Spatial and molecular determinants of fusion probability and timing

在化学突触中，神经元通过释放神经递质相互交流。 突触传递发生在两个动力学上不同的阶段。在动作电位的几毫秒内， 突触囊泡在多个突触中与质膜同步融合。以下 同步融合，突触囊泡继续与质膜融合数十至数百年， 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， 对突触囊泡融合位点的组织了解甚少。同步和异步 核聚变会发生在活跃区吗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？是否有一个单独的囊泡池， 异步聚变？还是两个阶段消耗的囊泡相同？解决这些 问题，我们已经开发了zap和冻结电子显微镜跟踪膜动力学毫秒- 以毫秒为单位这项技术将神经元的电刺激与高压冷冻结合起来， 在哺乳动物中央突触处的快速膜运输事件具有前所未有的空间（1 nm）和 时间（1 ms）分辨率。将这种方法与先进的遗传学，分子生物学， 和生物化学技术，我们将确定融合位点是如何组织在哺乳动物中央突触。 突触传递的缺陷在神经系统疾病中起着因果作用。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.