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Molecular mechanisms of electrical synapse formation in vivo

体内电突触形成的分子机制

基本信息

批准号：
8743313
负责人：
Adam C Miller
金额：
$ 9万
依托单位：
FRED HUTCHINSON CANCER RESEARCH CENTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2015-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8743313
关键词：
Adult Affect Animals Autistic Disorder Award Axon Back Behavior Behavioral Biochemical Biological Biological Models Biological Neural Networks Brain Cell Transplants Cells Cellular biology Chemical Synapse Chemicals Chromosome Mapping Cloning Communication Complement Confocal Microscopy Defect Dendrites Development Disease Electrical Synapse Electrophysiology (science)Embryo Ensure Epilepsy Equipment Fishes Foundations Fred Hutchinson Cancer Research Center Future Gap Junctions Genes Genetic Genetic Screening Genomics Goals Golgi Apparatus Human Image Individual Investigation Ions Knowledge Lead Learning Lesion Life Link Location Maps Mediating Mentors Methods Modeling Molecular Motor Motor output Multivesicular Body Mutation Neuraxis Neurons Pathway interactions Pattern Pennsylvania Perception Phase Physiological Physiology Process Property Proteins Recruitment Activity Research Role Sensory Signal Transduction Site Speed Stereotyping Stimulus Synapses Syndrome Technical Expertise Techniques Testing Therapeutic Training Universities Visit Washington Work Zebrafish base cellular imaging experience forward genetics gap junction channel gene cloning in vivo information processing insight medical schools mutant nervous system disorder neural circuit neuronal circuitry neurotransmitter release positional cloning postsynaptic processing speed protein transport public health relevance research study response skills small molecule synaptic function synaptogenesis theories tool trafficking transcriptome sequencing

项目摘要

DESCRIPTION (provided by candidate): All of brain function, from sensory perception to behavior, is derived from the pattern and properties of the synaptic connections among billions (in humans) of individual neurons. The long-term goal of this project is to understand molecular pathways that regulate synapse formation in vivo using a vertebrate model with a focus on the underappreciated electrical synapse. Electrical synapses are sites of direct communication between neurons that allow the passage of ions and small molecules. They are formed in a regulated manner between only a subset of potentially available partners and are composed of neuronal gap junction channels. Electrical synapses contribute extensively to neural circuits during development as well as to adult circuits from sensory perception to processing to motor output. However, the molecular mechanisms underlying the formation of the gap junction channels that form the electrical synapse are unknown. This proposal utilizes the Zebrafish Mauthner (M) circuit to investigate the genetics of electrical synapse formation. The M neurons are individually identifiable and their pre and postsynaptic partners, synapses, and function are exquisitely visualized in a living, vertebrate embryo. A forward genetic screen for mutations causing defects in the stereotyped M electrical synapses was performed that identified two distinct classes of mutations: 1) the Disconnect (Dis) class, which disrupts synapse formation, and 2) the Amped (Amp) class, which causes ectopic synapses to form along the M axon. Using an RNA-seq-based approach all three Dis mutations were positionally mapped, and one of the Dis mutants was found to be due to the loss of the autism- associated gene neurobeachin (nbea). This proposal will investigate Nbea's role in electrical synapse formation (Aim1), will clone the other Dis and Amp mutations identified in the pilot screen (Aim2), will examine the effect of the mutations on synapse function and behavior (Aim3), and will expand the pilot screen to elucidate further genes and pathways required for synaptogenesis (Aim4). During the two year mentored phase I will develop the model system by characterizing how the genes regulate electrical synapse formation in several ways: What are the temporal and spatial properties of synaptic cargo localization during in vivo synaptogenesis? How do the mutants affect the function of the synapse? How do the mutants affect neural network function and behavior? In Cecilia Moens' lab at the Fred Hutchinson Cancer Research Center (main mentor), I will learn to perform live cell imaging of fluorescently-tagged, synaptic proteins using spinning disc confocal microscopy. This technique will be applied to all mutants and will be the first live investigation of electrical synapse formation in vivo. To investigate M synapse and circuit function I will visit Joe Fetcho's lab at Cornell University to learn to perform electrophysiology n the M neural circuit and I will visit Michael Granato's lab at the University of Pennsylvania Perelman School of Medicine to learn behavioral analysis of the M-mediated escape behavior. The skills acquired will be brought back to Seattle where I will perform experiments on the mutants. For electrophysiology I will work with Rachel Wong at the University of Washington (main co-mentor) where I will receive ongoing training in electrophysiology and will have access to equipment for experiments. For behavior I will work in the Moens lab where we have the high- speed camera necessary to capture the M-mediated escape response. The electrophysiological and behavioral analysis will be applied to all mutants and will be essential for linking the cell-biological defects to functional deficits in the circuit. The training in the Fetcho and Granato las will be short and intensive, but both mentors will be available to me on an ongoing basis for technical expertise and guidance. The mentoring in the Moens and Wong labs will be ongoing, with extensive interaction and support. With this training I will have the necessary experience and a powerful set of tools and techniques to establish my own independent research group. During the independent phase of the project I will utilize the acquired skills to illuminate the molecular mechanisms that build gap junctions at the electrical synapse. The proposed studies will provide a detailed molecular, cellular, and functional view of how neural circuits form in a vertebrate in vivo. Disorders that cause neural circuit miswiring or synaptic imbalance are the basis of many neurological diseases including autism and epilepsy. In the case of autism, several molecular pathways (including Nbea examined here in Aim1) have been associated with the disorder. However a unifying theory explaining how these genes fit together to explain the syndrome remains elusive. Investigating the genetic pathways required for neural circuit wiring and synapse formation will lend insight into disease states that will ultimately allow for the identification of targets for therapy.

