Glu-Gly-Arg-Phe-NH2 (Antho-RFamide) a neuropeptide from sea anemones. Proceedings of the national Academy of Sciences, USA 83, 9817-21. GRIMMELIKHUIJZEN, C. J. P . , GRAFF, D., KOIZUMI, O., WESTFALL, J. A. & MCFARLANE, I. D. ( 1 9 9 0 ) .

Neuropeptides in Coelenterates. In Proceedings of the Vth International Conference on Coelenterates (in the Press). GRIMMELIKHUIJZEN, C. J. P . , HAHN, M., RINEHART, K. L. &

(1988). Isolation of < Glu-Leu-LeuGly-Gly-Arg-Phe-NH2 (Pol-RFamide), a novel neuropeptide from hydromedusae. Brain Research 475, 198-203.

SPENCER, A. N.

GUSTAFSSON, M. K. S., LEHTONEN, M. A. I. & SUNDLER, F.

(1986). Immunocytochemical evidence for the presence of ' mammalian' neurohormonal peptides in neurones of the tapeworm Diphyllobothrium dendriticum. Cell and Tissue Research 243, 41-9. GUSTAFSSON, M. K. S., WIKGREN, M. C , KARHI, T. J. &

p. c. (1985). Immunocytochemical demonstration of neuropeptides and serotonin in the tapeworm Diphyllobothrium dendriticum. Cell and Tissue Research 240, 255-60. HAAS, H. L. (1985). Histamine. In Neurotransmitter Actions in the Vertebrate Central Nervous System, pp. 321-337, (ed. Rogawski, M. A. & Barker, J. L.). New York: Plenum Press. HANKE, w. & BREER, H. (1987). Characterization of the channel properties of a neuronal acetylcholine receptor reconstituted into planar lipid bilayers. Journal of general Physiology 90, 855-79. HARDIE, R. c. (1987). Is histamine a neurotransmitter in insect photoreceptors ? Journal of Comparative Physiology 161, 201-13. HARDIE, R. c. (1988). Effect of antagonists on putative histamine receptors in the first visual neuropile of the housefly, (Musca domesticd). Journal of experimental Biology 138, 221-41. HARDIE, R. c. (1989). A histamine-activated chloride channel underlying synaptic transmission at a photoreceptor synapse. Nature, London 339, 704—6. SCHOT, L.

HARVEY, R. J., VREUGDENHIL, E., BARNARD, E. A. & DARLISON, M. G.

(1990). Cloning of genomic and

Evolutionary aspects of transmitter molecules cDNA sequences encoding an invertebrate yaminobutyric acidA receptor subunit. Biochemical Society Transactions 18, 438-9. HILDEBRAND, J. G., TOWNSEL, J. G. & KRAVITZ, E. A. ( 1 9 7 4 ) .

Distribution of acetylcholine, choline acetyltransferase and acetylcholinesterase in regions and single identified axons of the lobster nervous system. Journal of Neurochemistry 23, 951-63. HILLE, B. (1984). Ionic Channels in Excitable Membranes. Sunderland, MA, USA: Sinauer Associates. HILLE, B. (1988). Evolutionary origin of electrical excitability. In Cellular Mechanisms of Conditioning and Behavioural Plasticity, (ed. Wood, C. D., Alkon, D. L. & McGaugh, J. L.), pp. 511-518. New York: Plenum Press. HILLE, B. (1989). Ionic channels: Evolutionary origins and modern roles. Quarterly Journal of Experimental Physiology 74, 785-804. HIRATA, T., KUBOTA, I., TAKABATAKE, I., KAWAHARA, A.,

SHIMAMOTO, N. & MUNEOKA, Y. (1987). Catch-relaxing peptide isolated from Mytilus pedal ganglia. Brain Research 422, 374-6. HOLDEN-DYE, L., KROGSGAARD-LARSEN, P . , NIELSEN, L. &

R. j . (1989). GABA receptors on the somatic muscle cells of the parasitic nematode Ascaris suutn: Stereoselectivity indicates similarity to a GABA-A agonist recognition site. British Journal of Pharmacology 48, 841-50. HOLDEN-DYE, L. & WALKER, R. j . (1990). Classical GABA and glycine receptor antagonists preferentially block the response to acetylcholine in the nematode Ascaris. Neuroscience Letters. Suppl. 38, 5111. WALKER,

HOLLMAN, M., o'SHEA-GREENFIELD, A., ROGERS, S. W. & HEINEMANN, s. (1989). Cloning by functional expression of a member of the glutamate receptor family. Nature, London 342, 643-8. HORVITZ, H. R., CHALFIE, M., TRENT, C , SULSTON, J. E. &

S25

and distribution of some monoamines in tissues of the sunflower star, Pyconopodia hellanthoides (Echinodermata). Journal of Neurochemistry 28, 573-9. KANDEL, E. R-, FRAZIER, W. T., WAZIRI, R. & COGGESHALL,

R. E. (1967). Direct and common connections among identified neurones in Aplysia. Journal of Neurophysiology 30, 1352-76. KATZ, P. S. & HARRIS-WARRICK, R. M. ( 1 9 9 0 ) .

