Imaging Synaptic Transmission of Individual Active Zones

单个活动区的突触传递成像

• 批准号: 9883839
• 负责人: J. TROY LITTLETON
• 金额: $ 38.5万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9883839
• 关键词: Action Potentials; Acute; Address; Biological Models; Biology; Biosensor; Birth; Brain Diseases; Calcium Channel; Chronic; Communication; Complement; Data; Development; Docking; Drosophila genus; Elements; Event; Foundations; Generations; Glutamate Receptor; Glutamates; Heterogeneity; Image; Individual; Investigation; Link; Maps; Mental disorders; Methods; Molecular; Molecular Profiling; Molecular Structure; Motor Neurons; Neuromuscular Junction; Neurons; Pathway interactions; Population; Postsynaptic Membrane; Probability; Process; Property; Proteins; Recording of previous events; Research; Resolution; Set protein; Signal Transduction; Site; Structural Protein; Synapses; Synaptic Transmission; Synaptic Vesicles; Synaptic plasticity; Therapeutic; Time; Transgenic Organisms; Variant; base; density; experimental study; human disease; imaging approach; insight; intravital imaging; light microscopy; nervous system disorder; neural circuit; neurotransmitter release; presynaptic; presynaptic neurons; protein distribution; quantum; reconstruction; sensor; serial imaging; synaptic function; synaptogenesis; tool; trafficking; vesicular release; voltage

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.

我们建议使用果蝇作为模型系统，以确定神经递质释放 和可塑性在各个活动区（AZ）被调节。突触囊泡融合发生 通过一个高度概率性的过程，通常只有一小部分动作电位 触发从各个AZ释放。尽管AZ在很大程度上共享相同的互补性， 蛋白质，释放概率（Pr）在不同的神经元之间和不同的AZ之间是高度可变的。 同样的神经元。事实上，一些AZ特异性蛋白质是不均匀分布的， AZ的分子组成可经历快速变化。到目前为止，Ca2+通道丰度 和Ca2+流入与Pr异质性的联系最为密切，尽管其他因素 也可能做出贡献。果蝇的神经肌肉接头（NMJ）已经成为一种 通过转基因表达GCaMP来表征Pr的决定因素的鲁棒模型系统 Ca2+传感器靶向突触后膜，单个突触囊泡融合事件， 可以通过跟踪谷氨酸诱导的空间定位的Ca2+内流来对单个AZ进行成像 受体开放这使我们能够生成诱发和自发融合的Pr图， 所有的AZs，导致令人惊讶的观察，即由单个运动神经元形成的AZs具有 Pr的不均匀分布，相邻的AZ通常显示出约50倍的差异， 实力此外，10%的AZ群体仅支持自发释放，而 另外15%在诱发和自发融合方面功能沉默。在本提案中，我们 将确定Pr是如何为单个AZ唯一设置的，以及分子、结构和 发育变量控制Pr异质性。我们还将研究突触前Ca 2 + 通道和AZ之间的流量，以及可塑性如何改变这些过程。这些 这些方法应该为AZ蛋白的补充提供新的见解， 调节Pr、自发释放和沉默突触，以及它们如何与 突触前Ca2+通道，以设置跨功能多样的一组AZ的Pr。中断 突触的形成和功能与许多神经和精神疾病有关， 疾病，反映了这些过程的重要性。本文中描述的实验 提案将对定义优势和释放的重要元素产生新的见解 以前所未有的分辨率显示单个AZ模式。