This application is to study mechanisms of Ca2+ metabolism and transmitter release in vertebrate (lizard, mouse) nerve terminals innervating skeletal muscle. Stimulation-induced changes in cytosolic and mitochondrial [Ca2+] will be measured by monitoring fluorescence changes of indicator dyes at high spatial and temporal resolution with a confocal laser-scanning microscope. Phasic and asynchronous transmitter release will be measured electrophysiologically by recording end-plate potentials or voltage-clamped end-plate currents in the underlying muscle fiber. One group of experiments will study the relationship between intraterminal [Ca2+] ([Ca2+]i) and transmitter release, elevating [Ca2+]i by uncaging photolabile Ca2+ chelators. These experiments will test the hypothesis that quantal release from motor terminals result from two parallel mechanisms with differing Ca2+ affinities. Experiments using similar techniques will study the temperature sensitivity of the synaptic delay, to determine whether the interval between Ca2+ entry and the onset of transmitter release is Ca2+- dependent, and whether it is controlled by processes with low (e.g. diffusion) or high energy barriers. Another group of experiments will use simultaneous imaging of cytosolic and mitochondrial [Ca2+] and application of various inhibitors to study Ca2+ sequestering and extruding mechanisms in motor nerve terminals. These experiments will test two hypotheses. The first is that mitochondria sequester a major portion of the Ca2+ that enters the terminal during repetitive stimulation and effectively """"""""clamp"""""""" cytosolic [Ca2+] at a slightly elevated plateau level. The second hypothesis is that slow release of Ca2+ from mitochondria following stimulation has a major role in producing post-tetanic potentiation for transmitter release. We will also investigate mechanisms underlying the heterogeneity with which different boutons of the same motor terminal handle Ca2+ loads from trains of action potentials.
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