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.突触前终末的钙动力学我们将敏感染料注入蛙神经-肌肉接头的突触前神经末梢，并在刺激神经过程中和刺激后测定了钙的动力学。在1.8 mM钙离子林格液中，在100赫兹刺激10次时，游离钙离子浓度上升约1-2微米。破伤风结束后，Ca~(2+)浓度迅速下降，衰减时间常数约为50ms，约80%的玫瑰Ca~(2+)&gt；在200ms内被清除。这表明突触前终末存在快速、高容量的钙清除机制。CCCP可增加破伤风时钙离子浓度，延长钙离子清除时间常数。这表明线粒体摄取Ca~(2+)在很大程度上参与了这一清除机制。Ca^&lt；2+&gt；微域的可视化我们成功地拍摄了突触前Ca^&lt；2+&gt；动力学图像，时间分辨率为2ms。同时，我们还观察到了单次神经刺激后，神经末梢内钙离子浓度的空间异质性。BAPTA-AM和EGTA-AM对突触前终末Ca~(2+)&gt；动力学的影响以及100 Hz强直后递质释放的快速性和慢速性。BAPTA可显著消除快钙瞬变，降低快易化幅度。与应用BAPTA相比，慢运动钙缓冲剂EGTA可降低快速钙瞬变的幅度，但不能使其消失。EGTA对快速易化幅度的影响较小，但缩短了其时间常数。EGTA可显著降低慢促钙通道和SHOW钙通道的瞬时电流。这些结果提示神经末梢Ca~(2+)和Gt~(2+)之间的直接偶联和递质释放的易化。

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.