OPTICAL ANALYSIS OF SYNAPTIC VESICLE RECYCLING

本申请人合成并表征了新的荧光染料， 称为FM染料，它在一个活动中染色活的神经末梢， 依赖的方式。 这些染料似乎是通过标记细胞膜来起作用的， 回收突触囊泡 例如，在青蛙的运动神经末梢中， 用FM染料染色会产生一系列明亮的珠状荧光 沿着末端有一个点，每个点标记一簇突触。 囊泡 这些直径为1-2微米的斑点无限期地存在， 除非神经受到刺激，在这种情况下，它们会变暗并消失， 这明显反映了通过从标记的囊泡胞吐作用释放染料。 FM染料为研究突触功能提供了新的机会。在 在初步工作中，申请人已经使用它们来光学监测 突触活动在十几种不同的生物 准备。申请人也用它们来研究细胞生物学 突触囊泡的运输，这也是这个提议的主题。 申请人将使用染料研究突触囊泡再循环， 活的神经末梢，说明囊泡被固定的机制 在休息终端，和机制，他们被动员， 传递到突触前膜。 申请人还将学习 小泡胞吐后发生的过程，例如，如何以及何时 膜重新内化，以及囊泡如何及时再生， 空间 在其他项目中，申请人将研究 肉毒杆菌毒素，测试假设，它固定突触 囊泡申请人将探索使用新制剂， 联合收割机结合光学和电生理学（膜片钳/电容 测量）技术来研究胞吐作用和囊泡再循环。 这项工作的基本原理很简单：突触功能是所有 神经系统的整合过程，几乎所有的治疗 药物作用于突触 了解引导突触的机制， 囊泡，因为它们反复循环通过轮胞吐作用， 内吞作用，将有助于理解神经细胞的更广泛方面， 系统在健康和疾病中发挥作用。

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.