Optical Study of Secretion in Mammalian Nerve Terminals

哺乳动物神经末梢分泌物的光学研究

• 批准号: 6612613
• 负责人: BRIAN Matthew SALZBERG
• 金额: $ 39.33万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-24 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6612613
• 关键词: action potentials; calcium flux; calcium indicator; electron microscopy; electrophysiology; exocytosis; fluorescent dye /probe; genetically modified animals; green fluorescent proteins; immunologic assay /test; laboratory mouse; nephelometry; neural transmission; neurohypophysis; neuropeptides; neurotransmitter transport; secretion; spectrometry; synapses

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.

描述（由申请人提供）：分泌是最普遍的细胞过程之一，其机制的阐明仍然是一个挑战 给细胞生理学家。然而，越来越明显的是， 神经递质、神经肽的量子或囊泡释放， 神经调节剂和激素在不同细胞类型中的作用不同，并且 哺乳动物神经垂体是快速释放的优秀模型 神经分泌系统，包括突触前末梢。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， 神经末梢的电事件和细胞质钙变化 脊椎动物，以及一系列内在光学变化（IOC） 与分泌事件直接相关。因此，我们拥有非凡的 用于研究兴奋-分泌 (E-S) 耦合的一系列工具 提供对高级突触传递的更全面的理解 动物，以及一般的分泌事件。首先，我们将使用中等 角分辨率光散射方法以及荧光钙 指示剂、电压敏感染料、免疫金标记、电子显微镜、 和电子能量损失谱（EELS）来检验以下假设： 需要从终端内储存触发钙的释放 哺乳动物中神经肽的释放（并且储存可能是分泌 颗粒本身。）我们还将研究几种替代解释 IOC 的起源，包括 pH 依赖性的假设 分泌颗粒含量的溶解，通过增加介导 末端内钙，在胞吐作用之前并产生大量且快速的 光散射的变化。其次，我们将使用神经垂体 表达 pH 敏感绿色荧光蛋白的转基因小鼠， ecliptic-pHluorin，用于神经肽分泌的高时间分辨率研究。 最后，我们将广泛描述终端内钙的变化 在哺乳动物神经末梢的 E-S 耦合过程中，我们将识别 这个关键的第二信使的来源和汇，以及它们的具体作用 胞吐作用之前、期间和之后。这些实验应该有一个重大的 影响，并且应该增加我们对突触传递及其 人类神经系统疾病的失败或功能障碍。