Neurotransmitter Identification

To achieve NT status a substance must fulfil three main criteria:

(1) Presence. It perhaps goes without saying that the proposed transmitter must be shown to be present in the CNS and preferably in the area and at the synapses where it is thought to act.

(2) Release. Stimulation of the appropriate nerves should evoke a measurable release of NT.

(3) Identity of action. The proposed NT must produce effects postsynaptically which are identical physiologically (appropriate membrane potential changes) and pharmacologically (sensitivity to antagonists) to that produced by neuronal stimulation and the relased endogenous NT.

These criteria should be regarded as guidelines rather than rules. As guidelines they provide a reasonable scientific framework of the type of investigations that must be undertaken to establish the synaptic role of a substance. As rigid rules they could preclude the discovery of more than one type of neurotransmitter or one form of neurotransmission. Nevertheless, the criteria have been widely employed and often expanded to include other features which will be considered as subdivisions of the main criteria.

PRESENCE

Distribution and concentration

It is generally felt that a substance is more likely to be a NT if it is unevenly distributed in the CNS although if it is widely used it will be widely distributed. Certainly the high concentration (5-10 ^ol/g) of dopamine, compared with that of any other monoamine in the striatum or with dopamine in other brain areas, was indicative of its subsequently established role as a NT in that part of the CNS. This does not mean it cannot have an important function in other areas such as the mesolimbic system and parts of the cerebral cortex where it is present in much lower concentrations. In fact the concentration of the monoamines outside the striatum is very much lower than that of the amino acids but since the amino acids may have important biochemical functions that necessitate their widespread distribution, the NT component of any given level of amino acid is difficult to establish.

Nevertheless, useful information can be deduced from patterns of distribution. Glycine is concentrated more in the cord than cortex and in ventral rather than dorsal grey or white matter. This alone would be indicative of a NT role for glycine in the ventral horn, where it is now believed to be the inhibitory transmitter at motoneurons. GABA, on the other hand, is more concentrated in the brain than in the cord and in the latter it is predominantly in the dorsal grey so that although it is an inhibitory transmitter like glycine it must have a different pattern of activity.

Lesions in conjunction with concentration studies can also be useful. Section of dorsal roots and degeneration of afferent fibres produces a reduction in glutamate and substance P which can then be associated with sensory inputs. Temporary reduction of the blood supply to the cord causes preferential destruction of interneurons and a greater loss of asparate and glycine, compared with other amino acids and so links those amino acids with interneurons. Intrinsic neurons can also be destroyed through overactivity caused by kainic acid injections.

Subcellular localisation

A NT might be expected to be concentrated in nerve terminals and this can be ascertained since when nervous tissue is appropriately homogenised the nerve endings break off from their axons and surrounding elements and then reseal. Such elements are known as synaptosomes. They have been widely used to study NT release in vitro (Chapter 4) and some NT should always be found in them, at least if it is released from vesicles.

Synthesis and degradation

If a substance is to be a NT it should be possible to demonstrate appropriate enzymes for its synthesis from a precursor at its site of action, although peptides are transported to their sites of location and action after synthesis in the axon or distal neuronal cell body. The specificity of any enzyme system must also be established, especially if they are to be modified to manipulate the levels of a particular NT, or used as markers for it. Thus choline acetyltransferase (ChAT) may be taken as indicative of ACh and glutamic acid decarboxylase (GAD) of GABA but some of the synthesising enzymes for the monoamines lack such specificity.

After release there must be some way of terminating the action of a NT necessitating the presence of an appropriate enzyme and/or uptake mechanism. Such uptake processes can be quite specific chemically. Thus a high-affinity uptake (activated by low concentrations) can be found for glycine in the cord where it is believed to be a NT, but not in the cortex where is has no such action. This specific uptake can be utilised to map terminals for a particular NT, especially if it can be labelled, and also for loading nerves with labelled NT for release studies.

Of course, since CNS function depends on changes in the rate of neuronal firing, determined by a subtle balance between a number of different excitatory and inhibitory inputs, it may not always be necessary to destroy the NT rapidly. Excessive firing of a neuron may be controlled by activating a feedback inhibitory system or evoking presynaptic inhibition. There is also evidence for the release of the degrading enzyme together with NT at some purinergic (ATP) synapses (Kennedy et al. 1997) and possibly some cholinergic ones.

