Imaging Synaptic Transmission of Individual Active Zones

单个活动区的突触传递成像

基本信息

批准号：
10318177
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
项目类别：
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/10318177
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 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

项目摘要

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来确定神经递质释放方式和可塑性在各个活性区域（AZ）受到调节。发生突触小泡融合通过高度概率的过程，通常只有一小部分的动作潜力触发各个可用区的释放。尽管 AZ 大部分具有相同的互补性蛋白质的释放概率 (Pr) 在不同神经元之间以及 AZ 之间变化很大同一个神经元。事实上，一些 AZ 特异性蛋白质分布不均匀，并且 AZ 的分子组成可以发生快速变化。迄今为止，Ca2+通道丰度尽管还有其他因素，但 Ca2+ 流入与 Pr 异质性的联系最为密切。也有可能做出贡献。果蝇神经肌肉接头（NMJ）已成为一种稳健的模型系统来表征 Pr 的决定因素。通过转基因表达 GCaMP Ca2+ 传感器针对突触后膜，单突触小泡融合事件单个 AZ 可以通过谷氨酸诱导的空间局部 Ca2+ 流入进行成像受体开放。这使我们能够生成诱发和自发融合的 Pr 图所有 AZ，导致令人惊讶的观察结果，即由单个运动神经元形成的 AZ 具有 Pr 的异质分布，相邻的 AZ 通常表现出约 50 倍的差异力量。此外，10%的AZ群体仅支持自发释放，而另外 15% 对于诱发融合和自发融合在功能上保持沉默。在这个提案中，我们将确定如何为各个 AZ 独特地设置 Pr，以及什么分子、结构和发育变量控制 Pr 异质性。我们还将研究突触前 Ca2+ 引导流向可用区和可用区之间的流量，以及可塑性如何改变这些过程。这些方法应该为 AZ 蛋白的补充提供新的见解，这些蛋白在功能上调节 Pr、自发释放和沉默突触，以及它们如何与突触前 Ca2+ 通道在一组功能多样的 AZ 中设置 Pr。的干扰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。本文描述的实验提案将对定义强度和释放的重要元素产生新的见解单个 AZ 模式以前所未有的分辨率。