Molecular Physiological Analysis of Peptide Exocytosis and Pentidergic Neuromodulation.

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In the present research project, we obtained the following results concerning the peptide exocytosis and peptidergic neuromodulation of the gonadotropin-releasing hormone (GnRH) system..1. Radioimmunoassay (RIA) of GnRH release from brain-pituitary slicesTo measure GnRH release activities from multiple GnRH systems, we conducted a static incubation of brain- pituitary slices under various conditions, and GnRH released into the incubation medium was measured by RIA. We demonstrated that GnRH release was evoked by high [K+]_0 depolarizing stimuli via Ca^<2+> influx through voltage-gated Ca^<2+> channels. We revealed the presence of conspicuous sexual difference in the amount of GnRH release in the POA-GnRH slices. Immunohistochemical analysis using an antiserum specific to the GnRH specific to POA-GnRH neurons revealed presence of much larger number of POA-GnRH neurons in males than in females. This clear morphological sexual difference is suggested to underlie that of GnRH release in th … More e POA-GnRH slices.2. Exocytotic release of GnRHWe developed a method for real-time electrochemical recording of GnRH release using carbon fiber electrodes (CFE). The oxidation current of GnRH was measured by amperometry. We recorded the release activity in the pituitary of the teleost brain-pituitary slice. The CFE was located in the part of the pituitary that receives dense innervation from GnRH neurons in the preoptic area. A bulk amperometric current was recorded in response to high [K^+]_0 stimulation in a dose dependent manner. We suggested that the amperometric currents are mainly attributed to the oxidation currents of GnRH and reflect the GnRH release activity.3. Glutamatergic synaptic inputs to GnRHTo investigate the synaptic control of activities of the TN-GnRH neurons, we analyzed electrophysiologically the type of glutamate receptors (GluRs) in the TN-GnRH neurons. By using various specific GluR agonists and antagonists, we found that they have ionotropic GluRs (iGluR ; non-NMDAR and NMDAR) and group 3 metabotropic GluRs (mGluR). We also found evidence to suggest the presence of a novel type of iGluRs. Less