Transgenic tools for revealing the contributions of electrical synapses to neural circuits

摘要 虽然目前对神经回路的分析主要集中在化学物质介导的神经元间连接上 突触，人们对电子突触的贡献知之甚少。电力传输是通过以下方式进行的 神经元缝隙连接，广泛分布在脊椎动物的大脑中。然而，程度和 神经回路中电突触的亚细胞分布一直很难评估，因为：1) 针对连接蛋白(缝隙连接形成蛋白)的抗体的特异性不同，导致假阳性。 或负染色，因此可能生成错误或不完整的连接地图，以及2) 目前用于产生连接的电子显微镜方案不利于检测间隙 因此，对神经元与化学突触的相互连接的描述存在偏差。要克服这一点 问题，我们建议开发基于转基因的方法，将允许调查存在和 电突触在斑马鱼中的作用，斑马鱼是一种模式生物，已被确认为 有利于通过大脑倡议来分析神经回路。更具体地说，我们建议创建一个 转基因斑马鱼文库研究电突触传递将使 首次绘制出脊椎动物神经中电突触的完整分布图 系统。该提案涉及到对连接蛋白和/或其启动子进行标记的三种类型的鱼 与荧光蛋白或功能传感器相结合，将允许全面检查 电突触对具有细胞特异性的各种行为基础的回路的功能贡献。目标1 是产生转基因斑马鱼，神经元连接蛋白的启动子连接到报告 荧光蛋白。这些动物的存在将允许建立一个 在感兴趣的细胞或回路中的特定缝隙连接蛋白，这是一个众所周知的具有挑战性的问题，因为细胞表达 一种特定的连接蛋白将被荧光标记。目标2是产生转基因斑马鱼，斑马鱼 神经元连接蛋白用荧光蛋白标记。我们将设计内源性神经元连接蛋白 带有荧光蛋白或亲和标记的蛋白质以评估电子信号的数量和亚细胞位置 细胞与相邻细胞之间的突触。由于构建物的设计，标记的连接蛋白可以 通过多种方法成像，包括活体动物或组织的单光子或双光子成像，或化学成像 适用于电子显微镜分析的增强。最后，目标3是培育转基因斑马鱼 研究电突触对神经元回路的功能贡献。我们计划生产转基因鱼 其中神经元连接蛋白连接到钙传感器，这将使检测激活的电 突触以及那些正在经历可塑性变化的突触。拟议的方法代表了一个重要的 对当前分析方法的改进，如果成功地分析斑马鱼神经电路，可以 有可能应用于哺乳动物物种的电传输分析。

Abstract While current efforts in the analysis of neural circuits focus on interneuronal connectivity mediated by chemical synapses, less is known about the contribution of electrical synapses. Electrical transmission is mediated by neuronal gap junctions, which are widely distributed throughout the vertebrate brain. However, the extent and subcellular distribution of electrical synapses within neural circuits has been difficult to assess because: 1) antibodies targeting connexins (gap junction forming proteins) vary in their specificity, resulting in false positive or negative staining, and therefore potentially generating wrong or incomplete maps of connectivity, and 2) current electron microscopy protocols used to generate connectomes are unfavorable for detecting gap junctions, thus biasing the description of neuronal interconnection to chemical synapses. To overcome this problem, we propose to develop transgenic-based methods that will allow investigating the presence and contribution of electrical synapses in zebrafish, a model organism that has been identified as particularly advantageous for the analysis of neural circuits by the Brain Initiative. More specifically, we propose to create a Library of Transgenic Zebrafish to study Electrical Synaptic Transmission which will make it possible to generate, for the first time, a complete map of the distribution of electrical synapses in a vertebrate nervous system. The proposal involves generating three types of fish at which connexins and/or its promoters are tagged with fluorescent proteins or functional sensors that, combined, will allow comprehensive examination of the functional contributions of electrical synapses to circuits underlying various behaviors with cell specificity. Aim 1 is to generate transgenic zebrafish at which the promoters of neuronal connexins are linked to reporter fluorescent proteins. The availability of these animals will allow for the establishment of the presence of a particular gap junction protein in a cell or circuit of interest, a notoriously challenging problem, as cells expressing a particular connexin will be fluorescently labeled. Aim 2 is to generate transgenic zebrafish at which zebrafish neuronal connexins are tagged with fluorescent proteins. We will engineer the endogenous neuronal connexin proteins with fluorescent proteins or affinity tags to assess the number and subcellular location of electrical synapses of a cell with its connected neighbors. Because of the design of the constructs, tagged connexins can be imaged by diverse methods including single or 2-photon imaging of living animals or tissues, or chemical enhancements suitable for electron microscopic analysis. Finally, Aim 3 is to generate transgenic zebrafish to study functional contributions of electrical synapses to neuronal circuits. We propose to generate transgenic fish in which neuronal connexins are linked to Ca++ sensors that will make possible detecting active electrical synapses as well those undergoing plastic changes. The proposed approach represents a significant improvement over current methods of analysis and, if successful for analysis of zebrafish neural circuits, could be potentially applied to analysis of electrical transmission in mammalian species.