High time resolution imaging of CaィイD12+ィエD1 dynamics in the presynaptic nerve terminal : Investigation on the mechanisms of transmitter release and short-term plasticity

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1. CaィイD12+ィエD1 dynamics in the presynaptic terminalWe loaded CaィイD12+ィエD1 sensitive dyes into presynaptic nerve-terminals of the frog neuro-muscular junction and measured CaィイD12+ィエD1 during and after nerve stimulation. The free CaィイD12+ィエD1 concentration rose about 1-2 μM during 10 stimulus at 100 Hz in a 1.8mM CaィイD12+ィエD1 ringer solution. After the end of a tetanus, CaィイD12+ィエD1 concentration declined quickly with a decay time constant about 50 ms and about 80 % of rose CaィイD12+ィエD1 was cleared within 200 ms. This indicate that rapid and high capacity CaィイD12+ィエD1 clearance mechanism exist in the presynaptic terminal. CCCP increased the CaィイD12+ィエD1 concentration during tetanus and elongated the time constant of CaィイD12+ィエD1 clearance. This indicates that the uptake of CaィイD12+ィエD1 into mitochondria largely contributes as this CaィイD12+ィエD1 clearance mechanism.2. Visualization of CaィイD12+ィエD1 microdomainWe succeeded to take images of presynaptic CaィイD12+ィエD1 dynamics with a time res … More olution of 2 ms. And we visualized the spatial heterogeneity of CaィイD12+ィエD1 concentration in the terminal during increase transient phase of CaィイD12+ィエD1 after a single nerve stimulation.3. The dependency of the facilitation of transmitter release on presynaptic CaィイD12+ィエD1 dynamicsWe investigated the effect of BAPTA-AM and EGTA-AM on the CaィイD12+ィエD1 dynamics at the presynaptic terminal and the fast- and slow-facilitation of transmitter release after 10 tetanus at 100 Hz. BAPTA abolished the rapid CaィイD12+ィエD1 transient and reduced the amplitude of fast-facilitation significantly. The slow kinetic CaィイD12+ィエD1-buffer, EGTA, reduced the amplitude of the rapid CaィイD12+ィエD1 transient, but did not abolished in contrast to the case applied BAPTA. EGTA had a small effect on the amplitude of the fast-facilitation but shortened its time constant. EGTA reduced both the slow-facilitation and the slow CaィイD12+ィエD1 transient significantly. These results suggest a direct coupling between CaィイD12+ィエD1 nerve-terminal and the facilitation of transmitter release. Less