Neuromodulation of the crab pyloric central pattern generator by serotonergic/cholinergic proprioceptive afferents. Journal of Neuroscience 10, 1495-512. KEENAN, c. L. & KOOPOWITZ, H. (1982). Physiology and in situ identification of putative aminergic neurotransmitters in the nervous system of Gyrocotyle fimbriata, a parasitic flatworm. Journal of Neurobiology 13, 9-21. KEENAN, c. L. & KOOPOWITZ, H. (1984). Ionic basis of action potentials in identified flatworm neurones. Journal of Comparative Physiology A. 155, 197-208. KISIEL, M. J., DEUBERT, K. H. & ZUCKERMANN, B. M. ( 1 9 7 6 ) .

Biogenic amines in the free-living nematode Caenorhabditis briggsae. Experimental Aging Research 2, 37-44. KLEMM, N. (1985). The distribution of biogenic monoamines in invertebrates. In Neurobiology: Comparative Aspects of Aminergic Neurons, pp. 280-96, (ed. Gilles, R. & Balthazer, J.). Berlin: Springer. KLEMM, N. & AXELSSON, s. (1973). Determination of dopamine, noradrenaline and 5-hydroxytryptamine in the cerebral ganglia of the desert locust, Schistocerca gregaria Forsk. (Insecta. Orthoptera). Brain Research 57, 289-98. KNOCK, S. L., NAGLE, G. T., LIN, C-Y., MCADOO, D. J . &

(1989). II. Aplysia brasiliana neurones R3/R-14: Primary structure of the myoactive histidinerich basic peptide and its prohormone. Peptides 10, 859-67. KUROSKY, A.

p. D. (1982). Serotonin and octopamine in the nematode, Caenorhabditis elegans. Science. 216, 1012-14. HOUSE, c. R. (1973). An electrophysiological study of neuroglandular transmission in the isolated salivary glands of the cockroach. Journal of experimental Biology. 58, 29-43.

brains of platyhelminthes. Trends in Neuroscience 5, 77-9. KORN, H., SOTELO, c. & CREPEL, F. (1983). Electrotonic coupling between neurons in rat lateral vestibular nucleus. Experimental Brain Research 16, 255-75.

HUCHO, F., OBERTHUR, W. & LOTTSPEICH, F. ( 1 9 8 6 ) . T h e

KRAVITZ, E. A., BELTZ, B. S., GLUSMAN, S., GOY, M. F . ,

EVANS,

ion channel of the nicotinic acetylcholine receptor is formed by the homologous helices Mil of the receptor subunits. FEBS Letters 205, 137-42. JOHNSON, c. D. & STRETTON, A. o. w. (1985). Localization of choline acetyltransferase with identified motoneurones of the nematode, Ascaris. Journal of Neuroscience 5, 1984-92. JOHNSON, C. D. & STRETTON, A. O. W. (1987). G A B A - l i k e

immunoreactivity in inhibitory motor neurones of the nematode, Ascaris. Journal of Neuroscience 7, 223-35. JOHO, R. H., MOORMAN, J. R., VAN DONGEN, A. M. J., KIRSCH, G. E., SILBERBERG, H., SCHUSTER, G. & BROWN,

A. M. (1990). Toxin and kinetic profile of rat brain type III sodium channels expressed in Xenopus oocytes. Molecular Brain Research 7, 105-13. JUDGE, S. E., KERKUT, G. A. & WALKER, R. J. ( 1 9 7 7 ) .

Properties of an identified synaptic pathway in the visceral ganglion of Helix aspersa. Comparative Biochemistry and Physiology 57C, 101-6. JUORIO, A. v. & ROBERTSON, H. A. (1977). Identification

KOOPOWITZ, H. & KEENAN, L. (1982). The primitive

HARRIS-WARRICK, R. M., JOHNSTON, M., LIVINGSTONE, M. S., SCHWARTZ, T. & SIWICKI, K. K. ( 1 9 8 5 ) . T h e W e l l

modulated lobster: the roles of serotonin, octopamine and proctolin in the lobster nervous system. In Model Neurol Networks and Behaviour (ed. Selverston, A.), pp. 339-360. New York: Plenum Press. KRAVITZ, E. A., GLUSMAN, S., HARRIS-WARRICK, R. M . , LIVINGSTONE, M. S., SCHWARTZ, T. & GOY, M. F. ( 1 9 8 0 ) .

