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Ca 2+信号。当地 细胞溶质（Ca 2+）的时空差异，以及局部 细胞内Ca 2+受体分子，提供了一种选择性地 细胞内单个信使Ca 2+对多种细胞功能的调节 相同单元我们最近对Ryanodine受体（RyR）Ca 2+释放的共聚焦成像 细胞培养液中的钙通道、内质网（ER）钙泵和线粒体 分离的青蛙交感神经节神经元表明， 这些神经元中的细胞亚结构域：外周ER丰富的外壳， 下面的富甲壳，中央细胞质，周边和 中央核周ER和核极处的核。我们最近的视频 在这些神经元中的共聚焦荧光Ca 2+指示剂（fluo-4）成像， 揭示了不连续的亚质膜位点的优先启动钙 由咖啡因或单个动作电位激活的释放。的 外周Ca 2+瞬变可能通过外周ER的Ca 2+释放而发生 因为它们通过ER Ca 2+耗尽而被消除。我们现建议处理 的目标。(1)表征Ca 2+释放、Ca 2+摄取和Ca 2 + 在这些神经元中的每个所识别的子域中和子域之间的传播， 并模拟我们的观察与6互联子域模型， 神经元(2)以研究为基础，对当地周边站点进行优惠 在单个动作电位过程中的Ca 2+释放，并确定这些 优先释放位点也可产生离散的局部Ca 2+释放事件 (Ca2+）“火花”）。(3)为了确定发射机引起的 激活和其他生理扰动的神经元培养以及 就像神经节一样我们将结合联合收割机高速“波段扫描”（每波段4或8毫秒）， 线扫描（63 us/线）共聚焦荧光Ca 2+成像的神经元， 培养和部分解剖的新鲜神经节，电生理学，快速 细胞外灌注，笼状化合物的释放， 活的和固定的神经元的cDNA和/或蛋白质和组织化学研究。我们 结果将提供有关局部Ca 2+信号机制的新见解 在神经元疾病中可能受到损害。

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.