Generation of transgenic zebrafish to study electrical synaptic transmission

描述(由申请人提供)：缝隙连接(GJ)介导的电突触传递被认为是神经元间交流的一种基本形式。它在哺乳动物中枢神经系统不同区域的重要功能过程中起着至关重要的作用，并与多种神经疾病有关。电突触的可塑性通过重新配置电耦合神经元网络来支持重要功能，电耦合神经元网络的破坏可能导致神经功能障碍。与化学突触相比，人们对调节电突触强度的分子机制知之甚少。该建议侧重于了解在混合的电和化学突触观察到的GJ通讯可塑性变化的机制，突触将初级听觉传入耦合到硬骨Mauthner(M-)细胞，在M-细胞，GJS是由广泛存在的哺乳动物GJ蛋白Cx36(Cx36)的鱼类同源物形成的，并且有可能在体内分析细胞和亚细胞机制。我们在金鱼身上的研究表明，混合突触反应的两个组成部分都经历了各自强度的活性依赖增强。值得注意的是，我们最近的发现表明，调节功能GJ通道的周转和数量的因素可能是电传递强度的主要决定因素。我们建议研究GJ通道的贩运对调节电传递强度的可能机制的贡献。为此，我们将把这种独特的混合突触模型带到一个新的分析水平，通过研究它们在幼虫斑马鱼中的特性，它的透明度将使在活体细胞中追踪单个分子成为可能。支持这一可能性的初步结果表明，斑马鱼幼体的混合突触在分子和功能上与成年金鱼相似。该方案有两个目标：目标1是获得带有荧光蛋白的转基因斑马鱼，目标2是研究荧光标记的间隙连接通道在体内的周转及其在触发可塑性的条件下的特性。斑马鱼幼虫能够在体内成像荧光标记的GJ通道的运动，这应该允许监测经历可塑性的活跃突触，为分析这种传递方式提供了一个前所未有的窗口，通过结合电生理学和活体成像以及强大的基因操作可以研究详细的分子机制。因此，斑马鱼模型的发展将为研究Cx36介导的突触(在哺乳动物中普遍存在)的分子方面提供一个新的强大工具，从而为治疗各种神经疾病提供新的治疗机会。

DESCRIPTION (provided by applicant): Gap junction (GJ) mediated electrical synaptic transmission is considered an essential form of interneuronal communication. It critically contributes to important functional processes in diverse regions of the mammalian CNS and has been linked to a variety of neurological conditions. Plasticity of electrical synapses underlies important functions by reconfiguring networks of electrically coupled neurons, whose disruption might contribute to neurological dysfunction. In contrast to chemical synapses, less is known regarding the molecular mechanisms that regulate the strength of electrical synapses. This proposal focuses on understanding mechanisms underlying plastic changes in GJ communication observed at mixed, electrical and chemical, synapses that couple primary auditory afferents to the teleost Mauthner (M-) cells, at which GJs are formed by fish homologs of the widespread mammalian GJ protein connexin36 (Cx36) and where it is possible to analyze cellular and sub-cellular mechanisms in-vivo. Our studies in goldfish show that both components of the mixed synaptic response undergo activity-dependent potentiation of their respective strengths. Remarkably, our recent findings indicate that factors regulating the turnover and number of functional GJ channels might constitute major determinants of the strength of electrical transmission. We propose here to investigate the contribution of trafficking of GJ channels as a possible mechanism for regulating the strength of electrical transmission. For this purpose, we will take this unique model mixed synapse to a new level of analysis by investigating their properties in larval zebrafish, whose transparency will make it possible to track individual molecules within living cells, in vivo. Supporting this possibility, our preliminay results indicate that mixed synapses in larval zebrafish are molecularly and functionally analogous to those of adult goldfish. The proposal has two aims: Aim 1 is to generate transgenic zebrafish in which neuronal gap junction proteins are tagged with fluorescent proteins, and Aim 2 is to investigate the turnover of fluorescently tagged gap junction channels in-vivo and its properties under conditions that trigger plasticity. The amenability of zebrafish larvae to image the movement of fluorescently tagged GJ channels in-vivo should permit the monitoring of active synapses undergoing plasticity providing an unprecedented window for the analysis of this modality of transmission at which detailed molecular mechanisms could be investigated combining electrophysiology and live imaging with powerful genetic manipulations. Thus, the development of this zebrafish model will provide a new powerful tool to study molecular aspects of Cx36-mediated synapses (prevalent in mammals) that could lead to the identification of novel therapeutic opportunities for the treatment of various neurological conditions.