ELECTROPHYSIOLOGICAL ANALYSIS OF A C ELEGANS SYNAPSE

描述：（申请人摘要） 突触传递突触传递的过程似乎是高度保守的 物种，允许关于脊椎动物中枢神经系统的功能问题 系统将在更易处理的系统中处理。例如，超过30个基因 与突触传递有关的基因已经在线虫中被发现， 秀丽隐杆线虫，以及这些基因的同源物随后被 在脊椎动物中被发现。由于神经传递缺陷 神经变性可导致神经系统功能的严重缺陷 在帕金森病和阿尔茨海默病等疾病中很明显。之一 研究突触蛋白作用的最有力的方法是检查 缺乏这些蛋白质的突变动物的突触生理学。制备 适用于突触功能的详细电生理分析， C.elegans以前还没有，主要是由于技术上的原因。 解剖这么小的生物体很困难 本提案中提供的初步数据表明， 全细胞电压钳记录突触活动在一个新开发的C。 线虫体壁神经肌肉制剂。为了使用这种制剂 为了探测突变动物的突触传递， 需要野生型突触。 梭线虫神经肌肉接头受GABA能和 胆碱能运动神经元为了描述这个突触的释放， 有必要分别研究这两种投入。目标我会识别 分离两种突触输入的药理学工具。目标2将使用这些 表征自发GABA能和胆碱能的药理学工具 突触活动目标3将开发方法，以诱发GABA和ACh的释放， 神经肌肉接头目的4：研究诱发释放的钙依赖性， 将被量化。目的5将测试突触可塑性的 神经肌肉突触使用协议，以引起成对脉冲和短期 易化、增强和强直后增强。 这种制备有望提供一种有价值的新工具，其中联合收割机 C.线虫遗传学和分子生物学 电生理分析。该项目的完成预计将奠定 为将来评估30多种与突触相关的蛋白质奠定了基础。 传输

DESCRIPTION: (Applicant's Abstract) The process of synaptic transnuission. appears to be highly conserved across species, allowing functional questions about the vertebrate central nervous system to be addressed in more tractable systems. For example, over 30 genes implicated in synaptic transmission have been identified in the nematode, Caenorhabditis elegans, and homologs of these genes have subsequently been identified in vertebrates. Defects in neurotransmission due to neurodegeneration can lead to severe deficits in nervous system function evident in disorders such as Parkinson's and Alzeihmer's diseases. One of the most powerful approaches to study the roles of synaptic proteins is to examine synaptic physiology in mutant animals lacking these proteins. A preparation suitable for detailed electrophysiological analysis of synaptic function in C.elegans has not previously been available, primarily due to the technical difficulties of dissecting such a small organism. Preliminary data provided in this proposal demonstrate the feasibility of whole-cell, voltage-clamp recording synaptic activity in a newly developed C. elegans body wall neuromuscular preparation. In order to use this preparation to probe synaptic transmission in mutant animals, a baseline characterization of the wildtype synapse is required. The C. elegans neuromuscular junction is innervated by both GABAergic and cholinergic motor neurons. In order to characterize release at this synapse it will be necessary to study these two inputs separately. Aim I will identify pharmacological tools to separate the two synaptic inputs. Aim 2 will use these pharmacological tools to characterize spontaneous GABAergic and cholinergic synaptic activity. Aim 3 will develop methods to evoke GABA and ACh release at the neuromuscular junction. In aim 4, the Ca 2+-dependence of evoked release will be quantified. Aim 5 will test the synaptic plasticity of the neuromuscular synapse using protocols to elicit paired-pulse and short-term facilitation, augmentation and post-tetanic potentiation. This preparation is expected to provide a valuable new tool in which to combine the strengths of C. elegans genetics and molecular biology with electrophysiological analyses. Completion of this project is expected to lay the foundation for future evaluation of over 30 proteins implicated in synaptic transmission.