Molecular mechanisms of electrical synapse formation in vivo

体内电突触形成的分子机制

• 批准号: 9177889
• 负责人: Adam C Miller
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9177889
• 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); Ensure; Epilepsy; Equipment; Fishes; Foundations; Fred Hutchinson Cancer Research Center; Future; Gap Junctions; Genes; Genetic; Genetic Screening; Genomic Segment; Goals; Golgi Apparatus; Health; 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; experience; forward genetics; gap junction channel; gene cloning; in vivo; information processing; insight; live cell imaging; medical schools; mutant; mutation screening; nervous system disorder; neural circuit; neuronal circuitry; neurotransmitter release; postsynaptic; processing speed; protein transport; research study; response; reverse genetics; skills; small molecule; synaptic function; synaptogenesis; targeted treatment; theories; tool; trafficking; transcriptome sequencing; vertebrate embryos

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）：从感官感知到行为的所有大脑功能均来自单个神经元数十亿（人类）的突触连接的模式和特性。该项目的长期目标是了解分子途径，使用脊椎动物模型在体内调节突触形成，重点是不被征收的电气突触。电突触是神经元之间直接通信的部位，允许离子和小分子通过。它们仅在潜在可用的伙伴的一部分之间以调节方式形成，并由神经元间隙连接通道组成。电气突触在发育过程中以及从感觉感知到处理到运动输出的成人电路都对神经回路产生了广泛的贡献。但是，尚不清楚形成形成电气突触的间隙连接通道的基础的分子机制。该建议利用斑马鱼毛特（M）电路研究电突触形成的遗传学。 M神经元是单独识别的，其前和突触后伴侣，突触和功能在活的脊椎动物胚胎中被精美地可视化。进行了突变的正向遗传筛选，导致刻板印象的电气突触中缺陷，以鉴定出两种不同的突变类别：1）断开（DIS）类别，该突变（DIS）类，该类别破坏了突触形成，2）AMP（AMP）类别，这会导致沿M轴突形成的异位突触。使用基于RNA-Seq的方法，所有三个突变均进行了映射，发现其中一个是由于自闭症相关的基因神经裂（NBEA）的丧失所致。该提案将研究NBEA在电气突触形成中的作用（AIM1），将克隆试点屏幕中确定的另一个DIS和AMP突变（AIM2），将检查突变对突触功能和行为的影响（AIM3），并将扩展PILOT屏幕以阐明突触发生所需的进一步基因和途径（AIM4）。在两年的指导阶段，I将通过表征基因如何以多种方式调节电气突触形成的模型系统：突触货物在体内突触发生过程中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（Main Mentor）的塞西莉亚·莫恩斯（Cecilia Moens）实验室中，我将学习使用旋转的荧光标记的突触蛋白进行活细胞成像 椎间盘共聚焦显微镜。该技术将应用于所有突变体，并将是对体内电触突的首次实时研究。为了研究M突触和电路功能，我将访问康奈尔大学的Joe Fetcho实验室，以学习在神经巡回赛上进行电生理学，我将访问宾夕法尼亚大学佩雷​​尔曼大学医学院的Michael Granato实验室，以了解M介导的逃生行为的行为分析。所获得的技能将带回西雅图，在那里我将在突变体上进行实验。对于电生理学，我将与华盛顿大学（主要联合学）的瑞秋·黄（Rachel Wong）合作，在那里我将接受电生理学的持续培训，并可以使用实验设备。对于行为，我将在Moens实验室工作，在那里我们有需要高速摄像头来捕获M介导的逃生响应。电生理和行为分析将应用于所有突变体，对于将细胞生物缺陷与电路中的功能缺陷联系起来至关重要。在Fetcho和Granato Las进行的培训将是简短而密集的，但两位导师都将在我的技术专业知识和指导的基础上为我提供。在Moens和Wong Labs中的指导将进行，并具有广泛的互动和支持。通过此培训，我将拥有必要的经验以及一套强大的工具和技术来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照亮在电气突触处建立间隙连接的分子机制。拟议的研究将提供详细的分子，细胞和功能视图，以了解脊椎动物在体内如何形成的神经回路。导致神经回路误导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。就自闭症而言，几种分子途径（包括在AIM1中检查的NBEA）与该疾病有关。然而，一种统一的理论解释了这些基因如何融合来解释综合征仍然难以捉摸。研究神经回路接线和突触形成所需的遗传途径将有助于洞察疾病状态，最终将允许鉴定治疗靶标。