Optical Study of Secretion in Mammalian Nerve Terminals

描述(申请人提供)：分泌是最普遍的细胞过程之一，其机制的阐明(S)仍然是一个挑战 给细胞生理学家。然而，越来越清楚的是， 神经递质、神经肽的量子或囊状释放， 神经调节剂和激素在不同的细胞类型中的作用不同，并且 哺乳动物的神经垂体是快速释放的极佳模型 神经分泌系统，包括突触前终末。我们现在能够 监视器，使用具有亚毫秒时间分辨率的光学方法， 大鼠神经末梢的电活动和胞浆钙变化 脊椎动物，以及一系列固有的光学变化(IOC) 与分泌事件直接相关。因此，我们拥有一种非凡的 用于研究兴奋-分泌(E-S)耦合的一系列工具，并 更全面地了解高等语言中的突触传递 动物，以及一般的分泌活动。首先，我们将使用适度的 角分辨光散射法与荧光钙 指示剂、电压敏感染料、免疫金标记物、电子显微镜、 和电子能量损失谱(EELS)来检验这一假设 触发的钙从终端内存储释放是必需的 哺乳动物体内神经肽的释放(这些储藏物可能是 颗粒本身。)我们还将研究几种不同的解释 国际奥委会的起源，包括一种假设，即一种依赖于pH的 分泌颗粒内容物的增溶，通过增加 细胞内钙，先于胞吐作用，并迅速产生大量的 光散射的变化。第二，我们将使用 表达pH敏感绿色荧光蛋白的转基因小鼠， 黄道-PHluorin，用于神经肽分泌的高时间分辨率研究。 最后，我们将广泛描述肾小管内钙离子的变化。 在哺乳动物神经末梢E-S偶联过程中，我们将鉴定 这一关键的第二信使的来源和汇，以及它们的具体作用 胞吐前、中、后。这些实验应该有一个主要的 影响，并应该增加我们对突触传递的理解，及其 人类神经系统疾病中的故障或故障。

DESCRIPTION (provided by applicant): Secretion is one of the most ubiquitous of cellular processes, and the elucidation of its mechanism(s) remains a challenge to cell physiologists. It is becoming increasingly clear, however, that quantal, or vesicular release of neurotransmitters, neuropeptides, neuromodulators, and hormones proceeds differently in different cell types, and that the mammalian neurohypophysis is an excellent model for rapid-release neurosecretory systems including presynaptic terminals. We are now able to monitor, using optical methods having sub-millisecond time resolution, both the electrical events and cytoplasmic calcium changes in nerve terminals of vertebrates, as well as a sequence of intrinsic optical changes (IOC's) that are related directly to the secretory event. Thus, we possess an exceptional array of tools with which to study excitation-secretion (E-S) coupling, and to provide a more complete understanding of synaptic transmission in higher animals, and of the secretory event in general. First, we will use moderate angular resolution light scattering methods, together with fluorescent calcium indicators, voltage sensitive dyes, immuno-gold labeling, electron microscopy, and electron energy loss spectroscopy (EELS) to examine the hypothesis that the triggered release of calcium from intraterminal stores is required for the release of neuropeptides in mammals (and that the stores may be the secretory granules themselves.) We will also examine several alternative explanations for the origin of the IOC's, including the hypothesis that a pH-dependent solubilization of secretory granule contents, mediated by a rise in intraterminal calcium, precedes exocytosis and generates a large and rapid change in light scattering. Second, we will use the neurohypophyses of transgenic mice expressing the pH-sensitive Green Fluorescent Protein, ecliptic-pHluorin, for high time-resolution studies of neuropeptide secretion. Finally, we will characterize extensively the changes in intraterminal calcium during E-S coupling in mammalian nerve terminals, and we will identify the sources and sinks of this critical second messenger, and their specific roles before, during and after exocytosis. These experiments should have a major impact, and should add to our understanding of synaptic transmission, and its failure or malfunction in human neurological disease.