The goal is to fill in gaps concerning our electrophysiological understanding of GnRH neurons and gonadotropes in order to learn how they are stimulated to secrete and if they have intrinsic mechanisms that would explain the rhythmicity and pulse shape of secretion. Three cell types will be used initially: a neuron-like GnRH-secreting cell line derived from a transgenic mouse tumor; a gonado trope-like alphaT-3 cell line derived from a transgenic mouse pituitary tumor; and juvenile and adult gonadotropes enzymatically dissociated from salmon anterior pituitaries. Responses will be studied primarily by membrane biophysical techniques including: whole-cell and patch clamp methods to characterize the ionic channels and their response to GnRH in these cells; changes of membrane capacitance as an index of exocytosis of secretory vesicles; and intracellular calcium changes with indo 1 fluorescent dye.
The specific aims are: 1) To inventory the voltage-gated ionic channels and to determine the electrical activity they can support. 2) To inventory the fast ligand-gated channels. 3) To determine the actions of GnRH on channels, intracellular calcium, and secretion. 4) To determine modulatory actions of other peptides and neurotransmitters acting through G-protein- coupled receptors. 5) To explore the intracellular signaling pathways used for responses to GnRH and other modulatory transmitters. 6) To test hypotheses regarding the electrophysiological basis of excitation, inhibition, and ultradian rhythmicity.
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