A broad interdisciplinary research program is proposed that would concentrate on understanding one model neuroendocrine system that commands a reproductive act, the laying of eggs with associated behaviors. This research would combine the talents of individuals at six institutions, each specializing in a different branch of biology and chemistry (neurophysiology, immunology, receptor biochemistry, peptide structure and chemical synthesis). Diverse techniques are used including NMR, X-ray diffraction, the generation of monoclonal antibodies (MAbs) as function- interfering (FI) highly specific reagents, microsequencing and electrical techniques. We choose a marine mollusk, Aplysia, for this work because of certain advantages, over mammalian neuroendocrine systems, and the lessons of the past, that generalizable neurobiological principles are often obtained more efficiently from research on """"""""simple"""""""" systems. The command of egg-laying in Aplysia is mediated by the neuropeptide hormone, egg-laying hormone (ELH), synthesized by the bag cell (BC) neurons, and released in a 30 minute discharge of synchronous action potentials before spontaneous egg-laying. This """"""""afterdischarge"""""""" can be triggered in vitro by a few seconds of electrical stimulation. This long-lasting discharge is mediated by cAMP-dependent protein phosphorylation modulating at least two independent K+-channels. The research described in this application is concerned with: 1) Identifying, purifying and microsequencing those proteins, cytoplasmic and membrane, that mediate the special electrophysiological properties in two neuroendocrine pacemaker systems. We will develop MAbs as FI-probes, for intracellular injection, in order to identify the proteins mediating pacemaker mechanisms. 2) Determining the structure-activity relationships of the neuropeptide egg-laying hormone (ELH), made by the BCs, by using fragments and modifications of synthetic ELH, in conjunction with behavioral egg-laying assays, electrophysiological assays, and assays involving cubes of isolated gonad. In addition NMR spectroscopy of ELH and interesting analogs in solution and X-ray diffraction of crystals will be performed to gain information on the secondary and tertiary structure of these peptides. 3) Identifying, purifying and microsequencing the ELH receptor from non-neural tissue, the gonad, generating antibodies to it and using the antibody to determine binding sites in the nervous system.
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