Imaging Synaptic Transmission of Individual Active Zones

单个活动区的突触传递成像

• 批准号: 9229066
• 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/9229066
• 关键词: Action Potentials; Binding Proteins; Biological Models; Biological Process; Brain Diseases; Calcium; Cells; Closure by clamp; Communication; Complex; Cyclic AMP-Dependent Protein Kinases; Data; Docking; Drosophila genus; Electrophysiology (science); Event; Exocytosis; Foundations; Glutamate Receptor; Glutamates; 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; human disease; insight; nervous system disorder; neuromuscular; neurotransmitter release; postsynaptic; presynaptic; public health relevance; 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来确定如何调节各个活性区的神经递质释放。我们将对单个活性区的突触小泡融合进行成像，以定义释放概率、释放模式（自发与诱发）和释放可塑性在谷氨酸能突触中的调节方式。神经递质可以通过动作电位后的诱发融合来释放，或者在没有神经刺激的情况下通过囊泡（称为“微型”）的自发融合来释放。在大多数突触中都发现了两种囊泡释放模式，并且假定发生在相同的活跃区域群中，尽管这很难使用经典方法来定义。神经递质释放研究的一个主要技术限制是无法检查单个活性区域的囊泡融合。突触传递的电生理学研究测量神经递质释放在大量释放位点上的突触后效应，排除了对各个活性区如何参与和调节突触小泡融合的分析。我们开发了转基因工具，可以对单囊泡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果蝇 NMJ 中通过自发和诱发释放途径发生的所有胞吐事件，使我们能够定义单个活性区囊泡融合事件的一般规则。使用这些工具，我们已经开始表征自发发生或响应动作电位的胞吐事件的空间和时间动态。我们还开始分析这两种融合模式在单一释放点之间的关系。目前的数据表明，大多数活跃区都参与这两种融合模式，尽管两种释放模式之间的释放概率不相关，并且在整个群体中差异很大。事实上，活性区的一个子集专门用于自发释放，表明突触后受体群体被这种囊泡融合模式独特地激活。使用这些新的转基因工具来可视化单个活性区胞吐作用，我们将描述单个释放位点如何工作，它们如何经历可塑性，以及它们如何促进诱发和自发融合。