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: 6759446
• 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/6759446
• 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）：分泌物是最普遍的 细胞过程及其机制的阐明仍然是一个挑战 细胞生理学家。然而，越来越明显的是， 神经递质，神经肽， 神经调质和激素在不同的细胞类型中有不同的作用， 哺乳动物的神经垂体是一个很好的模型， 包括突触前末梢的神经分泌系统。我们现在能够 使用具有亚毫秒时间分辨率的光学方法， 电事件和细胞质钙变化的神经末梢 脊椎动物，以及一系列的内在光学变化（IOC）， 与分泌事件直接相关。因此，我们拥有一个特殊的 一系列研究兴奋-分泌（E-S）偶联的工具， 提供了一个更完整的了解突触传递在较高的 动物，以及一般的分泌事件。首先，我们将适度使用 角分辨率光散射法，连同荧光钙 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）来检查假设， 需要从终端内储存中触发钙的释放 哺乳动物神经肽的释放（以及储存可能是分泌性的） 颗粒本身）。我们还将研究几种不同的解释， IOC的起源，包括pH依赖性 分泌颗粒内容物的溶解，通过增加 终末内钙，先于胞吐作用，并产生一个大的和快速的 光散射的变化。第二，我们将使用 表达pH敏感性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，我们将广泛地描述终末内钙的变化， 在哺乳动物神经末梢的E-S耦合过程中，我们将确定 这一关键第二信使的来源和汇，以及它们的具体作用 在胞吐作用之前、期间和之后。这些实验应该有一个主要的 影响，并应增加我们对突触传递的理解， 人类神经系统疾病的失败或故障。