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

揭示电突触对神经回路贡献的转基因工具

基本信息

批准号：
10012410
负责人：
Adam C Miller
金额：
$ 309.59万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-10 至 2024-08-09
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10012410
关键词：
Affinity Animal Model Animals Antibodies Atlases Axon BRAIN initiative Behavior Brain Cells Chemical Synapse Chemicals Clustered Regularly Interspaced Short Palindromic Repeats Complement Connexins Dendrites Detection Electrical Synapse Electron Microscopy Electrons Electrophysiology (science)Engineering Enhancers Exhibits Fishes Gap Junctions Genes Image Interneurons Label Libraries Link Location Maps Mediating Methods Microscopic Molecular Negative Staining Nervous system structure Neurons Neurotransmitter Receptor Property Proteins Protocols documentation Reporter Research Research Personnel Specificity Synaptic Transmission Time Tissues Transgenic Organisms Zebrafish base connectome design and construction electrical property experience genomic locus interest neural circuit neural network neuronal cell body neuronal circuitry novel promoter sensor synaptic function tool transmission process two-photon

项目摘要

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.

抽象的虽然目前神经回路分析的重点是化学介导的神经元间连接对于突触，人们对电突触的贡献知之甚少。电传输是通过神经元间隙连接，广泛分布在整个脊椎动物大脑中。然而，程度和神经回路内电突触的亚细胞分布很难评估，因为：1) 针对连接蛋白（间隙连接形成蛋白）的抗体的特异性各不相同，导致假阳性或负染色，因此可能生成错误或不完整的连接图，以及 2) 当前用于生成连接体的电子显微镜协议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，以便调查其存在和斑马鱼中电突触的贡献，斑马鱼是一种模式生物，已被确定为特别有利于大脑计划对神经回路的分析。更具体地说，我们建议创建一个转基因斑马鱼文库用于研究电突触传递，这将使首次生成脊椎动物神经中电突触分布的完整图系统。该提案涉及培育三种类型的鱼，并在其上标记连接蛋白和/或其启动子与荧光蛋白或功能传感器相结合，将允许对电突触对具有细胞特异性的各种行为背后的电路的功能贡献。目标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一个感兴趣的细胞或电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。目标2是产生转基因斑马鱼，其中斑马鱼神经元连接蛋白带有荧光蛋白标记。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签的蛋白质，用于评估电信号的数量和亚细胞位置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功能。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。我们建议培育转基因鱼其中神经元连接蛋白与 Ca++ 传感器相连，这使得检测活性电成为可能突触以及那些正在经历可塑性变化的突触。所提出的方法具有重要意义对当前分析方法的改进，如果成功地分析斑马鱼神经回路，可以可能应用于哺乳动物物种的电传输分析。