Imaging Synaptic Transmission of Individual Active Zones

我们建议使用果蝇作为一个模型系统来确定神经递质如何释放 和可塑性在单个活动区(AZs)受到调节。突触小泡融合发生 通过一个高概率的过程，通常只有一小部分动作电位 触发从单独的AZE释放。尽管AZ在很大程度上分享了相同的补充 蛋白质，释放概率(Pr)在不同神经元和不同AZ之间是高度不同的 同样的神经元。事实上，一些AZ特有的蛋白质分布并不均匀，而且 AZs的分子组成可能会发生快速变化。到目前为止，钙离子通道丰富 而钙离子的内流与PR的异质性联系最强，尽管其他因素 可能也会做出贡献。果蝇神经肌肉接头(NMJ)是一种 描述公关决定因素的稳健模型系统。通过转基因表达GCaMP 定位于突触后膜的钙离子感受器，单个突触小泡融合事件 单个AZ可以通过跟随谷氨酸诱导的空间局部钙内流来成像 受体打开。这使我们能够生成PR图，用于诱发和自发融合 所有的AZ，导致了令人惊讶的观察到由单个运动神经元形成的AZ具有 Pr的异质性分布，邻近的AZ通常显示出约50倍的差异 力量。此外，10%的AZ人口只支持自发释放，而 另有15%的人在诱发和自发融合时功能上保持沉默。在这项提案中，我们 将决定如何为各个AZ唯一地设置PR，以及什么分子、结构和 发育变量支配着公共关系的异质性。我们还将研究突触前钙离子是如何 AZ之间的交通通道，以及可塑性如何改变这些过程。这些 这些方法应该为AZ蛋白的功能互补提供新的见解 调节PR、自发释放和无声突触，以及它们是如何配合的 突触前钙通道在一组不同功能的AZ上设置PR。中断 突触的形成和功能与许多神经学和精神病学 疾病，反映了这些进程的重要性。本文件中描述的实验 建议书将对定义实力和释放的重要元素产生新的见解 以前所未有的分辨率建立单个自贸区的模式。

We propose to use Drosophila as a model system for determining how neurotransmitter release and plasticity are regulated at individual active zones (AZs). Synaptic vesicle fusion occurs through a highly probabilistic process, often with only a small percent of action potentials triggering release from individual AZs. Although AZs largely share the same complement of proteins, release probability (Pr) is highly variable across different neurons and between AZs of the same neuron. Indeed, some AZ-specific proteins are non-uniformly distributed, and the molecular composition of AZs can undergo rapid changes. To date, Ca2+ channel abundance and Ca2+ influx have been most strongly linked to Pr heterogeneity, though other factors are likely to contribute as well. The Drosophila neuromuscular junction (NMJ) has emerged as a robust model system to characterize determinants of Pr. By transgenically expressing GCaMP Ca2+ sensors targeted to the postsynaptic membrane, single synaptic vesicle fusion events at individual AZs can be imaged by following spatially localized Ca2+ influx induced upon glutamate receptor opening. This enabled us to generate Pr maps for evoked and spontaneous fusion for all AZs, leading to the surprising observation that AZs formed by a single motor neuron have a heterogeneous distribution of Pr, with neighboring AZs often showing ~50-fold differences in strength. In addition, 10% of the AZ population supports only spontaneous release, while another 15% are functionally silent for both evoked and spontaneous fusion. In this proposal, we will determine how Pr is uniquely set for individual AZs and what molecular, structural, and developmental variables govern Pr heterogeneity. We will also examine how presynaptic Ca2+ channels traffic to and between AZs, and how plasticity alters these processes. These approaches should provide new insights into the complement of AZ proteins that functionally regulate Pr, spontaneous release, and silent synapses, and how they cooperate with presynaptic Ca2+ channels to set Pr across a functionally diverse set of AZs. Disruptions of synapse formation and function have been linked to a host of neurological and psychiatric diseases, reflecting the importance of these processes. The experiments described in this proposal will generate new insights into important elements that define the strength and release mode of individual AZs at an unprecedented resolution.