STRUCTURE, FUNCTION AND DEVELOPMENT OF THE ACTIVE ZONE

这项研究的长期目标是阐明细胞和 细胞递质释放和分化的分子机制 突触前神经末梢。目前的提案将重点放在活跃的 据信发生发射器释放的区域。一直以来 推测Ca~(2+)通道位于活动区。 这一假说将用W-芋螺毒素(CTX)作为钙离子的探针进行验证。 青蛙神经肌肉交界处的通道。此外，CTX将被 用于研究活动区形态计量学与突触的相关性 以及活跃区分化的机制。 (1)将使用薄层电子显微镜来揭示 神经末梢内钙通道的分布及钙离子通道的检测 2+通道优先位于活动区。(2) 冷冻断裂细胞化学技术--用金标记的环磷酰胺 粒子，将被用于研究钙通道是否与 活动区可见较大的膜内颗粒。(3)Quantal 已确定的神经肌肉连接的含量将通过 细胞内记录。相同交汇点的活动区将是 罗丹明标记的CTX染色和共聚焦显示 显微镜。量子含量与活动区大小的关系 将会被分析。(4)神经肌肉接头，在神经再支配过程中 在蛙类和蝌蚪的突触发生过程中，将用 罗丹明-环磷酰胺和荧光素α银环蛇毒素。是否存在钙离子通道 最初均匀分布在整个神经末梢，后来 将对积聚在活动区的区域进行调查。此外， 钙离子通道簇和钙离子通道簇的时空关系 本课程将对突触发生过程中的乙酰胆碱受体进行检测。 对突触中最重要的两个元素的拟议研究， 活动区和钙通道，将提供新的洞察 突触如何工作和形成的机制。这些结果可能会导致 对Lambert-Eaton肌无力综合征的进一步认识 学习和记忆的形态关联被认为涉及 突触效能的变化。

The long-term goals of this research are to elucidate the cellular and molecular mechanisms of transmitter release and differentiation of the presynaptic nerve terminal. The present proposal will focus on the active zone, where transmitter release is believed to occur. It has been postulated that Ca 2+ channels are strategically located at active zones. This hypothesis will be tested using w-conotoxin (CTX) as a probe for Ca 2+ channels at the frog neuromuscular junction . In addition, CTX will be used to study the correlation between active zone morphometry and synaptic efficacy as well as the mechanisms of active zone differentiation. (1) Thin-section electron microscopy will be used to reveal the distribution of Ca 2+ channels in the nerve terminal and to examine if Ca 2+ channels are located preferentially at the active zone. (2) Freeze-fracture cytochemical techniques, using CTX tagged with gold particles, will be applied to study whether Ca 2+ channels coincide with the large intramembrane particles seen at the active zone. (3) Quantal contents of identified neuromuscular junctions will be measured with intracellular recording. Active zones of the same junctions will be stained with rhodamine-labeled CTX and visualized with a confocal microscope. The correlation between quantal contents and active zone sizes will be analyzed. (4) Neuromuscular junctions, during reinnervation in the frog and during synaptogenesis in the tadpole, will be double-labeled with rhodamine-CTX and fluorescein alpha bungarotoxin. Whether Ca 2+ channels are initially distributed evenly throughout the nerve terminal and later accumulated at active zones will be investigated. In addition, the temporal and spatial relationship between clusters of Ca 2+ channels and acetylcholine receptors during synaptogenesis will be examined. The proposed research on two of the most important elements at the synapse, active zones and Ca 2+ channels, will provide new insights into the mechanisms on how the synapse works and forms. These results may lead to further understanding on Lambert-Eaton myasthenic syndrome and may provide morphological correlate of learning and memory which are thought to involve changes in synaptic efficacy.