Peptide secretion probably occurs from most neurons. It modulates CNS function as well as serving neuroendocrine regulation. Knowledge of peptide secretory mechanisms is limited to a very few experimentally tractable systems. This work proposes rigorous experiments on a uniquely suitable invertebrate preparation to understand how secretion of neuropeptides is related to physiological stimuli, the electrical responses and changes of cytoplasmic [Ca++] and [Na+] that these produce in the nerve terminals, and the possible modulation of these by hormones. The X-organ - sinus gland (XOSG) of the crab (Cardisoma carnifex) will be used. Analogous to the vertebrate hypothalamic-neurohypophyseal system in its role and physiology, it is purely peptidergic with 3 major hormonal activities that can be immunocytologically localized and quantified in perfusates by bioassay or ELISA. The XOSG is easily isolated; terminals and preterminal swellings form a neurohemal organ (the SG), and many are large enough for intracellular recording. Terminals can be dissociated for patch-clamping. Somata dissociated from the XO grow in defined culture. For each of the 3 hormonal activities, crustacean hyperglycemic hormone, red-pigment concentrating hormone and molt-inhibiting hormone, the following questions are to be addressed: Is the rate of basal secretion governed by [Ca++]i or [Na+]i, and what controls these? Is the response to stimulation accounted for by entry of Ca++, or is release from internal stores involved? Does [Na+]i influence evoked secretion? Does release occur only at contacts with blood spaces, or at other sites as well? Is secretion modulated by hormones, and at what sites and by what mechanisms? What limits the amount of releasable hormone, and is there peripheral restocking? In isolated XOSGs, for the 3 hormonal activities, basal secretion and secretion evoked by raised [K+]o and axonal stimulation will be followed by analysis of perfusate aliquots. The effects of ionic composition, pharmacological agents and temperature that alter these will then also be tested for effects on electrical responses. """"""""Whole terminal"""""""" patch-clamp will characterize ionic currents of isolated terminals which will then be identified immunocytologically. Fura-2 and SBFI will be used to optically assess [Ca]i and [Na]i in terminals and cultured neurons. Capacitance measurements and computer-aided, video-enhanced microscopy will be used to track secretion from terminals and growth cones. Immunocytologically identified terminals will be characterized at the EM level together with changes resulting from secretory activity.
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