This proposal concerns the development and application of electrochemical methods for measurements of single exocytosis events. The overall goal of the proposed work is to further understanding of neurotransmitter exocytosis. This is the basic process by which neurotransmitters and hormones are released from cells to initiate chemical communication. Yet, a great deal is still unknown about the function of the molecular mechanism of exocytosis. The applicants propose to develop new strategies for manufacturing smaller electrodes that can be placed in the synapse and should theoretically be useful to make enzyme electrodes with millisecond response time. The experiments proposed involve 1) investigation of events that involve cellular control of the timing of exocytosis; 2) investigation of sites of exocytosis outside the synapse and the target receptors for this release; 3) use of a recently developed electrochemical model of exocytosis to examine the effects of pharmacological agents on neurotransmitter concentration and vesicle size; 4) development of methodology to examine the role of vesicle-docking proteins on exocytotic release; 5) development of strategies to electrochemically measure exocytosis events in single synapses and 6) preparation of enzyme electrodes with submicron tip diameter. The successful completion of the work proposed will further develop the technology available for measurements of exocytotic events and will provide a significant step toward understanding the molecular basis of exocytosis.
