Communication between neurones: Current concepts

Neurochemistr~ lmernational, Vol. 3, No. 1, pp. 3 16. 1981

0197-0186/'81/010003-14502.00/0 © 1981 Pergamon Press Ltd.

Printed in Great Brilain

REVIEW

C O M M U N I C A T I O N BETWEEN NEURONES: CURRENT CONCEPTS N. N. OSBORNE Nuffield Department of Ophthalmology, The University of Oxford, Oxford, OX2 6AW, U.K.

Abstraet--Neurotransmitters are chemicals which have the specific function of transferring information from one neurone to another at specific sites called synapses. This concept is discussed in relation to the experimental evidence which suggests that neurotransmitters may be released non synaptically and that certain neurones may utilise more than one transmitter substance. The term 'modulator' is also discussed and compared with what is understood to be a "neurotransmitter'.

In the second half of the 19th century, a number of discussions took place between proponents of the ~cell theory', who considered that neurones were independent units, and those who thought that nerve cells were generally connected to one another by protoplasmic bridges. At the time it was difficult to obtain convincing histological evidence to show whether or not there was continuity between neurones. Only with the advent of e!ectron microscopy was it possible to show that each neurone is completely surrounded by its own membrane. During the period in which the issue of continuity vs contact between neurones remained unsettled, relatively little experimentation was done on how nerve impulses were transferred between cells. The concept that neurones communicate information among themselves by means of transmitter substances is generally attributed Elliott (1905). Langley (1901) was, however, the first to observe that injection of an extract of adrenal gland into an animal stimulated tissues which were innervated by the sympathetic nerves. This prompted Elliott (1905) to inject adrenaline into experimental animals. He found that, like the crude extract, it produced the response evoked by electrical stimulation of sympathetic nerves. Elliott (1905) made the then brilliant suggestion that adrenaline might be released from sympathetic nerves and thus cause a response in muscle cells with which the nerves form junctions. Langley discouraged further speculations by Elliott until more facts were available--that took quite a while. It was, in fact, 1921 when the first definite evidence for neurochemical transmission was obtained by Otto Loewi,

who was then working in Graz, Austria. Loewi (1921) put the heart of a frog into a bath in which the heart could be kept beating. The fluid bathing the heart was allowed to perfuse a second heart. When the heart's vagus nerve was stimulated, the beat of the second heart was slowed, showing that some substance had been liberated from the stimulated vagus nerve and transported by the fluid to influence the perfused heart. The substance was later identified by Dale (1937) as acetylcholine. Against this background, extracellular and intracellular recordings of synaptic potentials had been obtained, demonstrating unequivocally that transmission of information from cell to cell was chemical in nature. The analyses by Katz, Eccles and colleagues (Del Castillo and Katz, 1954; Eccles, 1957; Fatt, 1954) were particularly responsible for producing these compelling data. A landmark occurred, however, in 1959 when Furshpan and Potter (1959) demonstrated that transmission at the giant motor synapse of the crayfish was not chemically, but electrically, mediated. Electrical transmission between neurones has since been demonstrated by a number of authors (for review see Bennett, 1977), though there seems little doubt that the vast majority of synapses in the central nervous system are chemical in nature. This opinion is based primarily on morphological data; gap junctions are infrequent. Chemical communication between neurones would seem therefore to be the primary way of conveying information from one cell to another in nervous systems and the chemical involved in this transfer is known as a neurotransmitter substance. Table 1 3

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Table 1. Substances which have been proposed to be involved in the communication between neurones (partly from Barchas et aL 1978). Adrenalin Dopamine Norepinephrine Epinephrine Tyramine Octopamine Phenylethylamine Phenylethanolamine Dimet hoxyphenylet hylamine (DMPEA) Tet rahydroisoquinolines Serotonin (5-hydroxytryptamine) Melatonin Tryptamine Dimethyltryptamine (DMT) 5-Methoxytryptamine 5-Met hoxydimethyltryptamine 5-Hydroxydimet hyltryptamine (bufotenin) Tryptolines ATP Acetylcholine Carnosine Histamine ~,-Aminobutyric acid (GABA) 7-Hydroxybutyrate (GHB) Glycine Yaurine Purine Aspartate Glutamate Prostaglandins Corticosteroids Estrogens Testosterone Thyroid hormone Bombesin Cholecystokinin (CCK) fl-Endorphine Gastrin Neurotensin Proctolin Prolactin Oxytocin Substance P Somatostatin Angiotensin Luteinizing hormone releasing hormone (LHRH) Vasopressin Vasoactive intestinal polypeptide (VIP) Adrenocroticotropic hormone (ACTH) Thyroid releasing hormone (TRH) Sleep factor delta

whether a substance under question (i.e. putative transmitter) acts in an identical way to a neurotransmitter. The complexity of the vertebrate CNS makes it technically difficult or impossible, for example, to show release of a putative transmitter from a specific set of nerve terminals in response to stimulation. Inherent in the definition of a neurotransmitter was always the assumption that each neurone utilised only one transmitter substance. Neurotransmission was also assumed to take place at synapses and it was presumed that the neurotransmitter acts transynaptically. In recent years, however, it has become clear that brain function cannot be understood simply in terms of neurones acting as 'relaying stations in a chemically-transmitting telephone exchange'. For example, certain neurones (e.g. amacrine cells) in the retina, which is a part of the CNS, function almost entirely without the need for any propagated action potentials, suggesting that "local' electrical and chemical circuits exist. At particular retinal synapses, activation of the synapse may involve alteration (modulation) of a continuous release of a transmitter rather than triggering of a release of a burst of neurotransmitter. The transmitters may also be released from "non-synaptic' sites like dendrites.

