SYNAPTIC TRANSMISSION AND SINGLE VESICLE ANALYSIS

要确定滥用药物如何影响神经功能，需要我们进一步了解突触间隙的结构和运作。神经科学中测量大量样本平均特性的传统工具为实现这一目标贡献了大量信息。然而，为了充分了解突触，我们必须开发前所未有的灵敏度的新工具，这些工具能够分析单个突触成分并观察突触囊泡融合过程中的构象变化。本提案的目标是开发这样的工具和技术。我们计划利用毛细管电泳、光学捕获、可控囊泡融合、荧光共振能量转移、生物传感器检测、激光诱导荧光和单分子检测来研究：(a)单个突触囊泡的含量，(b)突触间隙中囊泡与细胞膜融合过程中涉及的单个蛋白质的结构和功能。除了提供有关突触囊泡周期和胞外分泌的宝贵信息外，该研究项目的目的是建立一套有价值的技术，可用于神经科学界对突触传递的研究。

Determining how abused drugs affect neurological function requires us to advance our basic understanding of the structure and operation of the synaptic cleft. Traditional tools in neuroscience, which measure averaged properties of bulk samples, have contributed an immense amount of information toward this goal. To understand fully the synapse, however, we must develop new tools of unprecedented sensitivity, tools that are capable of analyzing single synaptic components and observing conformational changes during vesicle fusion at the synapse. The objective of this proposal is to develop such tools and techniques. We plan to use capillary electrophoresis, optical trapping, controlled vesicle fusion, fluorescence resonance energy transfer, biosensor detection, laser induced fluorescence, and single-molecule detection to investigate: (a) the contents of individual synaptic vesicles, and (b) the structure and function of individual proteins involved in the process of vesicle fusion with the cell membrane in the synaptic cleft. In addition to yielding valuable information about the synaptic vesicle cycle and exocytosis, the purpose of this research program is to establish a valuable set of techniques that can be used by the neuroscience community in their investigations of synaptic transmission.