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. 项目总结/摘要 所有的大脑功能，从感觉知觉到行为，都源自大脑的模式和特性。 数十亿（人类）单个神经元之间的突触连接。该项目的长期目标是 使用有重点的脊椎动物模型了解体内调节突触形成的分子途径 关于未被充分认识的电突触。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的神经元。它们是按照规定的方式形成的 仅潜在可用伙伴的子集，并且由神经元间隙连接通道组成。电气 突触对发育过程中的神经回路以及感觉器官的成人回路有广泛的贡献 感知到处理到运动输出。然而，其形成的分子机制 形成电突触的间隙连接通道尚不清楚。 该提案利用斑马鱼 Mauthner (M) 电路来研究电突触的遗传学 形成。 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清晰可见。突变的正向遗传筛选 造成定型 M 电突触缺陷的研究确定了两类不同的 突变：1) Disconnect (Dis) 类，破坏突触形成，2) Amped (Amp) 类， 这导致异位突触沿着轴突形成。使用基于 RNA-seq 的方法，所有三个 Dis 突变被定位，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)。该提案将研究 Nbea 在电突触中的作用 形成 (Aim1)，将克隆在试点筛选 (Aim2) 中确定的其他 Dis 和 Amp 突变，将检查 突变对突触功能和行为的影响（目标3），并将试点屏幕扩展到 进一步阐明突触发生所需的基因和途径（目标4）。 在两年的指导阶段，我将通过描述基因如何调节来开发模型系统 电突触形成有多种方式：突触货物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？怎样做 突变体会影响神经网络功能和行为吗？在 Fred Hutchinson 癌症中心塞西莉亚·莫恩斯 (Cecilia Moens) 的实验室中 研究中心（主要导师），我将学习进行荧光标记、突触的活细胞成像 使用转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 首次对体内电突触形成的现场研究。为了研究 M 突触和电路功能，我将 参观 Joe Fetcho 在康奈尔大学的实验室，学习对 M 神经回路进行电生理学研究 将参观宾夕法尼亚大学佩雷​​尔曼医学院Michael Granato的实验室进行学习 M介导的逃避行为的行为分析。获得的技能将带回西雅图 我将在那里对突变体进行实验。对于电生理学，我将与 Rachel Wong 在 华盛顿大学（主要共同导师），我将在那里接受电生理学方面的持续培训，并将 有权使用实验设备。对于行为，我将在莫恩斯实验室工作，那里我们拥有高 测速摄像头是捕捉 M 介导的逃逸反应所必需的。电生理学和 行为分析将应用于所有突变体，对于将细胞生物学缺陷与突变体联系起来至关重要 电路中的功能缺陷。 Fetcho 和 Granato 实验室的培训将是短暂而密集的，但两者 导师将持续为我提供技术专业知识和指导。辅导中的 Moens 和 Wong 实验室将继续进行，并提供广泛的互动和支持。通过这次培训我将拥有 建立自己的独立研究所需的经验和一套强大的工具和技术 团体。在项目的独立阶段，我将利用获得的技能来阐明分子 在电突触处建立间隙连接的机制。 拟议的研究将为神经回路如何形成提供详细的分子、细胞和功能视图 在脊椎动物体内。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。就自闭症而言，有几种分子途径 （包括 Aim1 中检查的 Nbea）与该疾病有关。然而统一的理论 解释这些基因如何组合在一起来解释这种综合症仍然难以捉摸。研究遗传 神经回路布线和突触形成所需的途径将有助于深入了解疾病状态， 最终可以确定治疗靶点。