Dopamine (DA) release in the cortex and basal ganglia is strongly implicated in modulation of CMS functionand behavior and is thought to occur through a variety of potential secretory sites. Among these are axonalprojections where small clear synaptic vesicles appear clustered in varicosities that resemble presynapticterminals for typical fast-acting neurotransmitter secretion. Given that DA acts on much longer time scalesthan fast-acting neurotransmitters, the mechanism involved in controlling the presynaptic machinery maywell be different than for those more typical 'fast' synapses. Here we propose to examine details of thepresynaptic vesicle cycle for these dopaminergic release sites. The long term objective of this proposal is tocharacterize the mechanism that control the presynaptic vesicle cycle for small clear dopaminergicveshicles. We will make use of technologies previously developed in the lab to examine many aspects ofthe molecular and biophysical nature of the presynaptic vesicle cycle in cortical and hippocampal cultures.These approaches rely heavily on optical techniques using exogenous organic probes such FM dye familymembers as well as genetically-encoded tags of presynaptic proteins that allow dynamic and quantitativeinformation about the vesicle cycle to be obtained. These will be adapted to primary dissociated cell culturesof mid-brain neurons from the ventral tegmental area (VTA). We propose 3 specific aims to accomplish thisinitial characterization of the cell biological, physiological and biophysical aspects of the dopaminergicvesicle cycle. These include characterizing the properties of the vesicle pool in turns of depletion rates,replenishment rates, the sensitivity of pool turnover to stimulation at varied calcium concentrations, as wellas the kinetics of endocytosis. Finally we will take advantage of the ability to detect dopamine sectretiondirectly using carbon-fiber amperometry to examine how details of the vesicle cycle impact neurotransmitterrelease.
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