The goal of the research plan set forth in this application is to investigate uptake and release of neurotransmitters and related substances at the level of individual cells. The experimental approach will be two-fold: existing neurochemical tools will be used to further characterize and understand these two fundamental processes, and new tools will be developed that allow such characterization at the single cell level. This laboratory developed the first method to examine exocytotic secretion from single vesicles. We also developed the first technique to observe the very rapid time course of dopamine uptake into neurons from brain extracellular fluid. Thus, we have considerable expertise in quantifying and evaluating both release and uptake. The combined technological and mechanistic approach proposed here should lead to a more complete view of neuronal means of regulating neurotransmission by a variety of substances. The specific projects that will be investigated are: To characterize the interplay between storage and release in monoamine containing vesicles. This will be done by examining the time course of individual secretory events following exocytosis. To determine the role of the vesicular monoamine transporter (VMAT) on storage and release. These experiments will be done in mice that have been genetically altered so that they lack VMAT2. To characterize the relationship between intracellular Ca2+ and release. These experiments will be done at the single cell level with amperometry and fluorescent dyes to monitor Ca2+ entry. To characterize release from an acutely dissociated dopaminergic neuron. From such primary cultures, a clear view on the factors that affect exocytotic release will be obtained. To characterize dopamine uptake at the single cell level. Microsampling techniques will be adapted for this application.
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