Regulation mechanisms of transmitter release at the chemical synapse.

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1.We succeeded to load a Ca^<2+>-indicator, Indo-1, into the presynaptic nerve terminal of frog neuromuscular junction by making use of axonal trasport. We could then measured the of [Ca^<2+>] _i quantitatively at the presynaptic terminal in the time range of ms to s, analyzing the flaorecent ratio image of the terminal made by UV-lazer confocal microscope. When high-frequency nerve stimulation 100 times at 100 Hz was applied under the normal external Ca^<2+> (1.8mM), [Ca^<2+>] _i in the terminal increased to about 2muM,then decreased rapidly with decay time constant of several hundreds ms to the basal level. This result indicated the existance of strong Ca^<2+> sequestering mechanisms at the terminal and that augmentation and potentiation could not be accounted for by the classical residual Ca^<2+> hypothesis.2.We showed that multiplicative relationship holds between the fast and slow facilitations with regard to the amount of transmitter release at the frog neuromuscular synapse. The decay time constant of facilitation of EPP amplitude was about 5 times larger than that of MEPP frequency. This difference could be accounted for by the classical residual Ca^<2+> hypothesis under the assumption of 6th power law of [Ca^<2+>] _i dependence. Together with our former finding that BAPTA loaded into the terminal completely eliminated the fast facilitation, it is concluded that the fast facilitation is caused by the residual Ca^<2+>.3.We made the Indo-1 Ca^<2+> image of the synaptic bouton of the spiny lobster neuromuscular synapse using confocal laser macroscopy. We found hot spots of Ca^<2+> change which rapidly increased by nerve stimulation and decreased rapidly after stimulation within the bouton.