THE CONCEPT OF NEUROTRANSMISSION Figure 1 is an idealised representation of a serotonergic synapse which will help to define current concepts of a chemical neurotransmitter. Serotonin is

~.J¢Eyoked iRe-uptake presents a list of some compounds which have been suggested as having transmitter functions. A number of criteria have been formulated by various authors (e.g. Paton, 1958; Werman, 1966) to try to define a neurotransmitter substance, the main purpose being to provide an experimental basis for examining

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-synaptic cell Pig. 1. Schematic illustration of mechanisms existing at serotonergic synapses.

Review synthesised in two enzymatic steps from the amino acid tryptophan. The first enzyme, tryptophan-hydroxylase, present only in serotonergic neurones, is the rate-limiting step; the second, 5-hydroxytryptophandecarboxylase, is not an enzyme specific to serotonergic neurones. The formed serotonin occurs not only in the cytoplasm of the nerve terminal, but also in presynaptic structures called vesicles or granules. These organelles are specialised for the uptake, storage and release of serotonin. Through mechanisms which are still poorly understood, depolarisation of the presynaptic membrane by an action potential causes the release of serotonin into the synaptic cleft, where it diffuses across to the post-synaptic receptor. The transmitter interacts with receptors on the postsynaptic membrane and this interaction produces a change in the post-synaptic membrane's permeability to ions and initiates a complex chain of intracellular reactions. The signal is terminated by removal of the transmitter. For serotonin, there is a specific reuptake mechanism which actively transfers the substance from the synaptic cleft back into the pre-synaptic cell where it may be reused. The essential function of a neurotransmitter molecule may be summarised in the following way. The

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substance is stored and synthesised in the neurone, released during nerve activity and interacts with specific receptors on the post-synaptic membrane, thus causing a change in the post-synaptic cell's activity. Since it is, however, experimentally difficult or impossible to show the 'release' and 'interaction with receptors' of a substance, a number of criteria have been formulated which should be fulfilled before a substance can seriously be considered to have a neurotransmitter role. These criteria are shown in Table 2, which is according to Barchas et al. (1978).

CAN NEUROTRANSMITTERS BE RELEASED NON-SYNAPTICALLY? The classical view of neurotransmission is that it takes place in specific areas of the neurones known as the synapses. A synapse is, by definition the area where the ending of a neurone containing the transmitter substance comes into close contact with a part of the post-synaptic cell containing the receptors (see Fig. 1). The idea that neurotransmitters can be released from non-synaptic areas is new and has been suggested for several amine neurotransmitters, par-

Table 2. Possible criteria for distinguishing a neurotransmitter from a neuromodulator in the central nervous system (from Barchas et al., 1978) N eurotransmitter

The substance must be present in presynaptic elements of neuronal tissue, possibly in an uneven distribution throughout the brain. Precursors and synthetic enzymes must be present in the neuron, usually in close proximity to the site of presumed action. Stimulation of afferents should cause release of the substance in physiologically significant amounts. Direct application of the substance to the synapse should produce responses which are identical to those of stimulating afferents. There should be specific receptors present which interact with the substance; these should be in close proximity to presynaptic structures. Interaction of the substance with its receptor should induce changes in postsynaptic membrane permeability leading to excitatory or inhibitory postsynaptic potentials. Specific inactivating mechanisms should exist which stop interactions of the substance with its receptor in a physiologically reasonable time frame. Interventions at postsynaptic sites or through inactivating mechanisms. The effects of stimulation of afferents or of direct application of the substance should be equally responsive. N euromodulator

The substance is not acting as a neurotransmitter, in that it does not act transsynapticaUy. The substance must be present in physiological fluids and have access to the site of potential modulation in physiologically significant concentrations. Alterations in endogenous concentration of the substance should affect neuronal activity consistently and predictably. Direct application of the substance should mimic the effect of increasing its endogenous concentrations. The substance should have one or more specific sites of action through which it can alter neuronal activity. Inactivating mechanisms should exist which account for the time course of effects of endogenously or exogenously induced changes in concentrations of the substance. Interventions which alter the effects on neuronal activity of increasing endogenous concentrations are increased by exogenous administration.

