Molecular Physiological Analysis of Peptide Exocytosis and Pentidergic Neuromodulation.

在本研究项目中，我们获得了以下关于促性腺激素释放激素(GnRH)系统的多肽胞吐和多肽能神经调节的结果。GnRH释放的放射免疫测定法为了测定多个GnRH系统的GnRH释放活性，我们对不同条件下的脑垂体片进行了静态孵育，并用放射免疫法测定了GnRH释放到孵育液中的量。我们证明高[K]_0去极化刺激可通过电压门控性钙通道引起GnRH释放。我们发现在POA-GnRH切片中GnRH的释放量存在明显的性别差异。使用POA-GnRH神经元特异性GnRH抗血清的免疫组织化学分析显示，男性POA-GnRH神经元的数量比女性多得多。这种明显的形态性别差异被认为是TH…促性腺激素释放的基础更多的e POA-GnRH切片。GnRH的胞吐释放我们发展了一种使用碳纤维电极(CFE)实时电化学记录GnRH释放的方法。用安培法测定GnRH的氧化电流。记录硬骨鱼脑垂体片中脑垂体区的释放活动。CFE位于视前区GnRH神经元密集支配的垂体部。在高[K^]0刺激下，记录到一条体安培流，并呈剂量依赖关系。我们认为电流主要来源于GnRH的氧化电流，反映了GnRH的释放活性。谷氨酸能突触传入GnRH为了研究突触对Tn-GnRH神经元活动的控制，我们分析了Tn-GnRH神经元中谷氨酸受体(GluRs)的类型。通过使用各种特异性的GluR激动剂和拮抗剂，我们发现它们都有离子型GluR(iGluR；非NMDAR和NMDAR)和第3族代谢型GluR(MGluR)。我们还发现了一种新型的iGluR存在的证据。较少

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