Generation of transgenic zebrafish to study electrical synaptic transmission

产生转基因斑马鱼以研究电突触传递

• 批准号: 8735205
• 负责人: Alberto E Pereda
• 金额: $ 16.61万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-16 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8735205
• 关键词: Adult; Auditory; Behavior monitoring; Cells; Chemical Synapse; Chemicals; Chemosensitization; Communication; Connexins; Coupled; Coupling; Electrical Synapse; Electrophysiology (science); Fishes; Future; Gap Junctions; Generations; Glutamate Receptor; Glutamates; Goldfish; Homologous Gene; Image; Individual; Investigation; Larva; Lead; Life; Link; M cell; Mammals; Mediating; Microscopy; Modality; Modeling; Modification; Molecular; Monitor; Movement; Neurologic; Neurologic Dysfunctions; Neurons; Optics; Plastics; Process; Property; Proteins; Regulation; Resolution; Surface; Synapses; Synaptic Transmission; Transgenic Animals; Transgenic Organisms; Zebrafish; connexin 36; gap junction channel; genetic manipulation; high risk; in vivo; novel therapeutics; postsynaptic; presynaptic; public health relevance; research study; response; teleost; tool; trafficking; transmission process; zebrafish development

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.

描述（由申请人提供）：间隙连接（GJ）介导的电突触传播被认为是神经元通信的必要形式。它在哺乳动物中枢神经系统的各个区域中有助于重要的功能过程，并与多种神经系统疾病有关。电突触的可塑性是通过重新配置电耦合神经元网络的重要功能的基础，其破坏可能导致神经功能障碍。与化学突触相反，关于调节电突触强度的分子机制，知之甚少。该提案的重点是理解在混合，电气和化学物质上观察到的塑性变化的机制，它们将主要听觉传入与Teleost Mauthner（M-）细胞相对，这些传感将GJ通过广泛的哺乳动物GJ GJ蛋白Connexin36（CX36）和细胞分析而形成GJS。我们在金鱼中的研究表明，混合突触反应的两种组成部分都依赖于它们各自的强度的活性增强。值得注意的是，我们最近的发现表明，调节功能性GJ通道的周转和数量的因素可能构成电气传播强度的主要决定因素。我们在这里提议研究GJ通道的运输贡献，以此作为调节电气传播强度的可能机制。为此，我们将通过研究其在幼虫斑马鱼中的特性，将这种独特的模型混合突触列为新的分析水平，它们的透明度将使在Vivo中追踪活细胞内的个体分子成为可能。支持这种可能性，我们的预赛结果表明，幼虫斑马鱼中的混合突触在分子和功能上类似于成年金鱼的混合突触。该提案具有两个目的：目标1是生成转基因斑马鱼，其中神经元间隙连接蛋白用荧光蛋白标记为标记，而目标2是研究荧光标记的间隙连接通道的转换及其在触发可塑性的条件下的特性及其性能。斑马鱼幼虫在体内的荧光标记的GJ通道的移动应允许监测，这应允许监测有效的突触，从而提供一个前所未有的窗口，以分析这种传播方式，在该窗口中，可以调查详细的分子机制，并与功能强大的遗传相结合。因此，这种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）的分子方面提供新的强大工具，这可能导致鉴定新的治疗机会以治疗各种神经系统疾病。