Molecular mechanisms of electrical synapse formation in vivo

体内电突触形成的分子机制

• 批准号: 8618053
• 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
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8618053
• 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; 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

7. Project Summary/Abstract 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 on 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 labs 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.

7.项目摘要/摘要 大脑的所有功能，从感官感知到行为，都是从大脑的模式和属性中衍生出来的 数十亿(人类)单个神经元之间的突触连接。这个项目的长期目标是 利用有焦点的脊椎动物模型了解体内调节突触形成的分子途径 在被忽视的电子突触上。电突触是人与人之间直接交流的场所 允许离子和小分子通过的神经元。它们是以受监管的方式在 只有潜在可用的伙伴的子集，并由神经元缝隙连接通道组成。电气 突触对发育过程中的神经回路以及成人的感觉回路都有广泛的贡献 从感知到加工再到电机输出。然而，形成这种病毒的分子机制 形成电突触的缝隙连接通道尚不清楚。 这一建议利用斑马鱼Mauthner(M)回路来研究电突触的遗传学 队形。M个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 在一个活的脊椎动物胚胎中，功能被巧妙地可视化。基因突变的正向筛查 导致刻板印象中的M电突触缺陷的研究发现了两类不同的 突变：1)破坏突触形成的断开(Dis)类，和2)AMPED(Amp)类， 这会导致异位突触沿着M轴突形成。使用基于RNA-SEQ的方法所有三个DIS 突变被定位在地图上，其中一个Dis突变体被发现是由于自闭症的丧失- 相关基因Neurobeachin(NbeA)。这项提案将调查Nbea在电突触中的作用 形成(Aim1)，将克隆在Pilot Screen(AIM2)中发现的其他Dis和Amp突变，将检查 突变对突触功能和行为的影响(Aim3)，并将把飞行员屏幕扩大到 进一步阐明突触发生所需的基因和途径(Aim4)。 在为期两年的指导阶段，我将通过描述基因如何调节来开发模型系统。 电突触形成的几种方式：突触胞体的时间和空间特性是什么 体内突触发生过程中的定位？突变体如何影响突触的功能？你是如何 突变体会影响神经网络的功能和行为吗？在弗雷德·哈钦森癌症中心的塞西莉亚·莫恩斯的实验室里 研究中心(主要导师)，我将学习对荧光标记的突触进行活细胞成像 用旋转圆盘共聚焦显微镜观察蛋白质。这项技术将应用于所有突变体，并将成为 首次活体研究电突触的形成。为了研究M突触和电路功能，我将 参观乔·费乔在康奈尔大学的实验室，学习如何在M神经回路和我 将参观宾夕法尼亚大学佩雷尔曼医学院迈克尔·格拉纳托的实验室，了解 M中介逃逸行为的行为分析。所获得的技能将被带回西雅图 在那里我将在变种人身上做实验。在电生理学方面，我将与雷切尔·黄在 华盛顿大学(主要合作导师)，我将在那里接受持续的电生理学培训，并将 有权获得用于实验的设备。对于行为我将在莫恩斯实验室工作，在那里我们有很高的- 捕捉M介导的逃逸反应所需的速度相机。电生理学和 行为分析将应用于所有突变体，并将是将细胞生物学缺陷与 回路中的功能缺陷。Fetcho和Granato实验室的培训将是短暂而密集的，但两者 导师将为我提供持续的技术专长和指导。在学校里的辅导 Moens和Wong实验室将持续进行，并提供广泛的互动和支持。通过这次训练，我将拥有 必要的经验和强大的工具和技术来建立我自己的独立研究 一群人。在项目的独立阶段，我将利用所获得的技能来阐明分子 在电突触建立缝隙连接的机制。 拟议的研究将提供神经回路如何形成的详细的分子、细胞和功能视图。 在活体脊椎动物体内。导致神经回路错误连接或突触失衡的疾病是 许多神经系统疾病，包括自闭症和癫痫。在自闭症的情况下，有几个分子途径 (包括在Aim1这里检查的Nbea)与这种疾病有关。然而，一个统一的理论 解释这些基因是如何组合在一起来解释这种综合症的，仍然难以捉摸。研究基因 神经回路连接和突触形成所需的通路将使人们深入了解疾病状态， 最终允许确定治疗的目标。