Pathways

If a substance (or its synthesising or degradative systems) can be demonstrated in particular neurons with a distinctive pattern of distribution, or bunched together into a well-defined nerve tract and/or nucleus, then this is not only good evidence for its role as a NT but it tells us something of its function. Indeed the distinct patterns of distribution of ascending monoamine pathways from brainstem nuclei could probably be considered as adequate evidence alone for their neurotransmitter role. In practical terms we can, of course, only study the release (and actions) of an endogenous NT if it can be evoked by stimulating an appropriate nerve pathway. Also the neurological and behavioural consequences of lesioning such pathways can tell us much about the functions of the NT. It is therefore useful to try to map NT pathways.

Receptors

If a NT is to be effective, there must be receptors for it to act on. Thus demonstrating the presence of receptors for the proposed NT at sites where it is found is further proof of its NT role. This could be done by recording some effect of the NT, e.g. change in neuronal firing, by establishing specific binding sites for it using it in a labelled form, or showing the presence of its receptor mRNA. Unfortunately a substance can bind to sites other than a receptor (e.g. uptake sites) and not all receptors are innervated.

RELEASE

A substance cannot be considered as a NT unless it is released. Unfortunately, although it may be possible to show the presence of a substance and some effect when it is applied directly to neurons its release may not be measurable for technical reasons. This is even more true if one strives for the ideal of demonstrating the release of an endogenous substance by physiological stimuli.

In the CNS access to the site of release is a major problem and attempts to achieve it have led to the development of a wide range of techniques of varying complexity and ingenuity or to short-cuts of dubious value (see Chapter 4). The feasibility of release studies in the CNS is to some extent dependent on the type of NT being studied. If we are dealing with a straightforward neural pathway with a number of axons going from A to B then by stimulating A and perfusing B we should be able to collect the NT. Unfortunately such arrangements are rare in the CNS and where they exist (e.g. corticospinal tract) it is not easy to perfuse the receiving (collecting) area. Sometimes the origin of a pathway is clear and easy to stimulate, e.g. NA fibres in the locus coeruleus, but fibre distribution in the cortex is so widespread that collection of sufficient amounts for detection can be very difficult, although current methods are beginning to achieve it.

These approaches are, in any case, only suitable for classical neurotransmitters. Those with slow background effects will probably not be released in large amounts. For such substances we require a measure of their utilisation, or turnover, over a much longer period of time. With NTs released from short-axon interneurons there are no pathways to stimulate and it becomes necessary to activate the neurons intrinsically by field stimulation, which is of necessity not specific to the terminals of the interneurons.

Apart from actually demonstrating release it is important to consider how NTs are released and whether they all need to be released in the same way, especially if they do different things. The variable time-courses of NT action referred to previously may require NTs to be released at different rates and in different ways, only some of which are achievable by, or require, vesicular mechanisms and exocytosis (see Chapter 4).

It should be remembered that with the possible exception of voltammetry when the monitoring electrode is sufficiently small to reach synapses, it is not the actual release of the NT that is being measured in perfusion studies. It is overflow. As discussed previously, most of any released NT is either physically restricted to the synapse or destroyed before it can diffuse away.

Magic Mushrooms Species

Figure 1.9 Comparison of the effects of an endogenously released and exogenously applied neurotransmitter on neuronal activity (identity of action). Recordings are made either of neuronal firing (extracellularly, A) or of membrane potential (intracellularly, B). The proposed transmitter is applied by iontophoresis, although in a brain slice preparation it can be added to the bathing medium. In this instance the applied neurotransmitter produces an inhibition, like that of nerve stimulation, as monitored by both recordings and both are affected similarly by the antagonist. The applied neurotransmitter thus behaves like and is probably identical to that released from the nerve

Figure 1.9 Comparison of the effects of an endogenously released and exogenously applied neurotransmitter on neuronal activity (identity of action). Recordings are made either of neuronal firing (extracellularly, A) or of membrane potential (intracellularly, B). The proposed transmitter is applied by iontophoresis, although in a brain slice preparation it can be added to the bathing medium. In this instance the applied neurotransmitter produces an inhibition, like that of nerve stimulation, as monitored by both recordings and both are affected similarly by the antagonist. The applied neurotransmitter thus behaves like and is probably identical to that released from the nerve

IDENTITY OF ACTION

Many people consider this to be the most important of all the criteria. Obviously a substance must have an effect of some kind if it is to be a NT but not all substances that have an effect on neurons need to be NTs. It may seem unnecessary to say this but the literature contains many accounts of the study of various substances on neuronal activity from which a NT role is predicted without any attempt to compare its effect with that of physiologically evoked (endogenous NT) effects. The importance of this safeguard is highlighted by the ease with which both smooth muscle and neurons will respond to a range of substances that are not released onto them as NTs. Thus the value of this criterion depends very much on the rigour with which it is applied and on its own is no more or less important than any other approach.

