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

摘要 虽然目前在神经回路分析中的努力集中在由化学物质介导的神经元间连接上， 突触，对电突触的贡献知之甚少。电传输是由 神经元间隙连接，其广泛分布于整个脊椎动物脑中。然而，程度和 神经回路中电突触的亚细胞分布一直难以评估，因为：1） 靶向连接蛋白（间隙连接形成蛋白）的抗体在其特异性上变化，导致假阳性 或阴性染色，因此可能产生错误或不完整的连接图，以及2） 目前用于产生连接体的电子显微术方案不利于检测间隙 连接，从而使神经元互连的描述偏向于化学突触。为了克服这个 问题，我们建议开发基于转基因的方法，将允许调查的存在和 斑马鱼是一种模式生物，已被确定为特别是 这对大脑计划分析神经回路是有利的。更具体地说，我们建议建立一个 转基因斑马鱼库研究电突触传递，这将使人们有可能 产生，第一次，一个完整的地图分布的电突触在脊椎动物的神经 系统该提案涉及产生三种类型的鱼类，其中连接蛋白和/或其启动子被标记 与荧光蛋白或功能传感器，结合起来，将允许全面检查 电突触对具有细胞特异性的各种行为的电路的功能贡献。要求1 是产生转基因斑马鱼，其中神经元连接蛋白的启动子与报告基因连接， 荧光蛋白这些动物的可用性将允许建立存在 在细胞或感兴趣的回路中的特定间隙连接蛋白，这是一个众所周知的挑战性问题，因为细胞表达 特定的连接蛋白将被荧光标记。目的2是产生转基因斑马鱼， 用荧光蛋白标记神经元连接蛋白。我们将设计内源性神经连接蛋白 蛋白质与荧光蛋白或亲和标签，以评估电的数量和亚细胞位置 一个细胞与其相连的邻居的突触。由于构建体的设计，标记的连接蛋白可以 通过多种方法成像，包括活动物或组织的单光子或双光子成像，或化学成像。 增强适用于电子显微镜分析。最后，目标3是产生转基因斑马鱼， 研究电突触对神经元回路的功能贡献。我们建议培育转基因鱼 其中神经元连接蛋白连接到Ca++传感器，这将使得检测活性电 突触以及那些正在经历塑性变化的突触。所提出的方法代表了一个重要的 改进现有的分析方法，如果成功地分析斑马鱼神经回路， 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.