描述（由候选人提供）：所有的大脑功能，从感官知觉到行为，都来自于数十亿（人类）单个神经元之间突触连接的模式和特性。这个项目的长期目标是利用脊椎动物模型来了解体内调节突触形成的分子通路，重点是未被重视的电突触。电突触是神经元之间直接交流的场所，允许离子和小分子通过。它们仅在潜在可用伴侣的子集之间以受调节的方式形成，并且由神经元间隙连接通道组成。电突触在发育过程中对神经回路以及从感觉知觉到处理到运动输出的成人回路做出了广泛的贡献。然而，形成电突触的差距连接通道的分子机制尚不清楚。该提议利用斑马鱼Mauthner（M）电路来研究电突触形成的遗传学。M神经元是单独可识别的，它们的突触前和突触后伙伴、突触和功能在活的脊椎动物胚胎中被精细地可视化。对导致定型M电突触缺陷的突变进行正向遗传筛选，鉴定出两种不同的突变类别：1）断开（Disc）类别，其破坏突触形成，和2）安培（Amped）类别，其导致异位突触沿沿着M轴突形成。使用基于RNA-seq的方法，对所有三种Dis突变进行了定位，发现其中一种Dis突变体是由于自闭症相关基因神经海滩蛋白（nbea）的丢失。该提案将研究Nbea在电突触形成中的作用（Aim 1），将克隆在试点筛选中确定的其他Dis和Dis突变（Aim 2），将检查突变对突触功能和行为的影响（Aim 3），并将扩大试点筛选以阐明突触发生所需的进一步基因和途径（Aim 4）。在为期两年的指导阶段，我将开发模型系统，通过表征基因如何以几种方式调节电突触的形成：在体内突触发生过程中，突触货物定位的时间和空间特性是什么？突变体如何影响突触的功能？突变体如何影响神经网络的功能和行为？在弗雷德哈钦森癌症研究中心的塞西莉亚·莫恩斯实验室（主要导师），我将学习使用旋转技术对荧光标记的突触蛋白进行活细胞成像。圆盘共聚焦显微镜这项技术将应用于所有的突变体，并将是第一个活的调查电突触的形成在体内。为了研究M突触和回路功能，我将访问康奈尔大学的Joe Fetcho实验室，学习M神经回路的电生理学，我将访问宾夕法尼亚大学佩雷尔曼医学院的Michael Granato实验室，学习M介导的逃避行为的行为分析。学到的技能将被带回西雅图在那里我将对变种人进行实验。对于电生理学，我将与华盛顿大学的Rachel Wong（主要共同导师）合作，在那里我将接受电生理学的持续培训，并将获得实验设备。至于行为方面，我将在莫恩斯实验室工作，那里有高速摄像机，可以捕捉M介导的逃避反应。电生理和行为分析将应用于所有突变体，并将是必不可少的连接电路中的功能缺陷的细胞生物学缺陷。在Fetcho和Granato las的培训将是短期和密集的，但这两名导师将持续向我提供技术专长和指导。Moens和Wong实验室的指导将持续进行，并提供广泛的互动和支持。通过这次培训，我将获得必要的经验和一套强大的工具和技术，以建立自己的独立研究小组。在项目的独立阶段，我将利用获得的技能来阐明在电突触处建立间隙连接的分子机制。拟议的研究将提供一个详细的分子，细胞和功能的观点如何神经回路形成在脊椎动物体内。导致神经回路布线错误或突触失衡的疾病是许多神经系统疾病的基础，包括自闭症和癫痫。在自闭症的情况下，几种分子途径（包括在Aim 1中检查的Nbea）与这种疾病有关。然而，解释这些基因如何组合在一起来解释这种综合征的统一理论仍然难以捉摸。研究神经回路布线和突触形成所需的遗传途径将有助于深入了解疾病状态，最终可以确定治疗目标。