Ideally it should be shown that application of the proposed NT to a neuron, e.g. by iontopheresis (see Chapter 2), produces changes in membrane potential that are identical to and mediated by the same ionic mechanism as those produced by nerve stimulation and that the effects of both are equally overcome by an appropriate chemical antagonist. The basic system is outlined in Fig. 1.9. Clearly, changes in membrane potential can only be recorded if the neuron is large enough to take an intracellular electrode and even if it can be shown that the applied and released NT produce similar changes in membrane potential and share a common reversal potential and ionic mechanism this would not be so surprising, since the number of available ionic mechanisms is limited (i.e. both GABA and glycine produce hyperpolarisation by increasing chloride influx). Now that the properties of single ion channels can be recorded using modern patch-clamp techniques it will be necessary to show that application of the presumed NT produces identical changes in the frequency (ra), degree (y, amount of current conducted) and duration (r) of channel opening to that achieved by synaptic activation. Unfortunately such a detailed analysis is presently only applicable to relatively simple systems with restricted innervations.

The use of antagonists is absolutely vital but even they can give false positives. Thus GABA, B-alanine and glycine all produce hyperpolarisation of cord motoneurons by increasing chloride influx but only GABA is unaffected by strychnine. Since strychnine abolishes inhibition in the cord, GABA cannot be the inhibitory NT but other features (distribution, release) had to be satisfied before glycine rather than B-alanine was shown to have that role.

It must be remembered that a substance can only be shown to be identical in its action with that of a particular endogenous NT if the latter's precise mode of action is clearly established and easily studied. Thus it may be relatively easy to consider those NTs mediating classical postsynaptic excitation through distinct potentical change but more difficult for NTs which function over a much longer time-course and possible without producing recordable potentical changes. Nevertheless they are still NTs. Or are they?

The question is obviously an important one. Substances released from neurons are not always called neurotransmitters. Some of them are referred to as neuromodulators, neurohormones, neurotrophic factors or neurotoxins but since they all produce some effect on a neuron they could be said to have a transmitter role and justify the term neurotransmitter. On that basis every substance mentioned already and to be discussed further could be called a neurotransmitter. Even if we did try to distinguish between, say, a fast neurotransmitter and a slow neuromodulater effect, we have to realise that one substance can easily have both actions either at different synapses (ACh) or the same one (glutamate — ionotropic and metabotropic effects) and so could be both a neurotransmitter and a neuromodulater. If, however, the response they set up (transmit) has no reasonably quick and recordable effect, i.e. takes hours or days to develop (e.g. growth factors), or can actually kill the neuron (nitric oxide) it is difficult to conceive of them as neurotransmitters, however interesting they may be.

What this discussion does highlight, however, is that some modification is required to the standard dictionary definition of a neurotransmitter given in the introduction to this chapter, which sees a NT as a substance that transmits the impulse from one neuron to another neuron (or excitable cell). A more comprehensive definition of a NT might be

A substance preformed, stored and then released from a neuron by a calcium dependent exocytotic mechanism activated by invading action potentials which induces a change in excitability and function of an adjacent neuron without entering the bloodstream.

This description would cover the classical NTs such as glutamate, GABA, ACh, DA, NA, and 5-HT as well as some peptides and ATP. That is irrespective of whether the effect produced by them is basic to the actual process of transmitting an impulse from one neuron to another, as with glutamate and ACh, rapidly inducing inhibition (GABA) or just making the neuron more or less responsive to other inputs (monoamines, peptides).

There is no room within the definition for nitric oxide, the prostaglandins and steroids mainly because they are not released in a controlled manner by neuronal activity and only the last are preformed. Thus if they are to be classified as NTs then the definition must be simplified so that a NT becomes:

A substance produced in and released from a neuron to affect some aspect of neuronal function without being transported in the blood.

This would encompass those steroids synthesised in the CNS but not those entering it from the circulation (see Chapter 13).

While I feel that substances which only meet the abbreviated definition do not justify being called neurotransmitters, they will be treated as such in this text because this is the accepted practice and they are substances released from neurons to affect CNS function. Clearly, it is more important to distinguish between the different effects that a substance can produce when released from a nerve than to worry about what it is called. Nevertheless, it is unfortunate that the word neurotransmitter will inevitably be associated with the actual transmission of activity from one neuron to another and yet most of the substances we will be discussing do not actually do that.

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