SPATIAL AND TEMPORAL REGULATION OF NEUROTRANSMITTER RELEASE

神经递质释放的时空调节

• 批准号: 9696092
• 负责人: Vitaly A Klyachko
• 金额: $ 38.94万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9696092
• 关键词: Actins; Acute; Automobile Driving; Brain; Calcium; Communication; Complement; Dependence; Detection; Development; Disease; Event; Exhibits; Genetic; Goals; Hippocampus (Brain); Imaging technology; Individual; Location; Measurement; Modification; Mono-S; Motion; Myosin ATPase; Myosin Type V; Neurobiology; Neurosciences; Pharmacology; Play; Population; Probability; Process; Property; Protein Isoforms; Recycling; Regulation; Research; Resolution; Role; Site; Synapses; Synaptic Vesicles; Techniques; Time; Vesicle; base; computerized tools; imaging approach; information processing; innovation; insight; nanoscale; neuroregulation; neurotransmitter release; relating to nervous system; single molecule; spatiotemporal; synaptic function; tool; transmission process; vesicular release

ABSTRACT Neural communication is governed by the release of neurotransmitter-containing vesicles at the synaptic active zone (AZ). Several fundamental forms of neurotransmitter release are present at synapses, including synchronous mono- and multi-vesicular release, asynchronous and spontaneous release. Each of these release mechanisms plays important and distinct roles in synaptic development and function. However, despite several decades of research, how these canonical forms or release are organized and regulated in central synapses is poorly understood. This includes some of the most fundamental features of release, including the number, spatial organization and reuse of release sites supporting different forms of release, all of which remain largely undetermined because of the extremely small size and relative inaccessibility of central synapses to conventional recording techniques. Moreover, how the spatiotemporal properties and reuse of the release sites are regulated by neural activity is largely unknown. To overcome these limitations, we developed a nanoscale-resolution imaging approach that in combination with a pH-sensitive fluorescent indicator genetically tagged to the vesicle lumen, allows us to resolve individual vesicle fusion events at the AZ with ~27 nm precision. With this approach we have uncovered the presence of multiple distinct release sites in central synapses and demonstrated that their spatiotemporal properties are regulated by neural activity. By complementing this approach with computational single-molecule tools we are also able to robustly detect all other canonical forms of release. Our approach also permits us to visualize and track translocation of individual synaptic vesicles to the AZ, a critical time-limiting step in the refilling of the release sites during neural activity. Here we propose to combine this nanoscale-resolution imaging approach with advanced computational, genetic and pharmacological tools to study, at a single-vesicle level, the mechanisms governing organization and regulation of the canonical forms of neurotransmitter release at individual central synapses. We will further define the mechanisms governing vesicle translocation to the release sites and their activity-dependent regulation. These studies will provide major new insights into fundamental mechanisms of synaptic function.

摘要 神经通讯是由突触处含有神经递质的囊泡的释放控制的。 活动区（AZ）。神经递质释放的几种基本形式存在于突触中，包括 同步单泡和多泡释放、异步和自发释放。这一切成功都 释放机制在突触发育和功能中起着重要和独特的作用。但尽管 几十年的研究，这些典型的形式或释放是如何组织和调节中央 对突触了解甚少。这包括发布的一些最基本的特性，包括 支持不同形式释放的释放地点的数量、空间组织和再利用， 由于中区面积极小，交通不便， 突触到传统的记录技术。此外，如何时空属性和重用的 释放位点受神经活动的调节在很大程度上是未知的。为了克服这些限制，我们开发了 一种纳米级分辨率的成像方法，与pH敏感的荧光指示剂相结合， 基因标记的囊泡腔，使我们能够解决个别囊泡融合事件在AZ与~27 nm精度。通过这种方法，我们发现中环存在多个不同的释放地点 突触，并证明其时空特性是由神经活动调节。通过 用计算单分子工具补充这种方法，我们也能够稳健地检测所有 其他典型的释放形式。我们的方法还允许我们可视化和跟踪个体的易位， 突触囊泡的AZ，一个关键的时间限制的步骤，在神经活动期间的释放网站的再填充。 在这里，我们建议联合收割机这种纳米级分辨率成像方法与先进的计算， 遗传学和药理学工具，在单囊泡水平上研究组织管理机制 和调节在单个中枢突触处的神经递质释放的典型形式。我们将进一步 定义了囊泡移位到释放位点的机制及其活性依赖性 调控这些研究将为突触功能的基本机制提供重要的新见解。