SINGLE SYNAPTIC VESICLES FUNCTIONAL SPECTROSCOPY

总体目标：(1)确定单个突触囊泡（SVs）的化学成分并将其与生理学研究联系起来；(2)区分不同的胞吐模型；(3)在单个SV水平上研究学习过程中分子机制的变化。在多种病理中，谷氨酸受体的过度激活可介导神经元损伤或死亡。其中一个可能的原因是囊泡的异常释放。具体的研究目标包括：(1)识别单个SV的神经递质；(2)研究囊泡中的转运蛋白；(3)在体外胞吐过程中追踪SV膜蛋白和转运蛋白，并将其与上述(1)和SV膜蛋白的鉴定联系起来。在我的研究中将使用以下方法。光学陷阱将保持囊泡进行光谱分析，执行分子结合分析，并转移囊泡进行化学分析。小体积单个sv中电压和pH指标的光谱学可能需要单分子检测技术和非线性光学显微镜。融合将通过在模型膜中融合天然囊泡来模拟和研究，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物在其发育过程中的结合强度。用光学层析免疫分析法检测SV膜蛋白。通过这种方式，转运蛋白和囊泡的其他膜蛋白的功能可以与已识别的SV膜蛋白相关。用毛细管电泳法测定突触囊泡的含量，将囊泡裂解，其内容物衍生化，用激光光谱法分离和检测。

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.