The following hypotheses will be tested. First, that rapid onset of calcium-triggered neurotransmitter release results for calcium influx via channels that are located nanometers from synaptic release site. This hypothesis will be tested by determining how various intracellular buffers modify neurotransmitter release. The second hypothesis is that the rapid onset and termination of neurotransmitter release is mediated in a highly nonlinear fashion by a low-affinity calcium receptor. To examine this hypothesis, caged calcium and calcium indicator dyes will be used in conjunction with monitoring transmitter release to determine the dependence of release on calcium. The third hypothesis is that sustained release of neurotransmitter results from the availability of an exceptionally large pool of releasable vesicles. To test this hypothesis, the PI will estimate the readily releasable pools size by stimulating hair cells with pulses or trains of increasing during until exhaustion of the neurotransmitter release, and hence depletion of the pool, is observed. The final hypothesis tested is that this apparently large pool of releasable vesicles results from the mobilization of vesicles from cytoplasmic stores. To test this hypothesis, the rate of reserve pool vesicle mobilization will be measured using paired-pulse stimulation paradigms.
