Secretion is one of the most ubiquitous of cellular processes. Indeed, it is central to such diverse biological functions as information processing, reproduction, motility, temperature regulation, metabolism, the immune response, and signal transduction. However, despite significant progress in identifying components of the macromolecular machinery essential for release of neurotransmitters and neuropeptides, the full elucidation of its mechanism(s) remains a challenge to cell physiologists. This is because direct observation of the intraterminal events that follow cellular excitation, but precede fusion of secretory vesicles, has proven extremely difficult. This proposal seeks to identify some of the cellular events that define calcium-dependent excitation coupling in mammalian peptidergic nerve terminals, and, in particular, to begin to understand the relationship between two novel millisecond time-resolved phenomena that are related to secretion, viz., light scattering changes from nerve terminals and the mechanical """"""""spike"""""""" and the mechanical """"""""dip"""""""" that follows it. Our experimental model is unique, the mouse neurohypophysis, a neurosecretory organ that comprises some 40,000,000 nerve terminals and secretory swellings, and that releases oxytocin and arginine vasopressin into the local circulation. Because no single technique is capable of providing the spatial and temporal resolution required for a comprehensive description of the secretory process, we will employ a multi-disciplinary approach that includes optical measurement of rapid changes in membrane voltage (using potentiometric dyes), in intraterminal calcium concentration, and in light scattering. Also, a modified atomic force microscope will be used to detect minute, but very rapid changes in nerve terminal volume. We will also use a set of novel techniques to begin to characterize, spatially and temporally, the changes in intraterminal Ca2+ ([Ca2+]i), during excitation-secretion (E-S) coupling in mammalian peptidergic nerve terminals. This will entail the identification of the sources and sinks of [Ca2+]i, and the elucidation of their roles before, during, and after exocytosis, with particular emphasis on the dense core granules and their possible role as intraterminal Ca2+-stores and Ca2+-amplifiers.
The neurohypophysis is a model for the presynaptic component of chemical neurotransmission, and hormones secreted by the neurohypophysis are implicated in functions that vary from blood pressure regulation, water balance and milk ejection, to social cognition, sexual behavior, and mood disorders. Impairment of neurotransmission results in devastating neurological ailments including Parkinsonism, Alzheimer?s disease, schizophrenia and depression. This proposal will advance our understanding of synaptic transmission and its malfunction in human neurological disease.
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