SYNAPTIC TRANSMISSION AND SINGLE VESICLE ANALYSIS

确定滥用药物如何影响神经功能需要我们提高对突触裂隙结构和操作的基本理解。神经科学中测量大样本平均性质的传统工具为这一目标贡献了大量信息。然而，为了全面了解突触，我们必须开发出具有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。这项提议的目标是开发这种工具和技术。我们计划使用毛细管电泳法、光学捕捉法、可控囊泡融合技术、荧光共振能量转移技术、生物传感器检测技术、激光诱导荧光技术和单分子检测技术来研究：(A)单个突触小泡的含量，以及(B)突触裂隙中参与囊泡与细胞膜融合过程中单个蛋白质的结构和功能。除了获得有关突触小泡周期和胞吐作用的有价值的信息外，本研究计划的目的是建立一套有价值的技术，供神经科学界在突触传递研究中使用。

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.