neurotransmitters

neurotransmitters After Galvani had shown, in 1742, that electrical stimulation of the nerve to the muscle of a frog's leg caused the muscle to twitch, the idea gained ground that transmission from nerves to the ‘end organ’ was an electrical process. Today we know that only in very rare instances is transmission across a synapse — that is, between the end of a nerve and whatever it innervates — an electrical event. Virtually all neurotransmission is chemical. Nerves release one or more neurotransmitters, which act chemically on receptors in the membrane of the cells across the synaptic cleft. To detect neurotransmitters is a difficult task as the amounts released are minute and mechanisms exist that quickly remove the transmitter, leaving the system in a state of readiness for the arrival of the next nerve impulse. The first steps came from finding chemicals which could mimic the action of neurotransmitters. Muscarine was able to mimic the effects of stimulation of the heart by the vagus nerves, and its actions were blocked by atropine. Similarly, nicotine mimicked the effect of stimulating motor nerves to skeletal muscle, and this was blocked by curare.

Du Bois Reymond, in 1877, declared that chemical or electrical transmission were the only two real alternatives, although there was no data to decide unequivocally between the two. In 1904 Elliot suggested that adrenaline might be the transmitter in the sympathetic nervous system, and in the next year a nicotine-like substance was postulated by Langley as the transmitter in motor nerves to muscles. Dixon, in 1907, tried to extract from a heart the neurotransmitter released by the vagus, measuring the effect of the extract on another heart, but reached no conclusions. It was 1921 before Loewi performed the celebrated experiment in which two beating frog hearts were superfused in series, the perfusate of the first flowing over the second. Stimulation of the vagus nerve to the first heart caused its beat to slow and, after a brief interval, the second heart slowed too. While this experiment did not prove that the substance flowing over from the first heart was released from the vagus nerve, clearly something chemical rather than electrical slowed the second heart.

In the 1930s Dale and his school in England showed that acetylcholine was the transmitter at motor nerve endings in skeletal muscle (neuromuscular junctions) and Cannon and his colleagues in the US demonstrated that an adrenaline-like substance was the transmitter in the sympathetic nervous system. These discoveries depended on the development of sensitive bioassays for transmitter substances. Dale was not convinced that the sympathetic neurotransmitter was adrenaline itself, because he demonstrated subtle differences between the responses to the transmitter and to adrenaline. Von Euler, in 1946, finally showed that the sympathetic neurotransmitter was noradrenaline (non-methylated adrenaline).

After this period there was a lull in which many thought that acetylcholine and noradrenaline were the only two neurotransmitters. After all, the transmitter at the neuromuscular junction in skeletal muscle, in the synapses in ganglia of the autonomic nervous system, and at the ends of parasympathetic nerve fibres was shown to be acetylcholine, and noradrenaline was the transmitter at sympathetic nerve endings. So all types of synapse appeared to be accounted for. There was also evidence that acetylcholine was a transmitter in the brain. Furthermore, by then, criteria for showing that an agent was a neurotransmitter had been laid down and these were quite difficult to fulfill, especially in complex situations like the neural pathways in the brain. These five criteria are demonstrations that show:(i) the putative transmitter is released when the nerve trunk is stimulated;(ii) application of the transmitter to the post-synaptic structure causes the same effect as nerve stimulation;(iii) the nerve fibres have a mechanism for making, storing, and releasing the transmitter;(iv) a mechanism is present for rapidly terminating the actions of the neurotransmitter; and(v) an appropriate antagonist is equally effective at blocking both neurotransmission and exogenous application of the neurotransmitter.

Consider how these criteria are met for acetylcholine and for noradrenaline. We have already seen that these two substances were detected by sensitive bioassays in perfusates from neuronally-stimulated systems. To meet the second criterion it is necessary to show identity of action of the neurotransmitter and exogenously-applied transmitter substance. While this was straightforward with acetylcholine, Dale's insistence that adrenaline was not the neurotransmitter was based on differences between the responses to adrenaline and to nerve stimulation. This was an essential step leading to the discovery of noradrenaline.

The third criterion is satisfied because nerves that release acetylcholine (cholinergic nerves) have an enzyme (choline acetyltransferase) in the nerve terminals that synthesizes acetylcholine from choline, while noradrenergic nerves have a series of enzymes that synthesize noradrenaline from the amino acid tyrosine. The enzymes are produced in the nerve cell bodies and pass down the axon to the nerve terminals in the so-called axoplasmic flow (they can travel a considerable distance — for example, from the spinal cord to the foot muscles).

With respect to the fourth criterion: after release of acetylcholine from the nerve terminals it is attacked by acetylcholine esterase, which breaks down the neurotransmitter to choline and an acetate group, thus quickly terminating its action at the muscle membrane receptors. The choline is taken up into the terminal and recycled.

