Imaging Synaptic Transmission of Individual Active Zones

单个活动区的突触传递成像

• 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
• 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

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如何释放 在单个活动区域（AZS）调节可塑性。突触囊泡融合发生 通过高度概率的过程，通常只有一小部分动作电位 触发单个AZS的释放。尽管AZS很大程度上共享相同的补充 蛋白质，释放概率（PR）在不同的神经元中以及AZS之间的变化高度可变。 同一个神经元。实际上，某些AZ特异性蛋白是不均匀分布的，并且 AZS的分子组成可以快速变化。迄今为止，CA2+通道丰度 CA2+涌入与PR异质性最密切相关，尽管其他因素是 也可能做出贡献。果蝇神经肌肉连接（NMJ）已成为 强大的模型系统以表征PR的决定因素。通过传递的gcamp Ca2+传感器针对突触后膜，单个突触囊泡融合事件 可以通过遵循谷氨酸诱导的空间定位的Ca2+涌入来成像单个AZS 受体开口。这使我们能够生成诱发和自发融合的公关图 所有的AZS，导致令人惊讶的观察结果，即由单个电动机神经元形成的AZ具有 PR的异质分布，相邻的AZS通常显示出约50倍的差异 力量。此外，10％的亚利桑那州人口仅支持自发释放，而 对于诱发和自发融合，另有15％的功能在功能上保持沉默。在这个建议中，我们 将确定如何为单个AZS唯一设置PR，以及什么分子，结构和 发展变量控制PR异质性。我们还将研究突触前Ca2+如何 通道通道与AZS之间的交通以及可塑性如何改变这些过程。这些 方法应为AZ蛋白的补充提供新的见解 调节PR，自发释放和沉默的突触，以及它们如何与 突触前Ca2+通道将PR设置为功能多样的AZS。中断 突触的形成和功能已与许多神经和精神科有关 疾病，反映了这些过程的重要性。在此描述的实验 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的模式以前所未有的分辨率。