Molecular mechanisms of electrical synapse formation in vivo

体内电突触形成的分子机制

• 批准号: 9408653
• 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/9408653
• 关键词: Adult; Affect; Animals; Autistic Disorder; Award; Axon; Back; Behavior; Behavioral; Biochemical; Biological; Biological Models; Biological Neural Networks; Brain; Brain Diseases; 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; Human; Image; Individual; Investigation; Ions; Knowledge; Lead; Learning; Lesion; Link; Location; Maps; Mauthner' s neuron; Mediating; Mentors; Methods; Modeling; Molecular; Motor; Motor output; Multivesicular Body; Mutation; Neuraxis; Neurons; Pathway interactions; Pattern; Pennsylvania; Perception; Phase; Physiological; Physiology; Process; Property; Proteins; Reproducibility; Research; Role; Sensory; Signal Transduction; Site; Speed; Stereotyping; Stimulus; Synapses; Syndrome; Technical Expertise; Techniques; Testing; Therapeutic; Training; Universities; Visit; Washington; Work; Zebrafish; base; experience; experimental study; fluorescence imaging; forward genetics; gap junction channel; gene cloning; in vivo; in vivo imaging; information processing; insight; live cell imaging; medical schools; mutant; mutation screening; nervous system disorder; neural circuit; neuronal circuitry; neurotransmitter release; postsynaptic; protein transport; public health relevance; recruit; 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）：从感觉知觉到行为的所有大脑功能都源自数十亿（人类）单个神经元之间突触连接的模式和特性。该项目的长期目标是利用脊椎动物模型了解体内调节突触形成的分子途径，重点关注未被充分认识的电突触。电突触是神经元之间直接通信的场所，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。电突触对发育过程中的神经回路以及从感觉知觉到处理再到运动输出的成人回路做出了广泛的贡献。然而，形成电突触的间隙连接通道的分子机制尚不清楚。该提案利用斑马鱼 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清晰可见。对导致定型 M 电突触缺陷的突变进行了正向遗传筛选，确定了两种不同类型的突变：1）断开 (Dis) 类，它破坏突触形成；2) Amped (Amp) 类，它导致沿 M 轴突形成异位突触。使用基于 RNA 测序的方法对所有三种 Dis 突变进行了位置定位，其中一种 Dis 突变体被发现是由于自闭症相关基因 Neurobeachin (nbea) 的丢失所致。该提案将研究 Nbea 在电突触形成中的作用 (Aim1)，将克隆在试点筛选中发现的其他 Dis 和 Amp 突变 (Aim2)，将检查突变对突触功能和行为的影响 (Aim3)，并将扩大试点筛选以进一步阐明突触发生所需的基因和途径 (Aim4)。在两年的指导阶段，我将通过表征基因如何以多种方式调节电突触形成来开发模型系统：体内突触发生期间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​​何影响神经网络功能和行为？在 Fred Hutchinson 癌症研究中心的 Cecilia Moens 实验室（主要导师），我将学习使用旋转对荧光标记的突触蛋白进行活细胞成像 圆盘共焦显微镜。该技术将应用于所有突变体，并将是体内电突触形成的首次现场研究。为了研究 M 突触和回路功能，我将参观 Joe Fetcho 在康奈尔大学的实验室，学习在 M 神经回路中进行电生理学，我将参观 Michael Granato 在宾夕法尼亚大学佩雷​​尔曼医学院的实验室，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，在那里我将对突变体进行实验。对于电生理学，我将与华盛顿大学的 Rachel Wong（主要联合导师）合作，在那里我将接受持续的电生理学培训，并可以使用实验设备。对于行为，我将在莫恩斯实验室工作，那里有捕捉 M 介导的逃避反应所需的高速摄像机。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电路中的功能缺陷联系起来至关重要。 Fetcho 和 Granato las 的培训将是短暂而密集的，但这两位导师将持续为我提供技术专业知识和指导。莫恩斯和王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，我将拥有必要的经验和一套强大的工具和技术来建立我自己的独立研究小组。在该项目的独立阶段，我将利用所获得的技能来阐明在电突触处建立间隙连接的分子机制。拟议的研究将为脊椎动物体内神经回路如何形成提供详细的分子、细胞和功能视图。导致神经回路错误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）的基础。就自闭症而言，多种分子途径（包括 Aim1 中检查的 Nbea）与该疾病有关。然而，解释这些基因如何组合在一起来解释该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，最终确定治疗靶点。