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ticularly serotonin and noradrenatine (see Dismukes, 1979). The evidence for such a mode of release comes from a number of sources. Electron microscopy has revealed that noradrenergic and serotonergic varicosities contain aggregates of small agranular synaptictype vesicles ii.e. the machinery associated with synapsesi. Some of these axonal varicosities make synaptic contact but the majority do not {Ajika and H6kfelt, 1973; Calas et al., 1974: Chan-Patay, 1976; Descarries et al., 1977t. It is therefore possible for the release of a transmitter to take place in both synaptic and non-synaptic areas. Striking evidence was also produced by Descarries et al. (1957, 19771 that nonsynaptic areas can specifically take up exogenous serotonin or noradrenaline, a function previously thought to be associated with synapses Tritiated serotonin or noradrenaline (in conjunction with or without specific uptake inhibitors: for noradrenaline desmethylimipramine was used) was applied in rico to the neocortex, and then the specific uptake of [3H]-serotonin and [3H]-noradrenaline was analysed by electron microscopic autoradiography. Less than 55,1 of the [3H]-serotonin or [3H]-noradrenaline label associated with varicosities made typical synapses. In contrast, 50% of unlabelled boutons were seen in the immediate vicinity. It was not, however, possible to determine the percentage of labelled or unlabelled boutons which actually made synapses, because many junctions are missed in the sections. Nevertheless, these results by Descarries et al. (1975, 1977) do indicate that uptake of serotonin and noradrenaline is not restricted to synaptic areas, it has also been pointed out [Dismukes, 1979) that the small amount of varicosities demonstrating synaptic junctions could not account for the amount of serotonin and noradrenaline released following in vit,o stimulation [see Tanaka et al.. 1976: Reader et al.. 1976). More direct evidence for non-synaptic release has come from the work of Branton et al. (1978). These authors studied the release of a peptide from cells in the abdominal ganglion of Aplysia. The processes of these cells terminate in connective tissue and release the products directly into the haemolymph for diffusion within the ganglion. Evoked spike activity of these bag cells produces characteristic responses in nearby target cells, such as R 15. Direct application of bag cell extract elicited identical responses in R 15. It is thought that the active factor is a peptide. It is, of course, uncertain whether the peptide is acting as a transmitter or hormone. Nonsynaptic release of hormones is not unusual and their release directly into the cerebrospinal fluid or blood is a normal occurrence. Two good examples in the invertebrates are the

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neurosecretor) system in the heart of t f e l i ~ ((ottrell and Osborne, 1969) and the vcna cava ~,vstem of octopus (Alexandrowicz, 1964). Dunn 11978i has also pointed out that the cerebrospinal fluid is not iust a sewer for getting rid of "unwanted" subsLmces, but probably serves to transport molecules, particularly neuropeptides, to target cells. The new finding that neurones m the subsiantia nigra can release dopamine, not only from their axonal terminals, but also from their dendrites, can be taken to prove thal a nonsvnaptic release of Iransmitters occurs. Release of dopamme from the dcndrites and possibly, cell bodies from dopammergic neurones in the st|bstantia nigra can be brought about by depolarisation either itt rito (Nieloullon el al.. 1977) or in slices (Geffcn et al.. 1976) and it is blocked by the removal of calcium ions. The idea that dopamine might be released from dendrites was engendered by an important paper pt, blished by Bj6rklund and Lindwall (19751. Using the sensitive glyoxylic acid histofluorescence technique to localise monoamines, they showed that dopamine occurs not only in the axons of dopaminergic neurones, but also in their dendrites. Earlier studies by Parizek et aL t1971) had revealed that these dopammergic dendrites accumulated labelled dopamme. ;.uld immunocytochemical staining methods (H6kfch ct u/., 1973: Pickel el al.. 1975) disclosed the presence ol tyrosine hydroxylase in the dendrites. The dendrites in the dopamine neurones in the substantia nigra therefore contain dopamine and the enzymes necessary for its synthesis and like the dopamine synapses, possess a specialised transport system for the uptake of dopamine from the extracellular space. The dendrites do not apparently contain synaptic-type ves-icles {Cuello and Iversen, 1977: Sotelo, 1971 ), and so there is no morphological indication that dopamme can be released from the dendrites. Yet the experiments by Nieloullon et al. (19771 and Geffen et al. (1976} have shown that newly synthesised [-~H]dopamine or [3H]-dopamine taken up from the extracellular medium, can be released from the dendrites by depolarisation. Support for this comes from the studies of Korf et al. (1976), who observed an increase in the concentration of a dopamine metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), in the rat substantia nigra, when the nigrostriatal pathway, was antidromically stimulated. To summarise, non-synaptic release of a transmitter has not been established unequivocally, but from the evidence available it appears a likely occurrence. As stated by Dismukes (1979), "'non-synaptic release would tit rather nicely with the merging concepts of

Review the electrophysiological action of amine and peptides".

