Regulation mechanisms of transmitter release at the chemical synapse.

1.我们成功地利用轴突运输将Ca^<2+>-指示剂Indo-1加载到蛙神经肌肉连接处的突触前神经末梢。然后在ms ~ s的时间范围内定量测量了突触前末端的[Ca^<2+>] _i，并分析了紫外激光共聚焦显微镜下突触前末端的泛近比图像。在正常外源性Ca^<2+> (1.8mM)下施加100次100 Hz高频神经刺激，终末的[Ca^<2+>] _i增加到2muM左右，然后迅速下降，衰减时间常数为数百ms。这一结果表明在末端存在强Ca^<2+>的固存机制，并且不能用经典的Ca^<2+>残差假设来解释增强和增强。我们表明，在青蛙神经肌肉突触的递质释放量方面，快速和缓慢的促进之间存在乘法关系。EPP振幅的易化时间常数约为MEPP频率的5倍。这种差异可以用经典的残差Ca^<2+>假设来解释，假设[Ca^<2+>] _i依赖于第六次幂定律。结合我们之前的发现，加载到末端的BAPTA完全消除了快速促进作用，我们认为快速促进作用是由残留的Ca^<2+>.3引起的。用激光共聚焦显微镜对龙虾神经肌肉突触的突触扣进行了Indo-1 Ca^<2+>成像。我们发现钮扣内Ca^<2+>变化热点在神经刺激下迅速升高，刺激后迅速下降。

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.