Amines and a peptide as neurohormones in lobsters: actions on neuromuscular preparations and preliminary behavioural studies. Journal of experimental Biology 89, 159-75. KUPFERMANN, I. & WEISS, K. R. (1981). T h e role Of

serotonin in arousal of feeding behaviour of Aplysia. In Serotonin Neurotransmission and Behaviour, (ed. Jacobs, B. L. & Gelperin, A.), pp. 255-287; Cambridge, Mass: MIT Press. KUROKI, Y., KANDA, T., KUBOTA, I., FUJISAWA, Y., IKEDA, T., MIURA, A., MINAMITAKE, Y. & MUNEOKA, Y. ( 1 9 9 0 ) A

molluscan neuropeptide related to crustacean

R. J. Walker and L. Holden-Dye

S26

hormone, RPCH. Biochemical Biophysical Research Communications 167, 273—9. LEACH, L., TRUDGILL, D. L. & GAHAN, P . B. ( 1 9 8 7 ) .

Immunocytochemical localization of neurosecretory amines and peptides in the free-living nematode, Goodeyus ulmi. Histochemical Journal 19, 471-5. LEAKE, L. D. & WALKEH, R. j . (1980). Invertebrate

MACHADO, C. R. S., MACHADO, A. B. M. & PELLEGRINO, J.

(1972). Catecholamine-containing neurons in Schistosoma mansoni. Zeitschrift Zellforschung 124, 230-7. MCKAY, D. M., HALTON, D. W . , ALLEN, J. M. &

Neuropharmacology. Glasgow: Blackie. LEE, M. B . , BUEDING, E. & SCHILLER, E. L. ( 1 9 7 8 ) . T h e

occurrence and distribution of S-HT in Hymenolepis diminuta and H. nana. Journal of Parasitology 64, 257-64. LEES, G., BEADLE, D. J . , NEUMANN, R. & BENSON, J. A.

(1987). Responses to GABA by isolated insect neuronal somata: pharmacology and modulation by a benzodiazepine and a barbiturate. Brain Research 401, 267-78. LEROITH, D . , ROBERTS, C , LESNIAK, M. A. & ROTH, J .

(1986). Receptors for intercellular messenger molecules in microbes: Similarities to vertebrate receptors and possible implications for diseases in man. Experientia 42, 782-8. LEROITH, D . , SHILOACH, J . , BERELOWITZ, M . , FROHMAN, L. A., LIOTTA, A. S., KRIEGER, D. T. & ROTH, J. ( 1 9 8 3 ) . A r e

messenger molecules in microbes the ancestors of the vertebrate hormones and tissue factors. Federation Proceedings 42, 2602-7. LI, c. & CALABRESE, R. L. (1987). FMRFamide-like substances in the leech. III. Biochemical characterization and physiological effects. Journal of Neuroscience 7, 595-603. LIEBESWAR, G., GOLDMAN, J. E., KOESTE, J. & MAYERI, E.

(1975). Neural control of circulation in Aplysia III. Neurotransmitters. Journal of Neurophysiology 38, 769-79. LLINAS, R., BAKER, R. & SOTELO, c. (1974). Electronic coupling between neurons in the cat inferior olive. Journal of Neurophysiology 37, 560—1. LLOYD, p. E. (1982). Cardioactive neuropeptides in gastropods. Federation Proceedings of the Federated American Society of Experimental Biology 41, 2948-52. LLOYD, P . E., FRANKFURT, M . , STEVENS, P . , KUPFERMANN, I.

& WEISS, K. R. (1987). Biochemical and immunocytochemical localization of the neuropeptides FMRFamide, SCP-A, SCP-B, to neurons involved in the regulation of feeding in Aplysia. Journal of Neuroscience 7, 1123-32. LUETJE, C. W . , PATRICK, J. & SEGUELA, P . ( 1 9 9 0 ) .

synaptic transmission in the cerebral ganglion of Aplysia. Journal of Neurophysiology 53, 1016-37.

Nicotine

receptors in the mammalian brain. FASEB Journal 4, 2753-2760. LUMMIS, s. c. R. & SATTELLE, D. B. (1985). Binding of N[Propionyl-3H] propionylated a-bungarotoxin and L —[benzilic-4,4-3H] quinuclidinyl bezilate to CNS extracts of the cockroach, Periplaneta americana. Comparitive Biochemistry and Physiology 80C, 75-83. LUTZ, E. M. & TYRER, N. M. (1988). Immunohistochemical

localization of serotonin and choline acetyltransferase in sensory neurons of the locust. Journal of Comparative Neurology 267, 335—42. MCCAMAN, M. w. (1986). Uptake and metabolism of [3H]adenosine by Aplysia ganglia and by individual neurons. Journal of Neurochemistry 47, 1026—31. MCCAMAN, R. E. & WEINREICH, D. (1985). Histaminergic

