ELECTROPHYSIOLOGICAL ANALYSIS OF A C ELEGANS SYNAPSE

描述：(申请人摘要) 突触传递突触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的功能问题 系统将在更易处理的系统中寻址。例如，超过30个基因 已经在线虫中发现了与突触传递有关的信息， 秀丽隐杆线虫以及这些基因的同源物随后被 在脊椎动物中发现的。神经传递缺陷所致 神经退行性变会导致神经系统功能严重受损。 在帕金森氏症和阿尔茨海默氏症等疾病中很明显。其中一个 研究突触蛋白作用的最有效的方法是研究 缺乏这些蛋白质的突变动物的突触生理学。A准备工作 适用于突触功能的详细电生理分析 以前还没有线虫，主要是因为技术上的 解剖这么小的有机体很困难。 这份提案提供的初步数据证明了 全细胞电压钳记录新研制的C. 雅致体壁神经肌肉制剂。为了使用这种制剂 为了探索突触在突变动物中的传递，基线特征 需要野生型突触的。 线虫的神经肌肉接头由GABA能和 胆碱能运动神经元。为了表征在这个突触上的释放 将有必要分别研究这两个输入。Aim我会确定 分离两个突触输入的药理学工具。Aim 2将使用这些 表征自发性GABA能和胆碱能的药理学工具 突触活动。AIM 3将开发方法来激发GABA和ACh的释放 神经肌肉接头。在目标4中，诱发释放的钙依赖 将会被量化。目标5将测试突触的可塑性 神经肌肉突触诱导成对脉冲和短时程控 易化、增强和强直后增强。 这一准备工作有望提供一种有价值的新工具，在其中结合 线虫遗传学和分子生物学的优势 电生理分析。这一项目预计将竣工 未来对30多个与突触有关的蛋白质进行评估的基础 变速箱。

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.