DO NEURONES USE MORE THAN ONE NEUROTRANSMITTER? A classical view in neurobiology, that each neurone has the ability to synthesise, store and release only one transmitter substance, is generally attributed to Dale (see Burnstock, 1976). Dale's principle, as it has come to be known, is based on vast amounts of experimental data but has been questioned from time to time, e.g. the cholinergic link in adrenergic transmission (Burn and Rand, 1965; Koelle, 1962). Dale's principle is not, of course, a dogma, but a working hypothesis that should be abandoned or modified as new facts become known. The problem inherent in discussing the validity of Dale's principle is that of deciding what constitutes a transmitter substance. As discussed above, the essential features of a neurotransmitter are that it Ca) is located in the neurone, (b) is released from the neurone under physiological levels of stimulation and (c) induces an effect on the membrane potential of the post-synaptic cells. While it may be possible to make a case for certain neurones containing more than one "transmitter-type molecule', the evidence that a neurone can release more than one transmitter affecting post-synaptic cells is very weak.

CO-EXISTENCE OF TRANSMITTER MOLECULES IN INVERTEBRATE NEURONES Histochemical studies, using the fluorescence method of Falck and Owman (1965) to discriminate between dopamine and serotonin, allowed Kerkut et al. (1967) and Welsh and Williams (1970) to conclude that certain snail and planarian neurones contain both dopamine and serotonin. Such studies, based on the visual discrimination of the colours green (dopamine), yellow (serotonin) and yellow-green (serotonin and dopamine) are susceptible to serious criticism (see Osborne, 1979). Indeed, Kerkut and coworkers in later papers (Loker et al., 1975; Kerkut, 1976) refer to certain visceral neurones as serotonincontaining. These were the very cells which were supposed to contain both serotonin and dopamine (Kerkut et al., 1967). The injection of dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP), the precursors of dopamine and serotonin, into snails, and their subsequent examination, were used to substantiate the claim that these neurones contain both

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amines (Kerkut et al., 1967). Since it is generally accepted that DOPA-decarboxylase and 5-HTPdecarboxylase are the same enzyme, it is not surprising that dopamine or serotonin are formed following injection of DOPA or 5-HTP, and this cannot be taken as proving the conclusions made by Kerkut et al. (1967). The biochemical analysis of hand-dissected gastropod neurones has yielded several results suggesting that some neurones contain more than one transmitter-like substance. One of the first articles to appear along these lines was that of Brownstein et al. (1974). These authors analysed a number of hand-dissected Aplysia neurones for a whole spectrum of putative transmitter substances. They concluded that a number of cells contained more than one transmitter substance. For example, one cell designated R 14 was shown to contain approximately 10-5 M serotonin, 10- 5 M histamine, 10-4 M octopamine and 10 - 3 M glutamate (Brownstein et al., 1974). Another neurone, R 11, contained 10 -5 M serotonin, 10 - 6 M histamine, 10-5 M octopamine, 10 -4 M acetylcholine and 10-3M glutamate (see Brownstein et al., 1974; Saavedra, 1978). A re-examination of these observations made by a number of authors showed that the data published by these authors were questionable. Neither McCaman and McCaman (1978), who used radiochemical procedures, nor Farnham et al. (1978), who applied gas-chromatography mass spectrometry methods, could find any octopamine in the cell bodies, yet their methods would have detected less than 15~0 ~ of the concentration of the amine which Brownstein et al. (1974) reported as occurring in cell RI4. Another important work based on hand-dissected gastropod neurones and challenging the validity of Dale's principle arose from a study by Emson and co-workers (Emson and Fonnum, 1972; Hanley et al., 1974). These authors analysed a known serotonincontaining neurone (Osborne, 1978) for its choline acetyltransferase content and found that the cell had an enzyme activity of 21 pmole per cell per hour (Hanley et al., 1974). In an earlier paper, the choline acetyltransferase activity of the cell was described as being 'small' (Emson and Fonnum, 1972). These studies were carried out on the garden snail Helix; the same cell in Aplysia was reported to contain either trace amounts of (Brownstein et al., 1974), or no (Weinreich et al., 1973), choline acetyltransferase, i.e. whether these serotonin cells (GSCs) contain or lack choline acetyltransferase is undoubtedly due to the dissection of individual neurones (see Osborne, 1974, 1977). Glial investment, adhering satellite cells and

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exogenous substances from damaged neurones are all possible sources of error (Fig. 2) and have to be taken into consideration. Moreover, the actual dissection of the neurone is of major importance, since the chemical integrity of the cell must always be maintained (see Osborne, 1977). In a re-analysis of the GSCs from only trace quantities of choline acetyhransferase. Great care was taken in this study to dissect the cell cleanly and maintain its integrity. Osborne (1977) considered that the trace quantities of choline acetyltransferase associated with the GSCs were exogenous in origin, because of the overall morphology of the neurones and their relationship to other structures (see Fig. 2). The finding that the cells could not form acetylcholine from [14C]-choline was taken to support these conclusions (Osborne. 19771.

