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: 8623965
• 负责人: Alberto E Pereda
• 金额: $ 22.3万
• 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/8623965
• 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）介导的电突触传递被认为是神经元间通信的基本形式。它对哺乳动物中枢神经系统不同区域的重要功能过程发挥着至关重要的作用，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来实现重要功能的基础，电耦合神经元网络的破坏可能会导致神经功能障碍。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。该提案的重点是了解在混合、电和化学突触中观察到的 GJ 通讯塑料变化的机制，这些突触将初级听觉传入与硬骨鱼 Mauthner (M-) 细胞耦合，其中 GJ 由广泛存在的哺乳动物 GJ 蛋白的鱼类同源物形成connexin36 (Cx36) 以及可以分析体内细胞和亚细胞机制的地方。我们对金鱼的研究表明，混合突触反应的两个组成部分各自的强度都会经历活动依赖性增强。值得注意的是，我们最近的研究结果表明，调节功能性 GJ 通道的周转和数量的因素可能是电传输强度的主要决定因素。我们在此建议调查 GJ 通道贩运的贡献作为调节电传输强度的可能机制。为此，我们将通过研究斑马鱼幼虫的特性，将这种独特的混合突触模型提升到一个新的分析水平，其透明度将使在体内追踪活细胞内的单个分子成为可能。我们的初步结果表明，斑马鱼幼体的混合突触在分子和功能上与成年金鱼的混合突触相似，支持了这种可能性。该提案有两个目标：目标 1 是产生转基因斑马鱼，其中神经元间隙连接蛋白用荧光蛋白标记，目标 2 是研究体内荧光标记的间隙连接通道的周转及其在触发可塑性的条件下的特性。斑马鱼幼虫在体内对荧光标记的 GJ 通道运动进行成像的能力应该允许监测正在经历可塑性的活动突触，为分析这种传输方式提供了一个前所未有的窗口，在该窗口中可以结合电生理学和活体研究详细的分子机制。通过强大的基因操作进行成像。因此，这种斑马鱼模型的开发将为研究 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，这可能导致识别治疗各种神经系统疾病的新治疗机会。