FAIRWEATHER, I. (1989). The effects of cholinergic and serotonergic drugs on motility in vitro of Haplometra cylindracea (Trematoda: Digenea). Parasitology 99, 241-52. MADDRELL, s. H. p. & PHILLIPS, J. E. (1975). Secretion of hypo-osmotic fluid by the lower Malpighian tubules of Rhodnius prolixus. Journal of experimental Biology 62, 671-3. MAGEE, R. M., FAIRWEATHER, I., JOHNSTON, C. F., HALTON,

D. w. & SHAW, c. (1989). Immunocytochemical demonstration of neuropeptides in the nervous system of the liver fluke, Fasciola hepatica. Parasitology 98, 227-38. MALHERBE, P . , SIGEL, E., BAUR, R., PERSOHN, E., RICHARDS,

j . G. & MOHLER, H. (1990). Functional characteristics and sites of gene expression of the alt jix, y2-isoform of the rat GABA-A receptor. Journal of Neuroscience 10, 2330-7. MANCILLAS, J. R., MCGINTY, J. F . , SELVERSTON, A. I., KARTEN, H. & BLOOM, F. E. ( 1 9 8 1 ) .

Immunocytochemical localization of enkephalin and Substance P in retina and eyestalk neurones of lobster. Nature, London 293, 576-8. MANSOUR, T. E. (1984). Serotonin receptors in parasitic worms. Advances in Parasitology 23, 1-36. MARDER, E. (1976). Cholinergic motoneurones in the stomatogastric system of the lobster. Journal of Physiology 257, 63-86. MARDER, E. E., HOOPER, S. L. & EISEN, J. S. ( 1 9 8 7 ) .

Multiple neurotransmitters provide a mechanism for the production of multiple outputs from a single neuronal circuit. In Synaptic Function (ed. Edelman, G. M., Gall, W. E. & Cowman, W. M.), pp. 305-327. New York: Wiley. MAT JAIS, A. M . , SHARMA, R., PEDDER, S., KUBOTA, I . , MUNEOKA, Y. & WALKER, R. j . (1990). The actions of the Catch Relaxing Peptide, CARP, on identified Helix central neurones. Comparative Biochemistry and Physiology (in the Press). MAUE, R. A., KRANER, S. D., GOODMAN, R. H. & MANDEL, G.

(1990). Neuron-specific expression of the rat brain Type II sodium channel gene is directed by upstream regulatory elements. Neuron 4, 223-31. MAYERI, E., KOESTER, J., KUPFERMAN, I . , LIEBESWAR, G. & KANDEL, E. R. (1974). Neural control of circulation in Aplysia. I. Motoneurones. Journal of Neurophysiology 37,458-75. MOORE, G. J., THORNHILL, ] . A., GILL, V., LEDERIS, K. & LUKOWIAK, K. (1981). An arginine vasotocin-like neuropeptide is present in the nervous system of the marine mollusc Aplysia californica. Brain Research 206, 213-18. MORTON, D. B. & EVANS, p. D. (1984). Octopamine release from an identified neurone in the locust. Journal of experimental Biology 113, 269-87. NAMBU, J. R., MURPHY-ERDOSH, C , ANDREWS, P. C , FEISTNER,

G. j . &

SCHELLER, R. H.

(1988). Isolation and

Evolutionary aspects of transmitter molecules

S27

characterization of a Drosophila neuropeptide gene. Neuron 1, 55-61. NASSEL, D. R. (1988). Serotonin and serotoninimmunoreactive neurons in nervous system of insects. Progress in Neurobiology 30, 1-85.

PENZLIN, H.

NASSEL, D. R., MAYER, E. P. & KLEMM, N. (1985). M a p p i n g

PRICE, D. A., DAVIES, N. W., DOBLE, K. E. & GREENBERG, M.

and ultrastructure of serotonin-immunoreactive neurons in the optic lobes of three insect species. Journal of Comparative Neurology 232, 190-204.

(1989). Neuropeptides - Occurrence and functions in insects. Naturwissenschaften 76, 243-52. PRICE, D. A. (1986). The evolution of a molluscan cardioregulatory neuropeptide. American Zoologist 26, 1007-15. j . (1987). The variety and distribution of the FMRFamide-related peptides in molluscs. Zoological Science 4, 395-410.

