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

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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