Imaging Synaptic Transmission of Individual Active Zones

单个活动区的突触传递成像

• 批准号: 8852712
• 负责人: J. TROY LITTLETON
• 金额: $ 39万
• 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 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8852712
• 关键词: Action Potentials; Binding Proteins; Biological Models; Biological Process; Brain; Brain Diseases; Calcium; Cells; Communication; Complex; Cyclic AMP-Dependent Protein Kinases; Data; Docking; Drosophila genus; Event; Exocytosis; Foundations; Glutamate Receptor; Glutamates; Health; Human; Image; Individual; Link; Maps; Measures; Mediating; Mental disorders; Modeling; Nerve; Neuromuscular Junction; Neurons; Neurotransmitters; Pathway interactions; Phosphorylation; Population; Probability; Property; Proteins; Receptor Activation; Regulation; Research; SNAP receptor; Signal Transduction; Site; Synapses; Synaptic Transmission; Synaptic Vesicles; Synaptic plasticity; Testing; Therapeutic; Transgenic Organisms; Vesicle; Workplace; density; insight; nervous system disorder; neuromuscular; neurotransmitter release; postsynaptic; presynaptic; receptor; response; sensor; synaptic function; synaptogenesis; tool; vesicular release

DESCRIPTION (provided by applicant): We propose to use Drosophila as a model system for determining how neurotransmitter release at individual active zones is regulated. We will image synaptic vesicle fusion at single active zones to define how release probability, release mode (spontaneous versus evoked) and release plasticity is regulated at glutamatergic synapses. Neurotransmitters can be released through evoked fusion following an action potential, or by spontaneous fusion of vesicles (termed "minis") in the absence of nerve stimulation. The two modes of vesicle release have been found at most synapses and are assumed to occur across the same population of active zones, though this has been difficult to define using classical approaches. A major technical limitation for the study of neurotransmitter release has been the inability to examine vesicle fusion at individual active zones. Electrophysiological studies of synaptic transmission measure the postsynaptic effect of neurotransmitter release over a large population of release sites, precluding an analysis of how individual active zones participate in and regulate synaptic vesicle fusion. We have developed transgenic tools that allow Ca2+ imaging of postsynaptic glutamate receptor activation following single vesicle fusion to spatially visualize all exocytotic events occurring through both spontaneous and evoked release pathways at Drosophila NMJs, allowing us to define general rules for vesicle fusion events at single active zones. Using these tools, we have begun to characterize the spatial and temporal dynamics of exocytotic events that occur spontaneously or in response to an action potential. We have also begun analyzing the relationship between these two modes of fusion at single release sites. Current data indicate a majority of active zones participate in both modes of fusion, although release probability is not correlated between the two modes of release and is highly variable across the population. Indeed, a subset of active zones is specifically dedicated to spontaneous release, indicating a population of postsynaptic receptors is uniquely activated by this mode of vesicle fusion. Using these new transgenic tools to visualize single active zone exocytosis, we will characterize how single release sites work, how they undergo plasticity, and how they contribute to both evoked and spontaneous fusion.

描述（由申请人提供）：我们建议使用果蝇作为模型系统，以确定如何调节神经递质在单个活动区域的释放。我们将在单个活性区域对突触囊泡融合进行图像，以定义释放概率，释放模式（自发性与诱发）如何在谷氨酸能突触中调节释放可塑性。在没有神经刺激的情况下，可以通过诱发的融合或自发融合囊泡（称为“ Minis”）来释放神经递质。在大多数突触中发现了两种囊泡释放模式，并被认为是在同一活性区域中发生的，尽管这很难使用经典方法来定义。神经递质释放研究的主要技术局限性是无法检查单个活动区域的囊泡融合。突触传播的电生理研究测量神经递质在大量释放位点释放的突触后作用，从而排除了单个活性区域如何参与和调节突触囊泡融合的分析。我们开发了转基因工具，可以使单囊融合后的突触后谷氨酸受体激活进行Ca2+成像，从而在空间上可视化所有通过果蝇NMJ上的自发和诱发释放途径发生的所有胞胞菌事件，从而使我们能够在单个活性Zones处定义囊泡融合事件的常规规则。使用这些工具，我们已经开始表征自发或响应动作电位的胞吐事件的空间和时间动力学。我们还开始分析单个释放位点的这两种融合模式之间的关系。当前的数据表明，大多数活动区域都参与了两种融合模式，尽管释放概率在两种释放模式之间没有相关性，并且在整个种群中都高度变化。实际上，活动区域的一部分专门用于自发释放，表明突触后受体的种群被这种囊泡融合方式唯一激活。使用这些新的转基因工具来可视化单个活动区域胞吐作用，我们将表征单个释放位点的工作方式，它们如何实现可塑性以及它们如何促进诱发和自发融合。