NIEWIADOMSKA, K. & MOCZON, T. (1982). The nervous

PRICE, D. A. & GREENBERG, M. j . (1977). Structure of a

system of Diplostomum pseudospathaceum Niewiadomska, (Digenea, Diplostomatidae): I. Nervous system and caetotaxy in the cercaria. Zeitschrift fiir Parasitenkunde 68, 295-304. NISTRI, A. (1985). Glutamate. In Neurotransmitter Actions in the Vertebrate Central Nervous System, pp. 101-123, (ed. Rogawski, M. A. & Barker, J. L.). New York : Plenum Press.

molluscan cardioexcitatory neuropeptide. Science 197, 670-1. RAMIREZ, J-M. & ORCHARD, I. (1990). Octopaminergic modulation on the forewing stretch receptor in the locust Locusta migratoria. Journal of experimental Biology 149, 255-79.

NODA, M., SHIMIZU, A., TANABE, T., TAKAI, T., KANYANO, T., IKEDA, T., TAKAHASHI, H., NAKAYAMA, H., KANAOKA, Y., MINAMINO, N . , KANGAWA, K.., MATSUO, H., RAFTERY, M. A., HIROSE, T., INAYAMA, S., HAYASHIDA, H., MIYATA,

T. & NUMA, s. (1984). Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. Nature, London 312, 121-7. NODA, M., TAKAHASHI, H., TANABE, T., TOYOSATO, M., KIKOYOTANI, S., FURUTANI, Y., HIROSE, T., TAKASHIMA, H., INAYAMA, S., MIYATA, T. & NUMA, S. ( 1 9 8 3 ) .

Structural homology of Torpedo californica acetylcholine receptor subunits. Nature, London 302, 528-32. NUMA, S., NODA, M., TAKAHASHI, H., TANABE, T., TOYASATO, M . , FURATANI, Y. & KIKYOTANI, S. ( 1 9 8 3 ) .

Molecular structure of the nicotinic acetylcholine receptor. Cold Spring Harbor Symposia of Quantitative Biology 48, 57-69. O'CONNOR, E. F . , WATSON, W. H. & WYSE, G. A. ( 1 9 8 2 ) .

Identification and localization of catecholamines in the nervous system of Limulus polyphemus. Journal of Neurobiology 13, 49-60. O'DONOHUE, T. L., MILLINGTON, W. R., HANDELMANN, G. E., CONTRERAS, P. C. & CHRONWALL, B. M. (1985). ON

the 50th aniversary of Dale's law: multiple neurotransmitter neurons. Trends in Pharmacology 6, 305-8. OLSEN, R. w. & TOBIN, A. j . (1990). Molecular biology of

GABA-A receptors. FASEB Journal 4, 1469-80. ONO, j . K. (1989). Synaptic connections in the buccal ganglia of Aplysia mediated by an identified neuron containing a CCK/gastrin-like peptide co-localised with acetylcholine. Brain Research 493, 212-24. ORCHARD, I. (1987). Adipokinetic hormone - an update. Journal of Insect Physiology 33, 451-63. ORIDO, Y. (1989). Histochemical evidence of the catecholamine-associated nervous system in certain schistosome cercariae. Parasitology Research 76, 146-9. OSBORNE, N. N. (1984). Phenylethanolamine-AT-methyltransferase and dopamine-/?-hydroxylase immunoreactivity and the occurrence of noradrenaline and adrenaline in the nervous system of the snail, Helix aspersa. Cell and Tissue Research 237, 605-8. O'SHEA, M. & BISHOP, c. A. (1982). Neuropeptide proctolin associated with an identified skeletal motoneuron. Journal of Neuroscience 2, 1242-51.

RAMOA, A. S., ALKONDON, M., ARCAVA, Y., IRONS, J., LUNT, G. G., DESHPANDE, S. S., WONNACOTT, S., ARONSTAM, R.

s. & ALBUQUERQUE, E. x. (1990). The anticonvulsant MK-801 interacts with peripheral and central nicotinic acetylcholine receptor ion channels. Journal of Pharmacology and Experimental Therapeutics 254, 71-82. RETZLAFE, E. j . (1957). Mechanisms of Mauthner cells. Journal of Comparative Neurology 107, 209-25. REUTER, M., DARHI, T. & SCHOT, L P. C. ( 1 9 8 4 ) .

Immunocytochemical demonstration of peptidergic neurons in the central and peripheral nervous systems of the flatworm, Microstomum lineare with antiserum to FMRFamide. Cell and Tissue Research 238, 431-6. RIBEIRO, P. & WEBB, R. A. (1983 a) The

synthesis of 5-

hydroxytryptamine from tryptophan and 5hydroxytryptophan in the cestode Hymenolepis diminuta. International Journal for Parasitology 13, 101-6. RIBEIRO, P. & WEBB, R. A. (19836). The occurrence and synthesis of octopamine and catecholamines in the cestode Hymenolepis diminuta. Molecular and Biochemical Parasitology 7, 53-62. ROBB, s. & EVANS, P. D. (1990). FMRFamide-like peptides

in the locust: Distribution, partial characterization and bioactivity. Journal of experimental Biology 149, 335-60. ROBB, s., PACKMAN, L. c. & EVANS, P. D. (1989). Isolation, primary structure and bioactivity of SchistoFLRFamide, a FMRFamide-like neuropeptide from the locust, Schistocerca gregaria. Biochemical and Biophysical Research Communications 160, 850-6. ROBERTS, C. J., RADLEY, T., POAT, J. A. & WALKER, R. J.

