SPATIAL AND TEMPORAL REGULATION OF NEUROTRANSMITTER RELEASE

摘要 神经交流是由突触中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，包括 同步单囊和多囊释放，异步和自发释放。这其中的每一个 释放机制在突触的发育和功能中起着重要而独特的作用。然而，尽管 几十年的研究，这些规范形式或释放是如何在中央 人们对突触知之甚少。这包括版本的一些最基本的功能，包括 支持不同形式的发布的发布站点的数量、空间组织和重用，所有这些 由于中环面积极小，且相对难以到达，因此在很大程度上仍未确定 与传统记录技术的突触。此外，数据的时空属性和重用如何 释放部位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 一种与pH敏感的荧光指示剂相结合的纳米分辨率成像方法 基因标记到囊泡管腔，使我们能够解决AZ的单个囊泡融合事件与~27 NM精度。通过这种方法，我们发现在中央区域存在多个不同的发布站点 突触，并证明它们的时空特性受神经活动的调节。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 其他规范的释放形式。我们的方法还允许我们可视化和跟踪个体的移位 突触小泡连接到AZ，这是神经活动期间释放部位重新充盈的关键限时步骤。 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、 遗传学和药理学工具，用于在单个囊泡水平上研究支配组织的机制 以及调节单个中央突触的神经递质释放的规范形式。我们将进一步 确定控制囊泡转运到释放部位的机制及其活性依赖 监管。这些研究将为突触功能的基本机制提供重要的新见解。

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.