CO-EXISTENCE OF TRANSMITTER MOLECULES IN VERTEBRATE NEURONES All the important evidence for the co-existence of transmitter-like substances in the same neurones derives from studies which involve immuno-histochemical procedures. Chan-Palay and colleagues (1978) combined autoradiography, fluorescence microscopy. microspectrophotometry and immunofluorometry to study the serotonin and substance P-containing neurones in the rat brain. They found that certain neurones which displayed both an uptake-storage capacity for tritiated serotonin and a formaldehyde-induced fluorescence with spectral characteristics identical to those of the serotonin fluorophor, also exhibited detectable substance P-like immunoreactivity. The conclusion from their study was that neurones exist which contain either serotonin, substance P, or serotonin and substance P (Chan-Palay et al., 1978). H6kfelt et al. (1978), using immunohistochemical procedures, also produced evidence for the presence of substance P-like immunoreactivity in serotonin-containing neurones in the mammalian CNS. Furthermore, after intraventricular injection of the neurotoxins 5,6, or 5,7-dihydroxytryptamine (Baumgarten et al., 1971; 1973), certain serotonin fibres and fibres exhibiting substance P-like reactivity disappeared. The neurotoxins are generally considered to be fairly specific in destroying serotonin neurones, so these studies were interpreted as showing that substance P and serotonin co-exist, not only in certain neurones but in the endings of these neurones (H6kfelt et at., 1978). This is an important observation in view of the fact that a transmitter substance must be localised in the terminals of the neurones for participation in synaptic release.

The co-existence of neuropeptides and aminc-transmitter molecules has been demonstrated in a number of other neurones. H/Skfelt et al. (1977)showed that certain sympathetic ganglia neurones of the guineapig contain both noradrenaline and a somatostatinlike substance immunoreactivity. In the cells of the rat superior cervical ganglion, noradrenaline and enkephalin-like immunoreactivity are present (Schultzberg et al., 1979). Similarly, enkephalin-like immunoreactivity exists in the catecholamine-containing cells in the adrenal medulla (Schultzberg et aI., 1978). There is also evidence that cholecystokinin-like immunoreactivity exists in some nigral dopamine neurones (H6kfelt et al., 1978). Although the evidence as it stands (outlined above) suggests that certain vertebrate neurones contain monoamine transmitter molecules in conjunction with "transmitter-like' peptide molecules (indicating that Dale's principle could be faulty), the following points must be taken into consideration. H6kfelt et al. (1978j have stated that ~'interpretation of immunohistochemical results and subsequent conclusions should be met with caution, in view of the specificity of immunoreactions'. Antibodies may cross-react with substances (peptides) closely related by structure to those used as antigens. It is therefore possible that some peptide-like reactivity is not due to the peptide under question. Another point, is the evidence sufficiently convincing for any of the neuropeptides to be considered as transmitters in the same sense as acetylcholine or noradrenaline? It has been suggested that they may act as neuromodulators (discussed below). Is it also possible that some tissue sections have cells containing e.g. either serotonin and substance P and that they are superimposed on each other? Finally, in the study by Chan-Palay et al. (1978), certain neurones, for example, were shown to contain both serotonin and substance P, but the level of serotonin was very low compared with the amount present in cells which contain this amine alone. Is it then possible that some neurones which contain substance P accumulated some exogenous serotonin released from neurones which utilise it as a transmitter'? It is known that natural substances not synthesised by a given neurone, and different from its transmitter, may also be taken up sequentially by the transport system present in its plasma membrane and synaptic vesicles. neither of which shows absolute specificities (Shaskan and Snyder, 1970; Slotkin et al., 1978) and become cotransmitters. Serotonin produced in pineal cells can be taken up by synaptic vesicles of pineal sympathetic nerves and stored in the same vesicles containing noradrenaline iJaim-Etcheverry and Zieher, 1973, 1975j.

Review

Fig. 2. Electron micrograph showing a cross section of an axon from a giant serotonin cell situated in the metacerebral ganglion of Helix. The cell's somata was injected with [3H]-serotonin and following autoradiography grains can be seen to be restricted to the axon. The photograph illustrates the deep glial infolding into the cell's axon thus demonstrating the interrelationship between the serotonin cell and other structures. (Photograph by courtesy of Dr. V. W. Pentreath.)

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These authors have shown that oc/opamine, which is present in pineal nerves as a consequence of the unspecilicity of noradrenaline synthesis, also shares the vesicular storage sites with noradrenaline and serotonin (Jaim-Etcheverry and Zieher. 1975).