(1983). Occurrence of noradrenaline, dopamine and 5hydroxytryptamine in the nervous system of the horseshoe crab, Limulus polyphemus. Comparative Biochemistry and Physiology 74C, 437-40. ROBERTS, E. (1986). GABA: The road to neurotransmitter status. In Benzodiazepine/GABA Receptors and Chloride Channels - Structural and Functional Properties, pp 1-39, (ed. Olsen, R. W. & Venter, J. C.) New York: Alan R. Liss, Inc. ROGAWSKI, M. A. (1985). Norepinephrine. In Neurotransmitter Actions in the Vertebrate Central Nervous System, pp. 241-84, (ed. Rogawski, M. A. & Barker, J. L.) New York: Plenum Press. ROZENTAL, R., SCOBLE, G. T., ALBUQUERQUE, E. X., IDRISS,

R.J. Walker and L. Holden-Dye M . , SHERBY, S., SATTELLE, D. B . , NAKANISHI, K., KONNO, K., ELDEFRAWI, A. T. & ELDEFRAWI, M. E. ( 1 9 8 9 ) .

Allosteric inhibition of nicotinic acetylcholine receptor of vertebrates and insects by philanthotoxin. Journal of Pharmacology and Experimental Therapeutics 249, 123-9. SARGENT, P. B. (1977). Synthesis of acetylcholine by excitatory motoneurones in the central nervous system of the leech. Journal of Neurophysiology 40, 553-60. SATHANAN, A. H. & BURNSTOCK, G. (1976). Evidence for a non-cholinergic, non-aminergic innervation of the Venus clam heart. Comparative Biochemistry and Physiology 55C, 111-18. SATTELLE, D. B. (1985). Acetylcholine receptors. In Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 11, pp. 395-434, (ed. Kerkut, G. A. & Gilbert, L. I.) Oxford: Pergamon Press. SATTELLE, D. B. (1990). GABA receptors of insects. Advances in Insect Physiology 22, 1-113. SATTELLE, D. B., PINNOCK, R. D . , WAFFORD, K. A. & DAVID,

j . A. (1988). GABA receptors on the cell body membrane of an identified insect motor neuron. Proceedings of the Royal Society, London B, 232, 443-56. SAWADA, M. & CAGGESHALL, R. E. (1976). Ionic mechanism of 5-hydroxtryptamine-induced hyperpolarization and inhibitory junctional potential in bodywall muscle cells of Hirudo medicinalis. Journal of Neurobiology 7, 63-73.

S28

(1988). Neurobiology. 2nd Edn. Oxford: Oxford University Press. SHINOZAKI, H. (1980). The pharmacology of the excitatory neuromuscular junction in the crayfish. Progress in Neurobiology 14, 121-55. SIMMONS, P. j . & HARDIE, R. c. (1988). Evidence that histamine is a neurotransmitter of photoreceptors in the locust ocellus. Journal of experimental Biology 138, 205-19. SHEPHERD, G. M.

SITHIGORNGUL, P . , STRETTON, A. O. W. & COWDEN, C.

(1990). Neuropeptide diversity in Ascaris: An immunocytochemical study. Journal of Comparative Neurology 294, 362-76. SIWICKI, K. K., BELTZ, B. S. & KRAVITZ, E. A. ( 1 9 8 7 ) .

Proctolin in identified serotonergic, dopaminergic and cholinergic neurons in the lobster, Homarus americanus. Journal of Neuroscience 7, 522—32. SIWICKI, K. K. & BISHOP, C. A. (1986). Mapping of proctolin-like immunoreactivity in the nervous systems of lobster and crayfish. Journal of Comparative Neurology 243, 435-53. SOMBATI, s. & HOYLE, G. (1984). Glutamergic central nervous transmission in locusts. Journal of Neurobiology 15, 507-76. SMITH, c. u. M. (1989). Elements of Molecular Neurobiology. Chichester: John Wiley. STONE, B. F., BINNINGTON, K. C. & NEISH, A. L. ( 1 9 7 8 ) .

Norepinephrine as principal catecholamine in a specific neurone of an invertebrate. Experientia 34,

SAWADA, M . , GIBSON, D. & MCADOO, D. J. ( 1 9 8 4 « ) . L -

Glutamate acid, a possible neurotransmitter to anterior aorta of Aplysia. Journal of Neurophysiology 51, 375-86. SAWADA, M . , MCADOO, D. J . , ICHINOSE, M. & PRICE, C. H.

