Transmitter release

Botulinum neurotoxins (A-G), tetanus toxin Synaptic peptides a) Synapto-brevin b) Syntaxin c) SNAP25 Zinc dependent endoprotease Cleavage of synaptic peptides Inhibition of transmitter release (tetanus, botulism)... 

There are numerous transmitter substances. They include the amino acids glutamate, GABA and glycine acetylcholine the monoamines dopamine, noradrenaline and serotonin the neuropeptides ATP and NO. Many neurones use not a single transmitter but two or even more, a phenomenon called cotransmission. Chemical synaptic transmission hence is diversified. The basic steps, however, are similar across all neurones, irrespective of their transmitter, with the exception of NO transmitter production and vesicular storage transmitter release postsynaptic receptor activation and transmitter inactivation. Figure 1 shows an overview. Nitrergic transmission, i.e. transmission by NO, differs from transmission by other transmitters and is not covered in this essay. 

These cascades of reactions need time in the range of seconds synaptic transmission through GPCRs is slow. All further postsynaptic changes depend on the type of postsynaptic cell. For example activation of 32-adrenoceptors causes in the heart an increase of the rate and force of contraction in skeletal muscle glycogenolysis and tremor in smooth muscle relaxation in bronchial glands secretion and in sympathetic nerve terminals an increase in transmitter release. 

The findings recently reported can be relevant to understand the functional role of transmitter release from astrocytes in brain function. Interestingly, a recent report shows for the first time the occurrence in vivo of fast astrocytic calcium events in response to neuronal activity, peaking with millisecond time-scale from sensory stimulation (Winship et al. 2007). 

This finding suggests that the fast calcium events we have recently identified are not a peculiarity of astrocytes in cell culture but may correspond to events taking place in astrocytes of the living brain. Establishing whether fast calcium events in vivo are associated to transmitter release via SLMV exocytosis, becomes, therefore, of the outmost importance in order to define the type of modulatory influence exerted by astrocytes on neighboring neuronal circuits. 

Perea G, Araque A (2005) Properties of synapticaUy evoked astrocyte calcium signal reveal synaptic information processing by astrocytes. J Neurosci 25 2192-2203 Perea G, Araque A (2007) Astrocytes potentiate transmitter release at single hippocampal synapses. Science 317 1083-1086... 

When an action potential arrives at the axon terminal, it induces the release of a chemical transmitter. Transmitter release is a Ca +-dependent process (see Chapter 4) and requires a charge of Ca +. This is provided through the action potential-induced... 

Main functions Pacemaker Spike Transmitter release Transmitter release Transmitter release... 

Figure 2.4 Noradrenergic inhibition of Ca " currents and transmitter release in sympathetic neurons and their processes, (a) Inhibition of currents through N-type Ca " channels by external application of noradrenaline (NA) or by over-expression of G-protein P y2 subunits, recorded from the soma and dendrite of a dissociated rat superior cervical sympathetic neuron. Currents were evoked by two successive 10 ms steps from —70 mV to OmV, separated by a prepulse to -1-90 mV. Note that the transient inhibition produced by NA (mediated by the G-protein Go) and the tonic inhibition produced by the G-protein Piy2 subunits were temporarily reversed by the -1-90 mV depolarisation. (Adapted from Fig. 4 in Delmas, P et al. (2000) Nat. Neurosci. 3 670-678. Reproduced with permission), (b) Inhibition of noradrenaline release from neurites of rat superior cervical sympathetic neurons by the 2-adrenoceptor stimulant UK-14,304, recorded amperometrically. Note that pretreatment with Pertussis toxin (PTX), which prevents coupling of the adrenoceptor to Gq, abolished inhibition. (Adapted from Fig. 3 in Koh, D-S and Hille, B (1997) Proc. Natl. Acad. Sci. USA 1506-1511. Reproduced with permission)...

