STRUCTURE, FUNCTION, AND DEVELOPMENT OF THE ACTIVE ZONE

活动区的结构、功能和发展

• 批准号: 2268136
• 负责人: CHIEN-PING KO
• 金额: $ 14.8万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-01-15 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2268136
• 关键词: Rana; alternatives to animals in research; animal poison; calcium channel; cell differentiation; cholinergic receptors; confocal scanning microscopy; developmental neurobiology; electron microscopy; electrophysiology; fluorescent dye /probe; histochemistry /cytochemistry; nerve endings; nervous system regeneration; neurogenesis; neuromuscular junction; neurotoxins; neurotransmitter transport; synapses; synaptogenesis

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.

这项研究的长期目标是阐明细胞和 发射机释放和分化的分子机制 突触前神经末端。 目前的提议将集中于主动 区域，据信发射器释放发生。 它一直 假设Ca 2+通道在战略上位于活动区域。 该假设将使用W-持续毒素（CTX）作为Ca 2+的探针进行检验 青蛙神经肌肉连接处的通道。 此外，CTX将是 用于研究活动区形态计量学与突触之间的相关性 功效以及主动区分化的机制。 （1）薄片电子显微镜将用于揭示 在神经末端的Ca 2+通道的分布并检查CA是否是否 2+通道优先位于活动区域。 （2） 使用用金标记的CTX冻结裂纹细胞化学技术 颗粒将应用于研究Ca 2+通道是否与 在活动区域​​看到的大膜内颗粒。 （3）数量 将使用已鉴定的神经肌肉连接的内容进行测量 细胞内记录。 同一连接处的活动区域将是 用若丹明标记的CTX染色，并用共焦 显微镜。 数量内容与活动区大小之间的相关性 将进行分析。 （4）神经肌肉连接处，在加剧期间 青蛙和t的突触发生期间，将与 Rhodamine-CTX和荧光素αBungarotoxin。 CA 2+通道是否 最初在整个神经终端均匀分布 将研究在活动区域​​积累。 另外， Ca 2+通道的簇和空间关系和空间关系 将检查突触发生过程中乙酰胆碱受体。 关于突触中最重要的两个元素的拟议研究， 活动区域和CA 2+频道将为您提供新的见解 关于突触如何工作和形式的机制。 这些结果可能导致 进一步了解兰伯特 - 伊顿肌无力综合征，并可能提供 被认为涉及的学习和记忆的形态相关性 突触功效的变化。