Local Ca2+ signaling in sympathetic ganglion neurons

描述(由申请人提供)：本项目的总体目标是 进一步了解神经元中的局部钙信号。本地 胞浆(Ca~(2+))的时空差异及其局部化 细胞内钙受体分子，提供了一种选择性 细胞内单个信使钙离子对多种细胞功能的调节 同一间牢房。我们最新的Ryanodine受体(RyR)钙释放的共聚焦成像 培养细胞的通道、内质网钙泵和线粒体 分离的青蛙交感神经节神经元表明有六种 这些神经元中的细胞亚域：一个富含内质网的外周外壳，一个 下面是富含线粒体的外壳，中央细胞质，外周和 核周中央内质网和位于核极的核。我们最近的视频 这些神经元中的速率共聚焦荧光钙指示剂(Fluo-4)成像 揭示了钙离子优先启动的离散亚质膜部位 由咖啡因或单个动作电位激活的释放。这个 外周钙瞬变可能是通过外周内质网释放钙而发生的 因为它们被内质网钙离子耗竭所消除。我们现在建议解决 遵循目标。(1)表征钙离子释放、钙摄取和钙离子 在这些神经元中的每个识别的子域内和之间传播， 并用6个相互关联的子域模型来模拟我们的观察结果 神经元。(2)研究当地周边地区优先选址的依据 在单个动作电位过程中的钙释放，并确定这些 优先释放部位也可能产生离散的局部钙释放事件 (Ca2+)“火花”)。(3)确定递质诱导的影响 培养中神经元的激活和其他生理扰动 就像在神经节里。我们将结合高速“频段扫描”(每个频段4或8毫秒)和 线扫描(每行63us)共聚焦荧光钙离子成像 培养和部分解剖的新鲜神经节，电生理，快速 细胞外灌流，笼状化合物的释放，细胞内注射 活神经元和固定神经元的基因和/或蛋白质及组织化学研究。我们的 研究结果将为了解局部钙信号机制提供新的见解 这可能会在神经性疾病中受到影响。

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.