OPTICAL ANALYSIS OF SYNAPTIC VESICLE RECYCLING

突触小泡回收的光学分析

• 批准号: 6165405
• 负责人: WILLIAM J BETZ
• 金额: $ 28.06万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6165405
• 关键词: Rana; calcium flux; calcium indicator; electron microscopy; fluorescent dye /probe; intracellular transport; membrane activity; neurophysiology; synaptic vesicles

The applicants have synthesized and characterized new fluorescent dyes, called FM dyes, which stain living nerve terminals in an activity- dependent fashion. The dyes appear to work by labeling the membranes of recycled synaptic vesicles. In frog motor nerve terminals, for example, staining with FM dyes produces a series of bright, bead-like fluorescent spots along the terminals, each spot marking a cluster of synaptic vesicles. The spots, which are 1-2 um in diameter, persist indefinitely, unless the nerve is stimulated, in which case they dim and disappear, evidently reflecting release of dye by exocytosis from labeled vesicles. The FM dyes offer new opportunities for studying synaptic function. In preliminary work, the applicants have used them to monitor optically synaptic activity in more than a dozen different biological preparations.The applicants have also used them to study the cell biology of synaptic vesicle trafficking, which is the subject of this proposal. The applicant will use the dyes to study synaptic vesicle recycling in living nerve terminals, addressing mechanisms by which vesicles are held in resting terminals, and mechanisms by which they are mobilized and transported to the presynaptic membrane. The applicants will also study processes that occur after vesicle exocytosis, for example, how and when membrane is reinternalized, and how vesicles are regenerated in time and space. In other projects, the applicants will study the mechanism of action of botulinum toxin, testing the hypothesis that it immobilizes synaptic vesicles. The applicants will explore the use of new preparations to combine optical and electrophysiological (patch clamp/capacitance measurements) techniques to study exocytosis and vesicle recycling. The rationale for this work is simple: Synaptic function underlies all integrative processes in the nervous system, and nearly all therapeutic drugs work at synapses. Knowledge of mechanisms, which guide synaptic vesicles as they repeatedly cycle through rounds of exocytosis and endocytosis, will be useful for understanding broader aspects of nervous system function in health and disease.

申请人已经合成并表征了新的荧光染料， 被称为FM染料，在活动中对活的神经末梢进行染色- 依附时尚。染料的作用似乎是通过标记膜上的 回收的突触小泡。例如，在青蛙运动神经末梢中， 用FM染料染色可产生一系列明亮的珠状荧光 沿着终末的斑点，每个斑点标记一簇突触 水泡。这些直径为1-2微米的斑点会无限期地存在， 除非神经受到刺激，在这种情况下它们会变暗并消失， 明显地反映了通过胞吐作用从标记的囊泡释放染料。 FM染料为研究突触功能提供了新的机会。在……里面 前期工作，申请者已使用它们进行光学监控 十多种不同生物中的突触活动 申请者还用它们来研究细胞生物学 突触囊泡贩运，这是这项提案的主题。 申请者将使用这些染料来研究突触囊泡在 活的神经末梢，解决囊泡的保持机制 在休眠终端中，以及它们被动员和 被输送到突触前膜。申请者还将学习 囊泡胞吐后发生的过程，例如，如何以及何时发生 膜被重新内化，以及囊泡是如何及时和 太空。 在其他项目中，申请者将研究 肉毒杆菌毒素，测试它固定突触的假设 水泡。申请者将探索使用新的制剂来 结合光学和电生理(膜片钳/电容 测量)研究胞吐作用和囊泡循环的技术。 这项工作的基本原理很简单：突触功能是 神经系统的完整过程，几乎所有的治疗性过程 药物作用于突触。关于指导突触的机制的知识 囊泡重复地循环通过几轮胞吐作用和 内吞作用，将有助于理解神经的更广泛的方面 系统在健康和疾病中的功能。