SINGLE SYNAPTIC VESICLES FUNCTIONAL SPECTROSCOPY

总体目标：(L)确定单个突触小泡的化学成分并将其与生理学研究联系起来；(2)区分不同的胞吐模式；(3)在单个突触小泡水平上研究学习过程中分子机制是如何变化的。在多种病理过程中，谷氨酸受体的过度激活可能介导神经元的损伤或死亡。其中一个可能的来源是囊泡的异常释放。具体的研究目标包括：(1)确定单个SVS的神经递质；(2)研究囊泡中的转运蛋白；(3)跟踪体外胞吐过程中的SV膜蛋白和转运蛋白，并将其与上述(1)和已鉴定的SV膜蛋白联系起来。本研究将采用以下方法进行研究。光学捕捉器将保留用于光谱分析的小泡，执行分子结合分析，并将小泡转移用于化学分析。单个SVS小体积中的电压和pH指示剂的光谱可能需要单分子检测技术和非线性光学显微镜。融合将通过将天然囊泡融合在模型膜中来模拟和研究，同时进行光谱分析。我们将通过捕获囊泡来测量蛋白质复合体在其发育过程中的结合强度。用光学层析免疫分析法检测病毒膜蛋白。通过这种方式，囊泡的转运蛋白和其他膜蛋白的功能可以与已鉴定的SV膜蛋白相关。用毛细管电泳法探测突触小泡的含量，将小泡裂解，并对其含量进行衍生化、分离和激光光谱分析。

Broad Objectives: (l) Identify the chemical contents of individual synaptic vesicles (SVs) and relate this to the physiological study, (2) Discriminate between the different models of exocytosis, and (3) Study at the single SV level how the molecular machineries are changed during learning. In a variety of pathologies, excessive activation of glutamate receptors may mediate neuronal injury or death. One of the possible sources of this is abnormal release from vesicles. The specific research aims include: (1) Identifying the neurotransmitters of individual SVs, (2) Studying the transporters in vesicles, and (3) Following the SV membrane proteins and transporters during in vitro exocytosis and relating this to (1) above and to identified SV membrane proteins. The following methods will be used in my research. An optical trap will hold the vesicles for spectroscopy, perform a molecular binding assay, and transfer the vesicles for chemical analysis. Spectroscopy of voltage and pH indicators in the small volumes of single SVs may call for single molecule detection techniques and nonlinear optical microscopy. Fusion will be modeled and studied by the fusion of natural vesicles in a model membrane while spectroscopy is performed. We will measure the binding strength of the protein complex during its development by trapping the vesicle. The SV membrane proteins will be detected by optical chromatographic immunoassay. In this way the functionality of the transporters and other membrane proteins of the vesicle can be related to identified SV membrane proteins. To probe synaptic vesicle content by capillary electrophoresis the vesicle will be lysed and its contents derivatized, separated and detected by laser spectroscopy.