While this chapter is concerned primarily with the neurochemical mechanisms which bring about and control impulse-evoked release of neurotransmitter, some of the methods used to measure transmitter release are described first. This is because important findings have emerged from studies of the effects of nerve stimulation on gross changes in transmitter release and intraneuronal stores. The actual processes that link neuronal excitation and release of transmitter from nerve terminals have been studied only relatively recently. The neurochemical basis of this stimulus-secretion coupling, which is still not fully understood, is described next. The final sections will deal with evidence that, under certain conditions, appreciable amounts of transmitter can be released through Ca +-independent mechanisms which do not depend on neuronal activation. 

It is already evident that the turnover rate of a transmitter is only a crude measure of its release rate. Further limitations are that there is appreciable intraneuronal metabolism of some neurotransmitters notably, the monoamines. In such cases, turnover will overestimate release rate. Another problem, again affecting monoamines, is that some of the released neurotransmitter is taken back into the nerve terminals and recycled. This leads to an underestimate of release rate. Despite these drawbacks, studies of turnover rates uncovered some important features of transmitter release. In particular, they provided the first evidence for distinct functional pools of monoamines, acetylcholine and possibly other neurotransmitters a release pool, which could be rapidly mobilised for release, and a storage or reserve pool which had a slower turnover rate. 

Many early studies of transmitter release depended on measuring its concentration in the effluent of a stimulated, perfused nerve/end-organ preparation. This technique is still widely used to study drug-induced changes in noradrenaline release from sympathetic neurons and the adrenal medulla. However, it is important to realise that the concentration of transmitter will represent only that proportion of transmitter which escapes into the perfusate ( overflow ) (Fig. 4.2). Monoamines, for instance, are rapidly sequestered by uptake into neuronal and non-neuronal tissue whereas other transmitters, such as acetylcholine, are metabolised extensively within the synapse. Because of these local clearance mechanisms, the amount of transmitter which overflows into the perfusate will depend not only on the frequency of nerve stimulation (i.e. release rate) but also on the dimensions of the synaptic cleft and the density of innervation. 

The main advantage of using synaptosomes is that they are free from any influence of the parent axon. Another is that, since the volume of extracellular space (the incubation medium) is functionally infinite, transmitter will not accumulate near the synaptosomes. This means that reuptake of released transmitter is unlikely to occur and that, under drug-free conditions, transmitter release will not be modified by activation of auto- or heteroceptors (see below). 

Figure 4.2 The intraneuronal stores of monoamines are maintained by synthesis from precursors taken in with the diet. The pool is depleted by release of transmitter and some spontaneous metabolism of intraneuronal transmitter. Released monoamines are inactivated by reuptake on membrane-bound transporters. Following reuptake, some transmitter might be recycled while the remainder is metabolised. Some transmitter escapes the reuptake process and overflows from the synapse in the extracellular fluid...

A disadvantage of using synaptosomes is that they cannot be used to study transmitter release evoked by propagated nerve impulses, but the release, like that from intact neurons, is Ca +-dependent and K+-sensitive. Pharmacological studies using synaptosomes have also provided evidence that the amount of transmitter that is released following their depolarisation is regulated by the activation of presynaptic receptors. 

One approach, and the first to be adopted, is to study transmitter release from slices which have been preloaded with radiolabelled transmitter. In these experiments, drug-induced changes in the release of transmitter is usually monitored using the doublepulse technique. This involves comparing the effects of a test drug on the amount of transmitter released in response to a reference pulse and a second identical test pulse. If all the radiolabelled transmitter that overflows in the effluent is collected, and the amount which remains in the slice at the end of the experiment is also measured, it is possible to calculate not only how much radiolabelled transmitter was originally contained in the slice but also the effects of drugs on fractional release , i.e. the proportion of the store of radiolabelled transmitter which is released by nerve stimulation. As with... 

The cortical cup has been used for many years to monitor changes in transmitter release induced by physiological and pharmacological stimuli (Fig. 4.5). In the past, it was used most commonly to study release of amino acids and acetylcholine. More recently, it has... 

The rate at which the probes are perfused with aCSF is a compromise between the time required for the solutes in the CSF to reach equilibrium with those in the probe (the slower, the better) versus the ideal time-frame for studying changes in transmitter release (the shorter, the better). In general, flow rates of around 1-2 pl/min are used and the time which elapses between taking samples is determined by how much transmitter... 

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