WHY SHOULD A NEURONE NOT CONTAIN MORE THAN ONE TRANSMITTER'? Each neurone, supplied with a complete set of genes, possesses the potential to synthesise the complete enzymatic machinery for all transmitter substances. Is it possible, therefore, that the synthesis of certain substances is incompletely suppressed'? The small a m o u n t s of substance {Transmitter molecule) may simply not be sutficient to cause a functional effect and therefore, from the evolutionary point of view, a complete suppression of its synthesis was unnecessary. If this was the case, it could explain, for example, the neurones containing substance P-immunoreactivity (Chan-Palay et al., 1978) or the extremely low serotonin Ic,,els (2 x 10 s MI present in a cholincrgic cell lacetylcholine content 3.8 × 10"* M) {Osborne, 1977). It is known that isolated sympathetic neurones can be manipulated to have either noradrenergic or cholinergic characteristics (Reichardt and Patterson, 1977). It was shown that sympathetic neurones grown in the virtual absence of non-neuronal cells developed the ability to synthesise and accumulate radioactive noradrenaline from [3H]-tyrosine, but synthesised little [3H]-acetylcholine from labelled choline. In the presence of non-neuronal cells, or a medium conditioned by them, the neurones produced vast amounts of acetylcholine from choline, while the ability to accumulate noradrenaline was inversely related to the percentage of conditioning medium

present. Results from studies on single neurones were consistent with these findings tsee Table 3). The obvious question posed by the studies by Reichardt and Patterson (1977) is. what arc the factors respons i n e lor determining the nature of the tran~,mitters produced by sympathetic and other neurones? Hill and Hendry (1977)and Bunge and colleagues (Ross ct al., 19771 have shown thai the development of choimcrgic characteristics m adrenergic neurones is agedependent. Chun alld Patterson 11977) have also shown that oMer sympathetic neurones m culture lose their ability to respond to conditioning media. It is known that certain cells differentiating into cholinergic neurones become unresponsive to nerve growth factors and at the same time lose their ability to produce catecholamines {see Burnstock, 1978). It is also thought that the normal electrical activity imposed on a neurone during the first weeks after birth plays a part in determining the choice of transmitter. This receives support from experiments where it was found that when spinal impul to sympathetic ganglia of young mice is cut, further development of adrenergic metabolism is reduced (see Patterson et al., 19781. Experiments on the vertebrate retina are consistent with their observations. It has been shown by Lain and colleagues 11978) that different types of retinal nearones use different transmitter substances, depending on the cells with which these neurones synapse. For example, in the skate and goldfish retina, some horizontal cells which contact cones exclusively, possess an active transport system for G A B A and the ability to synthesise GABA, whereas other horizontal cells. which contact rods exclusively, possess neither of these mechanisms. These results could be interpreted as indicating that the transmitter used by horizontal cells is determined by the types of photoreceptors with which these cells make contact.

Table 3. Transmitter synthesis by single neurones grown in various conditions Growth conditions Control 20'!;i CM 50'!. CM Heart cells

ACH NA ACh and NA Negative neurones la) neurones (b) neurones (c) neurones (d) I) 2 15 26

!8 21 15 3

0 0 2 1

0 o 2 i)

ACH: NA ratio 0.002 0.15 2.4 v0

Neurones were grown from primary sympathetic neurones of the rat in control medium in 2(1 and 50'!; conditioned medium (CM), and on rat heart monolayers. Neurones grown with 2tl or 50% conditioned medium [conditioned with C6 cells) were labelled 25 28 days after plating. The values are the numbers of single neurones making only ACh(a), the number making only NA(b), the number making both ACh and NA(c), the number making neither transmitter (d}, and the average ratio of ACh to NA synthesis m the pooled results, including wells with more than one neurone (Reichardt and Patterson, 1977).

Review CAN NEURONES RELEASE MORE THAN ONE TRANSMITTER? The best evidence that a single neurone can release two transmitter substances and that each substance has an influence on postsynaptic cells comes from work on the giant serotonin cell (GSC) in the snail (see Cottrell, 1977). The evidence that the GSCs utilise serotonin as a neurotransmitter is very good and for present purposes need not be further discussed (see Cottrell, 1977; Osborne, 1979). However, the evidence that, in addition, these cells utilise acetylcholine as a transmitter, is open to criticism. As discussed above, the presence of choline acetylase in the GSCs could not be confirmed by Osborne (1977), nor could it be demonstrated that the cell could form acetylcholine from its precursor. This contrasts with the findings of Cottrell and co-workers, who not only demonstrated that choline acetylase occurs in the GSCs (Hanley et al., 1974), but used bioassay procedures to show that the cell apparently contains acetylcholine (Cottrell, 1977). Cottrell's group were also able to show that hexamethonium, the acetylcholine antagonist, changed the nature of the GSC's elicited response on follower cells in the buccal ganglia. Stimulation of the GSC to high frequencies of activity (up to 12 Hz) resulted in the appearance of biphasic depolarising potentials in the follower buccal cells. These were not blocked by high calcium, indicating that they were mediated by monosynaptic pathways. Hexamethonium selectively antagonised the first phase of this response, while the second phase was less affected. At the concentration used, hexamethonium did not alter the depolarising effect mediated by a known dopamine-containing neurone. Furthermore, iontophoretically applied serotonin depolarised the follower buccal cells and exposure of the cell to hexamethonium for periods of up to 20 min did not alter this response, but completely blocked the depolarising effect of applied acetylcholine. The data presented by Cottrell and collaborators (see Cottrell, 1977) are consistent with the view that the GSC can use serotonin and acetylcholine as neurotransmitters. Their data rely heavily, however, on hexamethonium having no effect on serotonin receptors and the GSC making monosynaptic contacts with certain buccal follower cells. Was it certain that the neuronal responses to application of acetylcholine and serotonin to the buccal follower cells were, in fact, recorded from the same neurone? It is also difficult to interpret the observation made by Cottrell and collaborators (see Cottrell, 1977; Cottrell and Macon, 1974) that the fast post-synaptic response

