Two separate lines of research led to the proposal that transmitter released in response to neuronal excitation is derived from a vesicle-bound pool rather than from the neuronal cytoplasm. One piece of evidence came from electron microscopy which showed that nerve terminals were packed with vesicle-like organelles (Fig. 4.10). Using differential centrifugation, these vesicles were soon identified as the major storage sites for neurotransmitters. The second was electrophysiological evidence that the effect of neuronal release of acetylcholine on the postsynaptic membrane potential at the neuromuscular junction was quantal in nature, suggesting that this transmitter, at least, was released in discrete packets.
Early neurochemical investigations of the source of released transmitter measured noradrenaline release from chromaffin granules in the adrenal medulla. Chromaffin granules are considerably larger (250 nm diameter) than the storage vesicles in noradrenergic nerve terminals (40-100 nm) and so their experimental use avoided the constraint imposed by the low sensitivity of early assay techniques (see Winkler 1993). Yet, like noradrenergic neurons, the adrenal medulla is derived from the developing neural crest and noradrenaline release is activated by stimulation of preganglionic cholinergic neurons. Chromaffin granules therefore provide a useful model for processes involved in the storage and release of noradrenaline from neurons. Subsequent refinements of assays for noradrenaline enabled studies of noradrenaline release to be extended to stimulated sympathetic nerve/end-organ preparations. These experiments confirmed that noradrenaline was released from vesicle-bound packets of transmitter contained within the terminal vesicles. This is because its release was paralleled by the
appearance of the proteins, dopamines-hydroxylase and chromogranins, which are found only in noradrenaline storage vesicles, whereas the cytoplasmic enzyme, lactate dehydrogenase, was not found in the perfusate.
Experiments of this kind have provided a great deal of evidence in favour of exocytotic release of vesicular noradrenaline. For example, by administering reserpine (which causes noradrenaline to leak out of the vesicles into the cytoplasm) together with an inhibitor of the enzyme monoamine oxidase (which will prevent metabolism of cytoplasmic noradrenaline), it is possible to redistribute the noradrenaline stored within nerve terminals because it leaks from the vesicles but is preserved within the neuronal cytoplasm. Under these conditions, the total amount of transmitter in the terminals is unchanged but impulse-evoked release rapidly diminishes.
Different evidence, mainly based on histological studies, suggested that acetylcholine is also released by vesicular exocytosis. Landmark experiments used a technique known as 'freeze-fracture' in which tissues are frozen rapidly during periods of intense transmitter release. It is then possible to fracture axolemma membranes in a way that separates their lipid bilayer. Electron microscopy reveals numerous pits in the membranes which are thought to reflect the vesicle/axolemma fusion pore of vesicles in the process of exocytosis. Subsequent studies, combining immunocytochemistry with electron microscopy, showed that proteins in the membranes of vesicles become incorporated into the axolemma during transmitter release. Furthermore, when neurons are stimulated in a medium containing an electron-dense marker, that does not penetrate the neuronal membrane, the marker later appears in vesicles inside the nerve terminals (Basbaum and Heuser 1979). This suggests that such markers are incorporated into the vesicles when they come into contact with the extracellular fluid during exocytosis. There is also some pharmacological evidence for exocytosis of acetylcholine. For instance, impulse-evoked release of this transmitter is prevented by the drug, vesamicol, which blocks uptake of acetylcholine from the cytoplasm into the terminal vesicles (Searl, Prior and Marshall 1991).
Although most evidence supports vesicular exocytosis of acetylcholine (see Ceccarelli and Hurlbut 1980), some researchers contest this view. An alternative suggestion is that an ATPase bound to the axolemma acts as a pore ('mediatophore'). According to this scheme, opening of the pore is triggered by an increase in the concentration of intracellular Ca2+ and allows gated release of aliquots of cytoplasmic acetylcholine. The vesicles are thought to serve merely as a reserve pool of transmitter and for sequestration of intracellular Ca2+ (Dunant 1994).
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