The mechanism for terminating transmitter action is different in the noradrenergic system. Seventy per cent of the released noradrenaline is taken back up into the nerve terminals and stored for later reuse. The rest is metabolized either extracellularly or after uptake into the end organ.

We have seen earlier that there are specific antagonists for acetylcholine, which also antagonize the effects of cholinergic stimulation. Similarly there are antagonists, such as the b-blockers, which block both noradrenaline and noradrenergic stimulation, thus meeting the fifth criterion.

In 1934 Dale enunciated a principle which stated that a nerve liberates the same neurotransmitter at all its terminations. Thus if a nerve branches, each branch will release the same neurotransmitter. This principle has remained a truism and it would be difficult to imagine a nerve cell that could exclusively send a different set of synthetic enzymes, in the axoplasmic flow, down different branches. However, until relatively recently it was assumed that any one nerve produced only one type of neurotransmitter. As usual in science, advances arose when observations were made which failed to fit the established dogma. It was recognized that in some systems antagonists that were able to block exogenously applied transmitter were not able to block nerve stimulation completely. There was a residual activity with nerve stimulation that was resistant to the block. The term NANC transmission was coined, standing for ‘nonadrenergic non-cholinergic transmission’. As the responses to nerve stimulation were partially blocked by antagonists with known specificities, the corollary was that the nerve must liberate more than one transmitter, but would do so from all its branches. Thus the concept of co-transmission was born, in which nerve stimulation could, in some instances, co-release more than one transmitter. In the peripheral autonomic nervous system — at the site where the nerves reach the tissue that they act upon — a great number of NANC transmitters have been claimed including, among others, ATP, VIP (vasoactive intestinal peptide), 5-HT, GABA, and dopamine. Undoubtedly some of these substances are transmitters, but few have yet met all the five criteria required to confirm their bona fides.

What advantages might accrue from co-transmission? First, the small molecular weight transmitters (amine transmitters) and one of the peptide transmitters are likely to have very different kinetics (fast and slow effects). Secondly, the receptor targets for the two transmitters may have different locations, for example one on the end organ and the other on the nerve terminal itself, allowing feedback control and finally ‘traffic neuromodulation’. This last results from the different ways in which the peptide transmitters and amine transmitters are handled. The ‘machinery’ needed to synthesize peptides like VIP is considerable. Consequently, these transmitters are synthesized in the nerve cell body and pass to the terminal in the axoplasmic flow, where they are stored ready for release. If there is heavy traffic in the nerve then the supply of the peptide neurotransmitter will soon be depleted. It is more difficult to deplete the supply of amine transmitters, which are made in the nerve terminal itself, so the ratio of the two transmitters released by nerve impulses will change.

While the criteria for proving that a chemical agent acts as a neurotransmitter in the periphery are not easy to achieve, the technical difficulties in the brain and spinal cord are much greater. Here there is a mass of neural tissue with intricate interconnections and ramifications within the brain, as well as the connections made with incoming and outgoing neural pathways. However, there is overwhelming evidence for many neurotransmitters in the central nervous system, even though not all the five criteria above have been satisfied. Histochemical methods have been used to demonstrate the localization of neurotransmitters in particular types of nerve cells, coupled with electrophysiological methods in which the physiology of a single identified cell is studied with microelectrodes. Finally, it is now possible to suck a few nanolitres of intracellular substance (cytosol) from a single, identified neuron in a brain slice and to determine which genes are activated, including those coding for proteins associated with neurotransmission (receptors, enzymes, etc.). Useful information can also be obtained by a study of disease states. For example, there can be no doubt that a lack of dopamine transmission gives rise to Parkinson's disease. Evidence from post-mortem brains and comparison of the dopamine concentrations in normal and diseased brains locates the dopaminergic pathways involved in the disease.

A potential forty neurotransmitters have been postulated to exist in the brain, of which ten are of the amine type with a small molecular weight. The amine types include acetylcholine, noradrenaline, dopamine, 5-HT, and histamine, and there are also excitatory and inhibitory amino acids. Glutamate and aspartate are the principal fast-acting excitatory transmitters in the brain, while GABA and glycine are the main inhibitory transmitters. Initially there was great reluctance to accept that these simple amino acids could act as neurotransmitters. Identified neurons were excited or inhibited when these amino acids were squirted onto them from very fine micropipettes. However, the presence of a pharmacological response does not prove physiological relevance. The development of selective agonists and antagonists has, subsequently, established that the four amino acids are true neurotransmitters. The remaining thirty-odd neurotransmitters in the central nervous system are mainly peptides, but much more evidence is needed before their true roles are unravelled.

Alan W. Cuthbert

See also autonomic nervous system; neuromuscular junction; peptides; synapse.