Visualization of Ca^<2+> in the presynaptic nerve terminal using a fast scan confocal microscope : Ca^<2+> microdomain and the short-term synaptic plastecity

1.突触前末梢的Ca^<2+>动态我们将Ca^<2+敏感染料负载于蛙神经-肌肉接头的突触前神经末梢，并在神经刺激期间和之后测量Ca^<2+>动态。在1.8mM Ca^<2 +>林格氏溶液中，在100 Hz的10次刺激过程中，游离Ca^<2 +>浓度升高约1-2 μ M。破伤风结束后，Ca^<2+>浓度迅速下降，衰减时间常数约为50 ms，约80%的玫瑰Ca^<2+>在200 ms内被清除，这表明突触前末梢存在快速、高容量的Ca^<2+>清除机制。CCCP可增加破伤风时的Ca^2+浓度，延长Ca^2+清除的时间常数.这表明线粒体对Ca^<2+>的摄取在很大程度上有助于这种Ca^<2+>清除机制。Ca^<2+>微区的可视化我们成功地获得了时间分辨率为2 ms的突触前Ca^<2+>动态图像，并在单次神经刺激后的Ca ^<2 +>增加瞬时相显示了终末Ca^<2+浓度的空间异质性.突触前Ca^<2+>动力学对递质释放易化的依赖性我们研究了BAPTA-AM和EGTA-AM对突触前末梢Ca^<2+>动力学的影响以及100 Hz强直刺激后递质释放的快易化和慢易化。BAPTA可显著阻断快速Ca^<2+>瞬变，降低快易化的幅度。慢动力学Ca^2+缓冲剂EGTA降低了快速Ca^2+瞬变的幅度，但与应用BAPTA的情况相反，EGTA对快速易化的幅度影响较小，但缩短了其时间常数。EGTA显著降低慢易化和显示Ca^2+瞬变。这些结果表明神经末梢Ca^<2+>与递质释放的促进作用之间存在直接耦合。

1. Ca^<2+> dynamics in the presynaptic terminalWe loaded Ca^<2+> sensitive dyes into presynaptic nerve-terminals of the frog neuro-muscular junction and measured Ca^<2+> dynamics during and after nerve stimulation. The free Ca^<2+> concentration rose about 1-2 muM during 10 stimulus st 100 Hz in a 1.8mM Ca^<2+> ringer solution. After the end of a tetanus, Ca^<2+> concentration declined quickly with a decay time constant about 50 ms and about 80% of rose Ca^<2+> was cleared within 200 ms. This indicate that rapid and high capacity Ca^<2+> clearance mechanism cxist in the presynaptic terminal. CCCP increased the Ca^<2+> concentration during tetanus and elongated the time constant of Ca^<2+> clearance. This indicates that the uptake of Ca^<2+> into mitochondria largely contributes as this Ca^<2+> clearance mechanism.2. Visualization of Ca^<2+> microdomainWe succeeded to take images of presynaptic Ca^<2+> dynamics with a time resolution of 2 ms. And we visualized the spatial heterogeneity of Ca^<2+> concentration in the terminal during increses transient phase of Ca^<2+> after a single nerve stimulation.3. The dependency of the facilitaion of transmitter release on presynaptic Ca^<2+> dynamicsWe investigated the effect of BAPTA-AM and EGTA-AM on the Ca^<2+> 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^<2+> transient and reduced the amplitade of fast-facilitation significantly. The slow kinetic Ca^<2+> -buffer, EGTA,reduced the amplitude of the rapid Ca^<2+> 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 show Ca^<2+> transient significantly. These results suggest a direct coupling between Ca^<2+> in nerve-terminal and the facilitation of transmitter releaes.