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

研制了一种全荧光采集双光子激光扫描显微镜，以较高的信噪比（S/N）记录细胞切片图像。一个带有附加物镜、单色计和光电倍增管的光学系统取代了倒置显微镜的原始聚光镜，并作为聚光镜、单目观察光学装置或视觉波长荧光的光度测量装置。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相似的分辨率，通过它们的总和产生更好的信噪比图像。Ca^<2+>通过长时间重复强直刺激进入青蛙运动神经末端，激活Ca^<2+>/钙调素依赖性激酶II，产生环adp核糖，激活Ca^<2+>诱导的Ca^<2+>释放（CICR），并最终使其失活。然而，CICR的失活受到蛋白激酶C的抑制，尤其是在刺激的初始阶段。Ca^<2+>通过末端IP_3受体进入牛蛙交感神经节节前末端，引发CICR，产生蛋白合成依赖性突触前和突触后的长时程增强。在牛蛙交感神经节细胞中，n型电压门控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，与sk型Ca^<2+>依赖性K^+通道松散偶联。在这些耦合中，CICR的激活和随后BK和SK通道的打开分别形成了尖峰再极化和缓慢的后超极化。在重复动作电位期间，由于CICR失活，尖峰复极化机制减弱，而由于Ca^<2+>进入的增加，由于尖峰持续时间的延长，后超极化缓慢增加。Ca^<2+>在细胞膜亚区被赋予ryanodine受体的网络扩散到核膜包膜中，导致Ca^<2+>在细胞核内释放。少

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