STRUCTURE FUNCTION AND DEVELOPMENT OF THE ACTIVE ZONE

长期的目标是阐明发射的机制。 突触前神经末梢的释放和分化。现在 提案将集中在电压敏感钙通道(VSCCs)和 神经肌肉接头的活动区(递质释放部位) (NMJ)。新型合成欧米茄螺环肽和二氢吡啶(DHP)将 用作探针以表征VSCC与发射机的关系 在发育、再生和患病的NMJ上释放。 (I)研究发育中哺乳动物VSCC亚型的个体发育 NMJ。将检验两个假说：(A)L型血管内皮细胞调制 发射机在开发中释放。但不是成熟的NMJ。脱氢表雄酮的作用 突触电位与钙门控K+的可能参与 L型VSCC调制递质释放中的通道和/或生长抑素 将会被研究。发育神经中L类血管SCCs的存在 终末将通过免疫细胞化学进行确认。(B)N型VSCCS 在发育阶段调节递质的释放，而不是在成熟的NMJ。这个 假说将通过生理和形态方法进行检验。 含有欧米茄椰子肽。此外，发展NMJ的概念是 也像成人一样使用PIQ类型的VSCCs来调节递质释放 将对肌肉进行检查。 (Ii)观察成人NMJ再生时VSCC亚型的变化 肌肉。成人NMJ损伤后再形成的假说 根据VSCC亚型的转换模拟胚胎发育 将会受到考验。 (Iii)检验Lambert-Eaton肌无力综合征的假设 (LEMS)抗体导致运动神经VSCCs减少 终点站。LEMS抗体将被动地转移到小鼠体内。这个 将用荧光法研究对血管干细胞数量的影响 显微镜和放射自显影。 这项拟议的研究将提供关于人类个体发育的第一个研究。 发展和再生NMJ的VSCC。由于缺乏具体的 探索VSCCs在过去，我们对突触前的认识 分化严重滞后于突触后分化。 差异化。因此，拟议的研究将产生新的见解。 关于突触如何工作、形成和修复的机制。这个 拟议中的工作也可能提供更好的理解 人类神经肌肉疾病。

The long-term goals are to elucidate the mechanisms of transmitter release and differentiation o the presynaptic nerve terminal. The present proposal will focus on voltage-sensitive calcium channels (VSCCs) and the active zone (site of transmitter release) at the neuromuscular junction (NMJ). Novel synthetic omega conopeptides and dihydropyridine (DHP) will be used as probes to characterize VSCCs in relation to transmitter release at developing, regenerating and diseased NMJs. (I) To examine the ontogeny of VSCC subtypes at developing mammalian NMJs. Two hypotheses will be tested: (A) L-type VSCCs modulate transmitter release at developing. but not mature NMJs. The effect of DHP on synaptic potentials, and the possible involvement of Ca2+-gated K+ channels and/or somatostatin in L-type VSCC-modulated transmitter release will be studied. The presence of L-type VSCCs at developing nerve terminals will be confirmed with immunocytochemistry. (B) N-type VSCCS mediate transmitter release at developing, but not at mature NMJs. The hypothesis will be tested by physiological and morphological approaches with omega conopeptides. In addition, the notion that developing NMJs also use PIQ-type VSCCs to mediate transmitter release as in adult muscles will be examined. (II) To examine the change in VSCC subtypes at regenerating NMJs in adult muscles. The hypothesis that re-formation of adult NMJs following injury mimics embryonic development with respect to the switch in VSCC subtypes will be tested. (III) To test the hypothesis that Lambert-Eaton Myasthenic Syndrome (LEMS) antibodies cause a reduction of VSCCs from the motor nerve terminal. LEMS antibodies will be passively transferred to mice. The effect on the number of VSCCs will be studied with fluorescence microscopy and autoradiography. The proposed research would provide the first study on the ontogeny of VSCCs at developing and regenerating NMJs. Due to the lack of specific probes for VSCCs in the past, our knowledge of presynaptic differentiation has considerably lagged behind that of postsynaptic differentiation. Thus, the proposed research would yield new insights into the mechanisms on how the synapse works, forms and is repaired. The proposed work may also provide a better understanding of the etiology of human neuromuscular diseases.