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 神经递质释放和分化的分子机制 突触前神经末梢 目前的建议将侧重于积极的 区域，其中发射器释放被认为发生。 已经 推测Ca 2+通道位于活性区。 本研究将用ω-芋螺毒素（CTX）作为Ca ~（2+）的探针来验证这一假说 青蛙神经肌肉接头处的通道。 此外，CTX将在 用于研究活动区形态计量学与突触 功效以及活性区分化的机制。 (1)薄切片电子显微镜将用于揭示 观察神经末梢钙通道的分布， 2+通道优先位于活性区。（二） 冷冻断裂细胞化学技术，使用金标记的CTX 粒子，将被应用于研究是否钙通道符合 在活性区看到的大的膜内颗粒。(3)Quantal 确定的神经肌肉接头的内容物将用 细胞内记录 同一路口的活动区域将 用罗丹明标记的CTX染色，并用共聚焦显微镜观察 显微镜 量子含量与活动带大小的关系 将被分析。(4)神经肌肉接头，在神经再支配过程中， 青蛙和蝌蚪的突触发生过程中，将被双重标记， 罗丹明-CTX和荧光素α银环蛇毒素。 钙通道是否 最初均匀分布在整个神经末梢，后来 将研究在活动区的累积。 此外该 Ca 2+通道簇的时空关系， 将检查突触发生期间的乙酰胆碱受体。 对突触中两个最重要的元素的研究， 活性区和Ca 2+通道，将提供新的见解 突触如何工作和形成的机制。 这些结果可能导致 进一步了解Lambert-Eaton肌无力综合征， 学习和记忆的形态学关联，被认为涉及 突触效能的变化。