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of the follower buccal neurone to repetitive stimulation of the GSC is blocked by hexamethonium. Beyond the possibility that this blockage is due to unknown, non-specific effects of the relatively high drug concentrations used (1 mM), it is possible that the block is only minor, given the problems inherent in reproducibly quantifying graded responses to repeated stimulation and the variability of neuronal reactions to acetylcholine in gastropod molluscs. It would have been nice had other anticholinergic drugs been used by Cottrelrs group to support the data with hexamethonium. Their data would also have been more persuasive if it had been possible to influence the serotonin or apparent acetylcholine levels in the GSC and then analyse the 'release' of these substances on follower buccal cells. This could be done by using the technique of Tauc et al. (1974) who injected acetylcholinesterase into a cholinergic cell and showed transmission to be abolished. A number of other studies have suggested that more than one transmitter may be released from the same neurone. Singh, in a series of papers (see Singh, 1964: Singh and Singh, 1966) claimed that no fewer than three putative transmitters (acetylcholine, serotonin and histamine) were released during stimulation of the vagus nerves supplying the stomach of the frog but that during the change of season one or more became the dominant transmitter. The main argument against the suggestion is that the vagus nerve in the frog contains a mixture of separate axons and that these fibres may be either histaminergic, serotonergic or cholinergic in function. There also appears to be good evidence for the simultaneous release of ATP and catecholamines from the adrenal medullary vesicles in perfused adrenal glands and in certain adrenergic nerves where noradrenaline is a transmitter (see Burnstock, 1976; Chubb, 1977). A case has been made out for ATP being a transmitter in the gastro-intestinal tract and in certain other organs too (Burnstock, 1975) and if this is accepted, then the data suggest that two transmitters, noradrenaline and ATP, are released from the same neurone. It still, however, remains to be shown that they have post-synaptic effects characteristic of transmitter molecules (see Table 1). In cholinergic cells, e.g. those associated with the electric organs of certain fish, the storage vesicles, like those from the adrenal medulla, contain a fixed amount of ATP in relation to its acetylcholine content (Dowdall et al., 1974). Indirect evidence suggests that acetylcholine and ATP are secreted from cholinergic neurones. As already pointed out, the release of two putative transmitters from the same neurone does not necess-

12

N. N OSBORNI,

arily challenge the validity of Dale's principle. It must yet be shown that the released substances act post-synaptically. It is known that neurones can release substances which, apparently, do not function in the manner of a transmitter (modulator? see below). For example, the chromogranins and dopamine-fl-hydroxylase are known to be secreted from adrenergic neurones (Smith et al., 1970). It also appears as if certain lysosomal hydrolases are secreted from the adrenal medulla (see Chubb, 1977). Nigral dopaminergic neurones have been shown to contain acetylcholinesterase (see Dray, 1979) and this enzyme is released from these cells (Greenfield et al., 1980). Acetylcholinesterase has been shown to be secreted from the adrenal medulla and other nigral neurones in the CNS (see Burnstock, 1976; Chubb, 1977). There is also evidence that adenosine can be released from central neurones, stimulate cyclic A M P formation through specific receptors and alter postsynaptic electrical activity (see Fox and Kelly, 1978). A transmitter role has been suggested for adenosine (see McIlwain, 1973). THE CONCEPT OF NEUROMODULATOR? The concept of neuromodulation is fairly new and, so far, incompletely developed. The term 'modulators' is being used increasingly to characterise the action of substances which do not fit within traditional concepts of neurotransmitters (see Table 1). In contrast to neurotransmitters, a neuromodulator is not responsible for direct transfer of a nerve signal from the preto post-synaptic element; instead it alters neuronal activity in other ways. Unlike neurotransmitters, a neuromodulator need not have specific receptors; instead it might affect neurotransmitter synthesis, release, receptor interactions, re-uptake or metabolism. Neuromodulators may be released from neurones, glial or true secretory cells to amplify or dampen local synaptic activity by altering the effect of the true neurotransmitters. Neuromodulators are not generally associated with synaptic vesicles and may be synthesised on demand, as are the prostaglandins. The term 'modulator' was described by Florey (1967) as "any compound of cellular or non-synaptic origin that affects the excitability of nerve cells and represents a normal link in the regulatory mechanism that governs the performance of the nervous system". The more modern definitions of modulators (Barchas et al., 1978; Chan-Palay, 1977) distinguish them more precisely from neurotransmitters as being compounds which are important in general communication between nerve cells, but which operate in a 'hormonelike fashion" rather than transynaptically. Neuromo-

