Spatial and molecular determinants of fusion probability and timing

在化学突触中，神经元之间通过释放神经递质进行交流。 突触传递发生在两个动力学上截然不同的阶段。在动作电位的毫秒内， 突触小泡跨多个突触与质膜同步融合。跟随 同步融合，突触小泡继续与质膜融合超过数十到数百个 毫秒。神经传递的这一阶段被称为异步释放，它变得更加明显 在病理生理条件下。尽管在过去的50年里进行了广泛的研究，但纳米尺度 突触小泡融合部位的组织结构还知之甚少。同步和异步在哪里？ 核聚变发生在活动区内？融合部位是由钙通道的位置决定的吗？ 哪些钙传感器负责异步融合？是否有单独的囊泡池用于 异步核聚变？或者两个阶段消耗的囊泡是相同的吗？要解决这些问题 问题，我们已经发展了电击和冷冻电子显微镜来跟踪膜动力学毫秒- 以毫秒计。这项技术将电刺激神经元与高压冷冻结合起来 哺乳动物中央突触的快速膜转运事件具有前所未有的空间(1 Nm)和 时间(1毫秒)分辨率。将这种方法与先进遗传学、分子生物学、 和生化技术，我们将确定融合部位是如何在哺乳动物的中央突触组织的。 突触传递的缺陷在神经疾病中起着因果作用。这项拟议的研究旨在 了解突触传递的分子机制，最终目标是 了解这些疾病的发病机制。

At chemical synapses, neurons communicate each other through release of neurotransmitters. Synaptic transmission occurs in two kinetically distinct phases. Within milliseconds of an action potential, synaptic vesicles fuse with the plasma membrane synchronously across multiple synapses. Following synchronous fusion, synaptic vesicles continue to fuse with the plasma membrane over tens to hundreds of milliseconds. This phase of neurotransmission is called asynchronous release, and it becomes more apparent in pathophysiological conditions. Despite extensive research over the last 50 years, the nano-scale organization of synaptic vesicle fusion sites is poorly understood. Where do synchronous and asynchronous fusions take place within an active zone? Are fusion sites determined by the locations of calcium channels? Which calcium sensors are responsible for asynchronous fusion? Is there a separate pool of vesicles for asynchronous fusion? Or are the same pool of vesicles consumed for both phases? To address these questions, we have developed zap-and-freeze electron microscopy to follow membrane dynamics millisecond- by-millisecond. This technique couples electrical stimulation of neurons with high-pressure freezing to capture rapid membrane trafficking events at mammalian central synapses with unprecedented spatial (1 nm) and temporal (1 ms) resolution. Using this approach in combinations with advanced genetics, molecular biology, and biochemical techniques, we will determine how fusion sites are organized at mammalian central synapses. Defects in synaptic transmission play a causal role in neurological disorders. The proposed research aims to understand the molecular mechanisms underlying synaptic transmission with the ultimate goal of understanding the pathogenesis of these diseases.