Project 6

Regulation of Vesicle Exocytosis by Nitric Oxide

This project is based on observations conducted in the Synapse Laboratory over the past several years that have identified a role for NO in modulating transmitter release from the motor nerve terminal. It has been found that NO is produced by the muscle following a short period of low frequency stimulation. End plate potential amplitudes become depressed in response to the NO and the frequency of spontaneous release is reduced. The postsynaptic membrane sensitivity to the transmitter is unchanged, suggesting that the reduced EPP amplitudes are the result of a decline in quantal content (Etherington and Everett, 2004, J. Physiol.). Unexpectantly, the level of paired pulse facilitation at these synapses is unaltered when it would be expected to increase as a result of the depressed transmission (unpublished).

We have therefore proposed that the NO is acting at a very late stage of vesicle exocytosis and ‘upstream’ of processes responsible for paired pulse facilitation. The aim of the project is to provide direct evidence to support this proposal.

The experiment

The objective of the work is to measure the level of exocytosis in ‘depressed’ terminals from the loss of a fluorescent dye (FM4.64) from prelabelled vesicles. When prelablelled vesicles undergo exocytosis, they lose the dye and the level of labelling in the terminal gradually declines. Thus, monitoring dye loss will provide a measure of the level of exocytosis. Toad neuromuscular junctions will be labelled with the dye by bathing the nerve-muscle preparation in the dye while the nerve is briefly stimulated. Dye incorporates into the vesicle membrane when the vesicles undergo a cycle of exocytosis/endocytosis. Such prelabelled terminals can now be stimulated once more to measure the rate of loss of the dye to provide a measure of exocytosis. Multiple images will be taken of the terminal during the stimulation to determine dye loss from the decline in fluorescent intensity of the terminals.