(19846). Influence of glycine and neuron R-14 on contraction of the anterior aorta of Aplysia. Japanese Journal of Physiology 34, 747-67. SCHILT, J., RICHOUX, J-P. & DUBOIS, M. P. ( 1 9 8 1 ) .

Demonstration of peptides immunologically related to vertebrate neurohormones in Dugesia lugubris (Turbellaria: Tricladida). General and Comparative Endocrinology 43, 331-5. SCHLESINGER, D. H . , BABIRAK, S. B. & BLANKENSHIP, J. E.

(1981). Primary structure of an egg laying peptide from the atrial gland of Aplysia californica. In Neurohypophyseal Peptide Hormones and Other Biologically Active Peptides, (ed. D. H. Schlesinger) pp. 138-150. North Holland, Amsterdam. SCHOFIELD, p. R. (1989). The GABA-A receptor: molecular biology reveals a complex picture. Trends in Pharmacological Science 10, 476-8. SCHOFIELD, P . R., DARLISON, M . G., FUJITA, N . , BURT, D. R., STEPHENSON, F. A., RODRIQUEZ, H . , RHEE, L. M., RAMACHANDRAN, J . , REALE, V., GLENCORSE, T. A.,

P. H. & BARNARD, E. A. (1987). Sequence and functional expression of the GABA-A receptor shows a ligand-gated receptor super-family. Nature, London 328, 211-27. SCHWARTZ, R. D. & MINDLIN, M. c. (1988). Inhibition of the GABA receptor-gated chloride ion channel in brain by noncompetitive inhibitors of the nicotinic receptor-gated cation channel. Journal of Pharmacology and Experimental Therapeutics 244, 963-70. SEEBURG,

T. w. (1989). Purine receptors and their pharmacological roles. Advances in Drug Research 18, 292-429.

STONE,

SULSTON, J., DEW, M. & BRENNER, S. ( 1 9 7 5 ) .

Dopaminergic neurons in the Nematode Caenorhabditis elegans. Journal of Comparative Neurology 163, 215-26. SWANN, j . w. & CARPENTER, D. o. (1975). Organisation of receptors for neurotransmitters on Aplysia neurons. Nature, London 258, 751-4. TAKEUCHI, A. & TAKEUCHI, N. (1964). The effect of crayfish muscle of iontophoretically applied glutamate. Journal of Physiology 170, 296-317. TAKEUCHI, A. & TAKEUCHI, N. (1966). A study of the inhibitory action of y-aminobutyric acid in neuromuscular transmission in the crayfish. Journal of Physiology 183, 418-32. THORNHILL, J. A., LUKOWIAK, K., COOPER, K. E. & VEALE,

w. L. (1981). Arginine vasotocin, an endogenous neuropeptide of Aplysia suppresses the gill withdrawal reflex and reduces the evoked synaptic input to central gill motor neurons. Journal of Neurobiology 12, 533-44. TOMOSKY-SYKES, T. K., JARDINE, I., JUELLER, J. F & BUEDING, E. (1977). Sources of error in neurotransmitter analysis. Analytical Biochemistry 83, 99-108.

TOYOSHIMA, c. & UNWIN, N. (1988). Ion channel

acetylcholine receptor reconstruction from images of postsynaptic membranes. Nature, London 336, 247-50. TRIMMER, B. A., KOBIERSKI, L. A. & KRAVITZ, E. A. ( 1 9 8 7 ) .

Purification and characterization of FMRFamide-like immunoreactive substances from the lobster nervous system: isolation and sequence analysis of two closely

Evolutionary aspects of transmitter molecules related peptides. Journal of Comparative Neurology 266, 16-26. TRITT, s. H. & BRYNE, j . H. (1982). Neurotransmitters producing and modulating opaline gland contraction in Aplysia californica. Journal of Neurophysiology 48, 1347-61. TURNER, j . D. & COTTRELL, G. A. (1977). Properties of an identified histamine-containing neurone. Nature, London 267, 447-8. USHERWOOD, P. N. R. & CULL-CANDY, S. G. ( 1 9 7 5 ) .

