Secretion is one of the most ubiquitous of cellular processes, and the elucidation of its mechanism(s) remains a challenge to cell physiologists. It is becoming increasingly clear, however, that quantal, or vesicular release of neurotransmitters, neuropeptides, neuromodulators, and hormones proceeds differently in different cell types, and that the mammalian neurohypophysis is an excellent model for rapid-release neurosecretory systems including presynaptic terminals. We are now able to monitor, using optical methods having sub-millisecond time resolution, both the electrical events and cytoplasmic calcium changes in nerve terminals of vertebrates, as well as a sequence of intrinsic optical changes (IOC's) that are related directly to the secretory event. Thus, we possess an exceptional array of tools with which to study excitation-secretion (E-S) coupling, and to provide a more complete understanding of synaptic transmission in higher animals, and of the secretory event in general. First, we will use moderate angular resolution light scattering methods, together with fluorescent calcium indicators, voltage sensitive dyes, immuno-gold labeling, electron microscopy, and electron energy loss spectroscopy (EELS) to examine the hypothesis that the triggered release of calcium from intraterminal stores is required for the release of neuropeptides in mammals (and that the stores may be the secretory granules themselves.) We will also examine several alternative explanations for the origin of the IOC's, including the hypothesis that a pH-dependent solubilization of secretory granule contents, mediated by a rise in intraterminal calcium, precedes exocytosis and generates a large and rapid change in light scattering. Second, we will use the neurohypophyses of transgenic mice expressing the pH-sensitive Green Fluorescent Protein, ecliptic-pHluorin, for high time-resolution studies of neuropeptide secretion. Finally, we will characterize extensively the changes in intraterminal calcium during E-S coupling in mammalian nerve terminals, and we will identify the sources and sinks of this critical second messenger, and their specific roles before, during and after exocytosis. These experiments should have a major impact, and should add to our understanding of synaptic transmission, and its failure or malfunction in human neurological disease.
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