dulation is not unlike a hormonal effect in the usual meaning of the word, i.e. on the one hand. an action at some distance from the point of release, and on the other, the effect lasting more than a few milliseconds (see Bloom, 1979). Barchas et al. (1978) have set up a number of preliminary criteria to characterise a neuromodulator and these are summarised in Table 1. Examples of what are considered neuromodulators by different authors in certain situations are numerous in literature. Barchas et aL (1978)considers the adrenal glucocorticoids as an example. These steroids are known to influence the steady-state levels of tyrosine-hydroxylase in the brain, which in turn affect (i.e. modulate) the activity of the catecholaminergic neurones (Ciaranello et al., 1975). Another example, quoted by Rotsztejn (1980), is the involvement of serotonin in the suckling-induced prolactin release. In this particular case, restoration of hypothalamic serotonin levels after inhibiting biosynthesis of the amine does not by itself induce the homonal response unless the adequate physiological stimulus, i.e. suckling reflex, is applied (Kordon et al., 1973). Florey (1967), in his review, has quoted numerous examples of what he considers to be neuromodulators originating from nerve cells, glial cells, gland cells. neurosecretory cells or ependyma cells, and occurring throughout the animal kingdom. A recent finding and what I consider a good example of a neuromodulator. is the observation that the benzodiazepines can interact with GABA-containing neurones. Here the studies by Costa and collaborators are particularly relevant (Guidotti et al., 1978). These authors found that fresh membranes from nervous tissue bound GABA very poorly compared with membranes prepared in the classical manner of freezing and detergent disposal. The reason for this was that the fresh tissue contains an inhibitor which blocks the GABA receptors, but the more drastic membrane techniques eluted this inhibitor. Further analysis showed the inhibitor to be thermostable, resistant to trypsin treatment and have a molecular weight of 15,000. The benzodiazepines were found to displace or block the action of this inhibitor and allow the fresh membranes to bind GABA with high affinity. Costa and colleagues have thus provided evidence for an endogenous regulator of GABA activity and an endogenous ligand for the bezodiazepine receptor, as well as a role for the benzodiazepines in interaction with this regulator. GENERAL CONCLUSIONS I have tried in this review to discuss the various types of chemical communication between neurones.

Review The separate terms 'neurotransmission', 'non-synaptic release' and 'neuromodulators' have been used, though clearly they overlap in meaning. For example, if non-synaptic release takes place, the substance may diffuse over a distance and affect or modulate a large population of cells on a given stimulus. Even the distinction between neurotransmission and neurosecretion of hormones is less sharp, and indeed overlaps, as pointed out by Sharrer more than a decade ago (Sharrer, 1969). Orrego (1979) states that a neurotransmitter is always present in vesicles (granules) and released by exocytosis. While it is true that they always appear to be associated with vesicles (granules), it is also known that these organelles contain non-transmitter molecules e.g. magnesium. It is also debatable whether transmitters are released by exocytosis or merely 'diffuse' out of the presynaptic end (Tauc, 1979; Israel et al., 1979; Zimmerman, 1979). Because of the overlapping nature of what we actually understand by neurotransmission, non-synaptic release and neuromodulator, Barchas et al. (1978) use the term 'neuroregulator' to cover them all. While the term 'neuroregulator' implies that definitions should not be dogmatically applied, it helps little in recognising the various forms of neuronal communication. Bloom (1979) has proposed that 'time course' 'spatial distribution' and 'energy' should be taken into consideration in categorising neuronal communication. These domains seem too ambiguous, however. Whereas the terms 'neurotransmission', 'non-synaptic release' and 'neuromodulator' are not ideal, they are nevertheless exceedingly useful for the understanding of the various forms of communication between cells. Most important is to recognise that one particular substance should not be thought of as functioning in one way. Even acetylcholine is not thought to function exclusively as a neurotransmitter. It is present, for example, in corneal epithelium where it seems to have a non-neurotransmitter function (see Fogle and Neufeld, 1979). One of the well-known 'hormone' actions of tuberoin-fundibular dopamine neurones consists of inhibiting prolactin secretion. The dopamine is released by nerve-endings located in the hypothalamus and is transported by the portal blood to affect prolactin secretion by the pituitary through specific receptor sites (Neill et al., 1979). It is known that dopamine acts additionally as a neurotransmitter in various parts of the central nervous system; thus dopamine can act as a hormone or a neurotransmitter. A 'hormone' is defined by Guillemin (1977) as "a substance released by a cell, carried by blood or extracellular fluid and which affects another cell near or far".

13

With the recognition that a classical neurotransmitter may function as a neurohormone or a neuromodulator it is clear that any debate on the validity of Dale's principle (see Osborne, 1979; Burnstock, 1976) approaches a circulatory argument. The demonstration that more than one transmitter exists in a neurone proves little. It has to be demonstrated that a single neurone utilises more than one transmitter substance. Even allowing for the controversy associated with the definition of a neurotransmitter, it is fair to conclude that the paucity of relevant data does not contradict the validity of Dale's principle. It would be equally wise to remember that the evidence to support Dale's principle is not as convincing as might have appeared in the 1950's.

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