Calcium control of Neurotransmitter Release

项目摘要 神经元之间通过大量释放储存在突触小泡中的神经递质进行交流 (SVS)，神经递质释放的强度和效果在生理过程中动态变化。 活动。这一过程对学习和记忆是必不可少的，在许多神经疾病中都会受到干扰。这个 神经递质的释放来自停靠在突触前活动区的SVS池，并受到严格控制 通过活动依赖的突触前钙离子浓度([Ca~(2+)])的变化。在神经末梢，钙离子- 传感蛋白(Synaptopagmins)将囊泡释放机制(SNARs)与钙信号偶联，从而 协调神经细胞的交流。尽管进行了多年的研究，但仍有很大差距 定性描述了囊泡融合机的工作原理和毫秒级精度和钙离子。 在神经元突触观察到神经递质释放的依赖动力学。 在这项提案中，我们描述了第一次系统和全面的努力，以协调分子 囊泡融合的生物化学与钙诱导的神经元神经递质释放的“生理学” 使用体外和体内实验系统相结合的互补突触。具体来说，我们的目标是 为了确定synaptopagmin亚型是否具有不同的生化特性(Syt1和Syt7)，以及 突触圈套代表了不同神经递质释放模式的最小蛋白质机制 短期可塑性。我们还建议对Syt1和Syt1的“双重结合”假设进行定量检验 Syt7 to SNARES允许囊泡释放动力学的协同调节。对于体外分析，我们将部署 生物化学定义的高通量融合系统，能够跟踪单个囊泡对接和钙离子 毫秒级触发融合，与快速钙离子传递协议集成以生成 [CA2]模拟突触前钙动力学的瞬变。这将得到生理分析的补充 用快速荧光谷氨酸感受器(IGluSnFR)和钙离子染料对培养的神经元进行定量成像 2-4毫秒单个突触前突触的谷氨酸释放和突触前钙动力学 决议。我们期待这个项目将为分子机制提供重要的见解。 潜在的神经递质释放和构建钙离子诱发突触的详细机制模型 囊泡融合。总体而言，这将大大增强我们对神经传递及其调节方式的理解 跨不同的神经细胞，允许在神经元网络中进行特定而多样的通信。

Project Summary Neurons communicate with each other by the quantal release of neurotransmitters stored in synaptic vesicles (SVs), and the strength and efficacy of neurotransmitter release are dynamically altered during physiological activity. This process is essential for learning and memory and is disrupted in many neurological disorders. The neurotransmitter release occurs from a pool of SVs docked at the presynaptic active zone and is tightly controlled by activity-dependent changes in the presynaptic calcium ions concentration ([Ca2+]). At nerve terminals, Ca2+- sensing proteins (Synaptotagmins) couple vesicular release machinery (SNAREs) to Ca2+ signals, thus orchestrating neuronal communication. Despite years of research, there remains a substantial gap between a qualitative description of how the vesicle fusion machinery operates and the millisecond-precision and Ca2+- dependent kinetics of neurotransmitter release observed at the neuronal synapses. In this proposal, we describe the first systematic and comprehensive effort to reconcile the `molecular biochemistry' of vesicle fusion with the `physiology' of Ca2+-evoked neurotransmitter release in the neuronal synapses using a combination of complementary in vitro and in vivo experimental systems. Specifically, we aim to resolve whether Synaptotagmin isoforms with distinct biochemical properties (Syt1 and Syt7), along with the synaptic SNAREs represent the minimal protein machinery for different modes of neurotransmitter release and short-term plasticity. We also propose to quantitatively test the hypothesis that the `dual-binding' of Syt1 and Syt7 to SNAREs allows for synergistic regulation of vesicular release kinetics. For in vitro analysis, we will deploy a biochemically-defined, high-throughput fusion system capable of tracking individual vesicle docking and Ca2+ triggered fusion on a millisecond timescale, which is integrated with fast Ca2+-uncaging protocols to generate [Ca2+] transients mimicking presynaptic calcium dynamics. This will be complemented by physiological analysis in cultured neurons utilizing fast, fluorescent glutamate sensor (iGluSnFR) and Ca2+ dyes to image quantal glutamate release and presynaptic Ca2+ dynamics in individual presynaptic boutons with 2-4 millisecond temporal resolution. We anticipate that this project will provide important insights into the molecular mechanisms underlying neurotransmitter release and build toward a detailed mechanistic model of Ca2+-evoked synaptic vesicle fusion. Overall, this will greatly enhance our understanding of neurotransmission and of how it is tuned across different nerve cells allowing specific yet diverse communication in neuronal networks.