Local Ca2+ signaling in sympathetic ganglion neurons

交感神经节神经元中的局部 Ca2 信号传导

• 批准号: 6753539
• 负责人: MARTIN F SCHNEIDER
• 金额: $ 27.92万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-03 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6753539
• 关键词: Rana; action potentials; calcium flux; confocal scanning microscopy; electrophysiology; endoplasmic reticulum; histochemistry /cytochemistry; imaging /visualization /scanning; intracellular; mitochondria; neurons; perfusion; sympathetic ganglion; tissue /cell culture

DESCRIPTION (provided by applicant): The overall goal of this project is to gain further insight into local Ca2+ signaling in neurons. Local spatio-temporal differences in cytosolic (Ca2+), together with localized intracellular Ca2+ receptor molecules, provide one mechanism for selective modulation of diverse cellular functions by the single messenger Ca2+ in the same cell. Our recent confocal imaging of ryanodine receptor (RyR) Ca2+ release channels, endoplasmic reticulum (ER) Ca2+ pumps and mitochondria in cultured dissociated frog sympathetic ganglion neurons has indicated six specialized cellular sub-domains in these neurons: a peripheral ER-rich shell, an underlying mitochondria-rich shell, a central cytoplasm, a peripheral and a central perinuclear ER and the nucleus at the nuclear pole. Our recent video rate confocal fluorescent Ca2+ indicator (fluo-4) imaging in these neurons has revealed discrete sub-plasma membrane sites of preferential initiation of Ca2+ release activated either by caffeine or by a single action potential. The peripheral Ca2+ transients presumably occur via Ca2+ release from peripheral ER since they are eliminated by ER Ca2+ depletion. We now propose to address the following aims. (1) To characterize Ca2+ release, Ca2+ uptake and Ca2+ propagation in and between each of the identified sub-domains in these neurons, and to simulate our observations with a 6 interconnected sub-domain model of the neurons. (2) To study the basis for local peripheral sites of preferential Ca2+ release during single action potentials, and to determine whether these preferential release sites may also generate discrete local Ca2+ release events (Ca2+) "sparks"). (3) To determine the effects of transmitter-induced activation and other physiological perturbations in neurons in culture as well as in ganglia. We will combine high speed "band scan" (4 or 8 ms per band) and line scan (63 us per line) confocal fluorescent Ca2+ imaging of neurons in culture and in partially dissected fresh ganglia, electrophysiology, rapid extracellular perfusion, release of caged compounds, cytosolic injection of cDNA and/or proteins and histochemical study of live and fixed neurons. Our results will provide new insights regarding local Ca2+ signaling mechanisms that could be compromised in neuronal disease.

描述（由申请人提供）：该项目的总体目标是 进一步了解神经元中的局部 Ca2+ 信号传导。当地的 胞浆 (Ca2+) 的时空差异，以及局部 细胞内 Ca2+ 受体分子，提供了一种选择性机制 单一信使 Ca2+ 对多种细胞功能的调节 同一个细胞。我们最近对兰尼碱受体 (RyR) Ca2+ 释放的共聚焦成像 培养物中的通道、内质网 (ER) Ca2+ 泵和线粒体 分离的青蛙交感神经节神经元表明有六个专门的 这些神经元中的细胞亚结构域：富含 ER 的外周外壳、 底层富含线粒体的外壳，中央细胞质，外周细胞质和 中央核周 ER 和核极处的细胞核。我们最近的视频 这些神经元中的共焦荧光 Ca2+ 指示剂 (fluo-4) 成像的速率 揭示了优先启动 Ca2+ 的离散亚质膜位点 由咖啡因或单个动作电位激活的释放。这 外周 Ca2+ 瞬变可能是通过外周 ER 释放 Ca2+ 发生的 因为它们会因 ER Ca2+ 耗尽而被消除。我们现在建议解决 以下目标。 (1) 表征Ca2+释放、Ca2+吸收和Ca2+ 在这些神经元中每个已识别的子域内和之间的传播， 并用 6 个互连的子域模型来模拟我们的观察结果 神经元。 （二）研究当地周边站点优惠依据 单次动作电位期间 Ca2+ 的释放，并确定这些是否 优先释放位点也可能产生离散的局部 Ca2+ 释放事件 （Ca2+）“火花”）。 (3) 确定发射器引起的影响 培养物中神经元的激活和其他生理扰动 就像神经节一样。我们将结合高速“频带扫描”（每个频带 4 或 8 毫秒）和 线扫描（每线 63 us）神经元的共焦荧光 Ca2+ 成像 培养和部分解剖的新鲜神经节，电生理学，快速 细胞外灌注、笼中化合物的释放、胞质注射 活体和固定神经元的 cDNA 和/或蛋白质以及组织化学研究。我们的 结果将为局部 Ca2+ 信号传导机制提供新的见解 这可能会在神经元疾病中受到损害。