Molecular mechanisms of electrical synapse formation in vivo

描述(由Candiate提供)：大脑的所有功能，从感觉到行为，都来自于(在人类中)数十亿个神经元之间的突触连接的模式和属性。该项目的长期目标是使用脊椎动物模型了解在体内调节突触形成的分子路径，重点是被低估的电突触。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。它们仅在潜在可用伙伴的子集之间以受调控的方式形成，并由神经元缝隙连接通道组成。电突触对发育过程中的神经回路以及从感觉到加工到运动输出的成人回路都有广泛的贡献。然而，形成电突触的缝隙连接通道形成的分子机制尚不清楚。这一建议利用斑马鱼Mauthner(M)回路来研究电突触形成的遗传学。M神经元是可以单独识别的，它们在突触前和突触后的伙伴、突触和功能在活的脊椎动物胚胎中被精美地可视化。对导致刻板印象中的M电突触缺陷的突变进行了正向遗传筛查，发现了两类不同的突变：1)断开(Dis)类，破坏突触形成；2)AMPed(Amp)类，导致异位突触沿M轴突形成。使用基于RNA序列的方法，所有三个Dis突变都被定位，其中一个Dis突变被发现是由于自闭症相关基因Neurobeachin(Nbea)的丢失所致。这项提案将研究NbeA在电突触形成中的作用(Aim1)，将克隆在试点屏幕(AIM2)中发现的其他Dis和Amp突变，将检查这些突变对突触功能和行为的影响(Aim3)，并将扩大试点筛选以阐明进一步的突触发生所需的基因和途径(Aim4)。在为期两年的指导阶段，我将开发模型系统，通过几种方式描述这些基因如何调控电突触的形成：在体内突触形成过程中，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·莫恩斯的实验室(主要导师)，我将学习使用Spin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突变体，并将是第一次活体研究电突触的形成。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神经回路中执行电生理学，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，了解M介导的逃逸行为的行为分析。获得的技能将被带回西雅图，在那里我将在变种人身上进行实验。在电生理学方面，我将在华盛顿大学与Rachel Wong合作(主要合作导师)，在那里我将接受持续的电生理学培训，并可以获得实验设备。至于行为，我将在莫恩斯实验室工作，在那里我们有必要的高速摄像机来捕捉M中介的逃逸反应。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。Fetcho和Granato LAS的培训将是短暂而密集的，但这两位导师将持续向我提供技术专业知识和指导。Moens和Wong实验室的指导工作将持续进行，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，我将利用所获得的技能来阐明在电突触建立缝隙连接的分子机制。拟议的研究将提供关于脊椎动物体内神经回路如何形成的详细的分子、细胞和功能视图。导致神经回路错误连接或突触失衡的疾病是包括自闭症和癫痫在内的许多神经系统疾病的基础。在自闭症的案例中，几个分子途径(包括在Aim1中检测到的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症的统一理论仍然难以捉摸。研究神经回路连接和突触形成所需的遗传路径将有助于洞察疾病状态，最终将允许识别治疗的靶点。

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.