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. 我们将Ca^<2+>敏感染料加载到蛙神经-肌肉连接处的突触前神经末梢，并测量了神经刺激期间和之后Ca^<2+>的动态。在1.8mM Ca^<2+>林格溶液中，在100 Hz、10次刺激下，游离Ca^<2+>浓度升高约1 ~ 2个muM。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。这表明突触前末端存在快速、高容量的Ca^<2+>清除机制。CCCP增加了破伤风期间Ca^<2+>的浓度，延长了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Ca^<2+>的清除机制。我们成功地以2 ms的时间分辨率拍摄了突触前Ca^<2+>的动态图像。我们观察了单次神经刺激后Ca^<2+>瞬时升高时终末Ca^<2+>浓度的空间异质性。我们研究了BAPTA-AM和EGTA-AM对突触前末端Ca^<2+>动力学的影响以及100 Hz 10次破伤风后递质释放的快慢促进作用。BAPTA消除了快速Ca^<2+>瞬态，显著降低了快速促进的振幅。慢动力学Ca^<2+>缓冲剂EGTA降低了快速Ca^<2+>瞬态的振幅，但与应用BAPTA的情况相比没有消除。EGTA对快速促进的振幅影响较小，但缩短了其时间常数。EGTA显著降低了慢化和Ca^<2+>瞬态。这些结果表明神经末梢Ca^<2+>与促进递质释放之间存在直接耦合。

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.