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: 6917143
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6917143
• 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）：分泌是蜂窝过程中最普遍的一种，其机制的阐明仍然是一个挑战 致细胞生理学家。然而，越来越清楚的是 神经递质，神经肽的量化或囊泡释放， 神经调节剂和激素在不同的细胞类型中的进行不同，并且 哺乳动物的神经噬菌管是快速释放的绝佳模型 神经分泌系统，包括突触前末端。我们现在能够 监视，使用具有亚毫秒时间分辨率的光学方法 电气事件和细胞质钙的神经末端变化 脊椎动物，以及一系列内在的光学变化（IOC） 与分泌事件直接相关。因此，我们拥有一个非凡的 使用的一系列工具来研究激发 - 分泌（E-S）耦合，并 在更高的 动物，以及一般的分泌事件。首先，我们将使用中等 角度分辨率光散射方法，以及荧光钙 指标，电压敏感染料，免疫金标记，电子显微镜， 和电子能量损失光谱（EELS），以检查以下假设。 触发钙从偏源储存中释放的释放是需要的 在哺乳动物中释放神经肽（并且商店可能是分泌物 颗粒本身。）我们还将检查一些其他解释 IOC的起源，包括依赖pH的假设 分泌颗粒含量的溶解化，介导 内部钙，先于胞吐作用，并产生较大而快速的 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， 黄道磷脂，用于神经肽分泌的高度分辨率研究。 最后，我们将广泛地表征内血钙的变化 在哺乳动物神经终端的E-S耦合期间，我们将确定 这个关键的第二使者的来源和水槽及其特定角色 胞吐作用前，期间和之后。这些实验应该有一个专业 影响，应该增加我们对突触传播的理解，及其 人类神经疾病的失败或故障。