Pharmacology of somatic nerve muscle synapses. In, Insect Muscle (ed. Usherwood, P. N. R.), pp. 207-280, London: Academic Press. USHERWOOD, p. N. R. & GRUNDFEST, H. (1964). Inhibitory postsynaptic potentials in grasshopper muscle. Science 143, 817-18. VANDER MAELEN, c. P. (1985). Serotonin. In Neurotransmitter Actions in the Vertebrate Central Nervous System, pp. 201-240 (ed. Rogawski, M. A. & Barker, J. L.). New York: Plenum Press. VENTER, J. C , DI PORZIO, U., ROBINSON, D. A., SHREEVE, S. M., LAI, J., KERLAVAGE, A. R., FRACEK, S. P., LENTES, K-U. & FRASER, c. M. (1988). Evolution of neurotransmitter

receptor systems. Progress in Neurobiology 30, 105-69. VENTURINI, G., CAROLEI, A., PALLADINI, G., MARGOTTA, V.

& LAURO, M. G. (1983). Radioimmunological and immunocytochemical demonstration of methionine encephalin in planaria Dugesia gonocephala. Comparative Biochemistry and Physiology 74C, 23-6. VENTURINI, G., STOCCHI, F., MARGOTTA, V., RUGGIERI, S., BRAVI, D., BELLA, P . & PALLADINI, G. ( 1 9 8 9 ) . A

pharmacological study of dopaminergic receptors in planaria. Neuropharmacology 28, 1377-82. WACHTEL, H. & KANDEL, E. R. (1971). Conversion of synaptic excitation to inhibition at a dual chemical synapse. Journal of Neurophysiology 34, 56-68. WALKER, R. j . (1986). Transmitters and modulators. In The Mollusca, vol. 9, pp. 279-485, (ed. Willows, A. O. D.). Orlando: Academic Press. WALKER, R. J., BOYD, P. J. & OSBORNE, N. N. ( 1 9 8 7 ) .

Immunocytochemical and physiological studies involving Substance P (SP), Arg-Vasotocin (AVT) and FMRFamide on central neurones and other tissues of Helix aspersa. In Neurobiology: Molluscan Models; (ed. Boer, H. H., Geraerts, W. P. M. & Joosse, J.) Proceedings of the Second Symposium of Molluscan Neurobiology. Amsterdam: North-Holland. WALKER, R. j . & HOLDEN-DYE, L. (1989). Commentary on the evolution of transmitters, receptors and ion channels in invertebrates. Comparative Biochemistry and Physiology 93A, 25-39.

S29 WANG-BENNETT, L. T . , SOVAN, M. L. & GLANTZ, R. M.

(1988). Immunocytochemical studies of the distribution of acetylcholine in the crayfish brain. Journal of Comparative Neurology 273, 330—43. WATSON, A. H. D. (1990). Ultrastructural evidence for GABAergic input onto cereal afferents in the locust (Locusta migratorid) Journal of experimental Biology 148,509-15. WEBB, R. A. (1985). The uptake and metabolism of 5hydroxytryptamine by tissue slices of the cestode Hymenolepis diminuta. Comparative Biochemistry and Physiology 80C, 305-12. WEBB, R. A. (1986). The uptake and metabolism of Lglutamate by tissue slices of the cestode Hymenolepis diminuta. Comparative Biochemistry and Physiology 8SC, 161-2. WEBB, R. A. (1988). Endocrinology of Acoelomates. In Endocrinology of Selected Invertebrate Types, pp. 31-62, (ed. Laufer, H. & Downer, R. G. H.). New York: Alan R. Liss. Inc. WEBB, R. A. & EKLOVE, H. (1989). Demonstration of intense glutamate-like immunoreactivity in the longitudinal nerve cords of the cestode, Hymenolepis diminuta. Parasitology Research 75, 545-8. WEBB, R. A. & MIZUKAWA, K. (1985). Serotonin-like immunoreactivity in the cestode Hymenolepis diminuta. Journal of Comparative Neurology 234, 431-40. WELSH, j . H. & MOORHEAD, M. (1960). The quantitative distribution of serotonin in invertebrates, especially in their nervous system. Journal of Neurochemistry 6, 146-69. WERMAN, R., DAVIDOFF, R. A. & APRISON, M. H. ( 1 9 6 8 ) .

The inhibitory action of glycine on spinal neurons in the cat. Journal of Neurophysiology 31, 81-95. WIKGREN, M. c. & REUTER, M. (1985). Neuropeptides in a microturbellarian-whole mountimmunocytochemistry. Peptides 6, Suppl. 3, 471-5. WILLETT, J. D. (1980). Control mechanisms in Nematodes. In Nematodes as Biological Models, vol. 1, 197-225, (ed. Zuckerman, B. M.). New York: Academic Press. WILLETT, J. D . , TURNER, R. A., MCRAE, M. & BOLLINGER, J.

A. (1979). Catecholamine sensitive adenylate cyclase activity during development and senescence in the nematode Panagrellus redivivus. Age 2, 126. WRIGHT, D. j . & AWAN, F. A. (1978). Catecholaminergic structures in the nervous system of three nematode species, with observations on related enzymes. Journal of Zoology London 185, 477-89.