Neurotransmitter release occurs as a result of axonal spikes invading nerve terminals. Although there is considerable evidence that depolarization of neuronal somata leads to the entry of Ca+ and to the subsequent secretion of neurotransmitters and / or neurohormone, the molecular details of how ionic currents control the release of neuroactive substances from nerve terminals remain undetermined. This proposal takes advantage of a mammalian system in which these questions can be directly addressed. Much is already known about the electric activity of the rat neurohypophysis and the subsequent release of the peptide hormones, vasopressin and oxytocin,has been well characterized. It is now uniquely possible to prepare isolated nerve terminals from this neuroendocrine structure which respond to depolarization by releasing identified peptides via Ca2+ - dependent exocytosis. Furthermore, release from the neurohypophysis is regulated by specific patterns of electrical activity. Thus, these nerve terminals have all the properties necessary to analyze in detail the process of depolarization-secretion coupling (how neuropeptides are released in response to depolarization of the nerve terminal membrane). We propose to study, using sophisticated patch-clamp, imaging, and biochemical methodology, the physiology of identified, in terms of peptide secreted, nerve terminals in conjunction with their differential release of neuropeptides. In particular we propose to characterize in detail the Ca2+ channels, including verification, at the single channel level, of the kinetics and pharmacology of the controversial 'Q' -type, found at these nerve terminals. We will localize the different neurohypophysial Ca2+ channels between types of terminals and within individual terminals in relation to possible release sites. Finally, we will determine which types are important for the release of vasopressin vs oxytocin. Elucidation of the molecular mechanisms underlying such interactions will represent a major advance in the understanding of how neuronal communication is regulated.
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