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.

抽象的 神经通信受突触处的神经递质囊泡的释放来控制 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 同步的单一和多动物释放，异步和自发释放。每个 释放机制在突触发育和功能中起着重要而独特的作用。但是，尽管如此 数十年的研究，这些规范形式或释放如何在中央组织和调节 突触知之甚少。这包括发行的一些最基本功能，包括 数字，空间组织和支持不同形式发行形式的发行站点的重复使用，所有这些都 由于中央的尺寸极小和相对无法访问，因此在很大程度上保持不确定 突触到传统记录技术。此外，时空特性和重复使用 释放位点受神经活动的调节，在很大程度上未知。为了克服这些限制，我们发展了 纳米级分辨率成像方法与pH敏感的荧光指示器结合 遗传标记在囊泡管腔上，使我们能够在AZ处解决〜27 NM精度。通过这种方法，我们发现了中央的多个不同释放位点的存在 突触并证明其时空特性受神经活动调节。经过 通过计算单分子工具来补充这种方法，我们也能够稳健地检测所有 其他规范的释放形式。我们的方法还允许我们可视化和跟踪个人的易位 突触囊泡到AZ，这是在神经活动期间释放位点补充释放位点的关键时间限制步骤。 在这里，我们建议将这种纳米级分辨率成像方法与先进的计算相结合， 遗传和药理学工具，用于在单维水平上研究机制 以及在单个中央突触中神经递质释放的规范形式的调节。我们将进一步 定义管理囊泡转运到释放位点及其活动依赖性的机制 规定。这些研究将为突触功能的基本机制提供主要的新见解。