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 神经通讯由突触处含有神经递质的囊泡的释放控制 活动区域 (AZ)。突触处存在神经递质释放的几种基本形式，包括 同步单囊泡和多囊泡释放、异步和自发释放。这些中的每一个 释放机制在突触发育和功能中起着重要而独特的作用。然而，尽管 经过几十年的研究，这些规范形式或发布是如何在中央组织和监管的 人们对突触知之甚少。这包括发行版的一些最基本的功能，包括 支持不同形式发布的发布站点的数量、空间组织和再利用，所有这些 由于面积极小且中心相对难以到达，这些问题在很大程度上仍未确定。 突触到传统的记录技术。此外，如何利用时空属性和重用 释放位点是否受神经活动调节尚不清楚。为了克服这些限制，我们开发了 纳米级分辨率成像方法与 pH 敏感荧光指示剂相结合 对囊泡腔进行基因标记，使我们能够用 ~27 解决 AZ 处的个体囊泡融合事件 纳米精度。通过这种方法，我们发现了中部地区存在多个不同的释放站点。 突触并证明它们的时空特性受到神经活动的调节。经过 通过计算单分子工具补充这种方法，我们还能够稳健地检测所有 其他规范的发布形式。我们的方法还允许我们可视化和跟踪个体的易位 突触小泡到达 AZ，这是神经活动过程中释放位点再填充的关键限时步骤。 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， 在单囊泡水平上研究组织调控机制的遗传和药理学工具 以及单个中枢突触神经递质释放的规范形式的调节。我们将进一步 定义控制囊泡易位到释放位点的机制及其活性依赖性 规定。这些研究将为突触功能的基本机制提供重要的新见解。