Development of a total fluorescence collecting-two photon laser scanning microscope and analysis of intracellular Ca^<2+> dynamics

全荧光采集双光子激光扫描显微镜的研制及细胞内Ca^2动态分析

• 批准号: 10357001
• 负责人: KUBA Kenji
• 金额: $ 20.8万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10357001/
• 关键词: Total fluorescence collecting-two photon laser scanning microscope; Ca^<2+> -dependent K^+ channel; Frng motor nerve terminal; Ryanodine receptor; Intracellular Ca^<2+> release; Exocytosis; Short-term plasticity; Bullfrog sympathetic panglion cell

A total fluorescence collecting-two photon laser scanning microscope that records the sliced images of a cell at a higher signal to noise ratio (S/N) was developed. An optical system with an additional objective, a monochrometer and a photomultiplier replaced the original condenser of an inverted microscope and functioned as a condenser, a monocular viewing optics or a photometric device for visual wavelength fluorescence. The fluorescence images recorded through the original and additional objectives showed similar resolutions in horizontal and axial planes, yielding an image of a better S/N by their summation.Ca^<2+> entry into frog motor nerve terminals by long-lasting repetitive tetanic stimulation primed ryanodine receptors via activation of Ca^<2+>/calmodulin-dependent kinase II and production of cyclic ADP-ribose, subsequently activated Ca^<2+>-induced Ca^<2+> release (CICR) through ryanodine receptors and finally inactivated it. The inactivation of CICR, however, was inhibited … More by the activation of protein kinase C especially during the initial phase of stimulation. Repetitive Ca^<2+> entry into the preganglionic terminals of bullfrog sympathetic ganglion primed CICR via IP_3 receptors at the terminals and produced the protein synthesis-dependent, pre- and postsynaptic long-term potentiation.In bullfrog sympathetic ganglion cells, N-type voltage-gated Ca^<2+> channels were found to functionally couple with ryanodine receptors and BK-type Ca^<2+> -dependent K^+ channels, forming a functional triad, and loosely with SK-type Ca^<2+>-dependent K^+ channels. The activation of CICR and subsequent opening of BK and SK channels in these couplings shapes the spike repolarization and the slow afterhyperpolarization, respectively. The mechanism of spike repolarization wanes due to the inactivation of CICR during repetitive action potentials, while the slow afterhyperpolarization increases for the increase in Ca^<2+> entry due to the prolongation of the spike duration. The network of Ca^<2+> stores endowed with ryanodine receptors in the submembrane regions spread into the nuclear membrane envelopes and caused Ca^<2+> release in the nucleus. Less

研制了一种能以较高信噪比记录细胞切片图像的全荧光采集双光子激光扫描显微镜。一个额外的物镜，单色仪和光电倍增管的光学系统取代了原来的聚光镜的倒置显微镜，并作为一个聚光镜，单目观察光学或测光装置的可见波长荧光。通过原始物镜和附加物镜记录的荧光图像在水平和轴向平面上显示出相似的分辨率，通过它们的总和产生了更好的S/N图像。通过长时间重复强直刺激，Ca^2+进入蛙运动神经末梢，通过激活Ca^2+/钙调蛋白依赖性激酶II和产生环ADP-核糖来启动ryanodine受体，随后通过ryanodine受体激活Ca^<2 +>诱导的Ca^<2+>释放（CICR），最后使其失活，但CICR的失活被抑制 ...更多信息 通过蛋白激酶C的激活，特别是在刺激的初始阶段。在牛蛙交感神经节，N型电压门控性Ca^<2+>通道与Ryanodine受体和BK型Ca^<2+>依赖性K^+通道在功能上偶联，形成功能性三联体，并与SK型Ca^<2+>依赖性K^+通道松散结合。CICR的激活和随后的BK和SK通道的开放在这些耦合形状的尖峰复极和缓慢的后超极化，分别。在重复动作电位过程中，CICR的失活使放电复极机制减弱，而放电持续时间的延长使Ca^2+内流增加，使慢后超极化增强。在膜下区域，被赋予ryanodine受体的Ca^2+储存网络扩展到核膜被膜中，并引起细胞核中的Ca^2+释放。少

项目成果

K.Kuba: "Two-photon laser-scanning microscopy : Tests of objective lenses and Ca^<2+> probes"Neurosci. Res.. 32. 281-294 (1998)

K.Kuba：“双光子激光扫描显微镜：物镜和Ca ^ 2 探针的测试”Neurosci。

Liu, C., Shirasaki, T., Tanaka, S. & Kuba, K.: "Activity-dependent enhancement of miniature excitatory postsynaptic current amplitude and its modulation by protein kinase C in cultured rat sympathetic neurons"Neurosc. Lett.. 280. 57-60 (2000)

刘 C.、白崎 T.、田中 S.

K.KUBA: "Ca^<2+> dynamics and modulation. In slow synaptic responses and modulation"Springer. 10 (1999)

K.KUBA：“Ca^<2>动力学和调制。在缓慢的突触反应和调制中”施普林格。

Kuba, K.: "Ca^<2+> dynamics and modulation : Introductory review. In Slow synaptic Responses and Modulation, eds. Kuba, K., Higashida, H., Brown, D. A. & Yoshioka, T."Springer Verlag, Tokyo. 163-172 (2000)

Kuba, K.：“Ca^<2> 动力学和调制：介绍性综述。慢突触反应和调制，编辑。Kuba, K.、Higashida, H.、Brown, D. A.

F.-M.Lu: "Synchronized Ca^<2+> signals mediated by Ca^<2+> action potentials in the hippocampal neuron network in vitro"Cell Calcium. 29. 379-394 (2001)

F.-M.Lu：“体外海马神经元网络中由 Ca^2 动作电位介导的同步 Ca^2 信号”细胞钙。