STRUCTURE FUNCTION AND DEVELOPMENT OF THE ACTIVE ZONE

长期目标是阐明递质的作用机制 突触前神经末梢的释放和分化。本 该提案将重点关注电压敏感性钙通道（VSCC）和 神经肌肉接头处的活动区（递质释放部位） （NMJ）.新的合成ω-芋头肽和二氢吡啶（DHP）将 用作探头，以表征与变送器相关的VSCC 在发育、再生和患病的NMJ中释放。 (I)研究发育中哺乳动物VSCC亚型的个体发生 NMJ将检验两个假设：（A）L型VSCC调节 发射机在显影时释放。但不是成熟的NMJ DHP的效果 对突触电位的影响，以及Ca 2+门控K+的可能参与 L型VSCC调节的递质释放中的通道和/或生长抑素 将被研究。发育中的神经中存在L型VSCC 将用免疫细胞化学确认终末。(B)N型VSCCS 在发育中介导递质释放，但在成熟的NMJ中不介导递质释放。的 将通过生理学和形态学方法来检验假设 ω-芋螺肽此外，发展NMJ的概念 也使用PIQ型VSCC来介导递质释放，如在成人中一样 将检查肌肉。 (II)研究成人再生NMJ时VSCC亚型的变化 肌肉.假设成年NMJ在损伤后的重新形成 在VSCC亚型转换方面模仿胚胎发育 会得到考验 (III)为了验证Lambert-Eaton肌无力综合征 （LEMS）抗体导致运动神经VSCC减少 终端LEMS抗体将被动转移至小鼠。的 将用荧光法研究对VSCC数量的影响 显微镜和放射自显影。 这项拟议的研究将提供第一个研究个体发育的 VSCC在NMJ发育和再生中的作用。由于缺乏具体 在过去，我们对突触前神经元的认识 分化明显落后于突触后的分化。 分化因此，拟议的研究将产生新的见解 了解突触如何工作、形成和修复。的 拟议的工作也可能提供一个更好的理解的病因， 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.