Investigation on the mechanisms of transmitter release and its regulation : Imaging of ion dynamics and release process in the presynaptic nerve terminal

1.在无钙条件下，破伤风引起的镁离子积聚使MEPP频率增加。100赫兹破伤风作用50秒，可使含有EGTA的无镁离子外液中的MEPP频率增加约30倍。[Ca^&lt；2+]-成像显示，强直性脊柱炎时突触前终末[Ca^&lt；2+]_i无明显升高。当外界浓度从5 mM降至2 mM时，破伤风诱发的MEPP频率增强峰值降低约一半。用ω-芋螺毒素GVIA阻断N型Ca~(2+)&gt~(2+)通道，可使增强峰值降低约25%。[mg^&lt；2+]-显像显示，破伤风后末梢[mg^；lt；2+]_i增加约1.5倍，并恢复到静息状态，时间常数约为1min。破伤风引起的I…升高经ω-芋螺毒素GVIA处理后，n[mg^&lt；2+&gt；]_i减少到约1/3。这些结果表明，通过N型钙通道在突触前终末积聚镁离子是强直性刺激诱发MEPP增强的主要原因。2.一种不依赖于钙离子和镁离子的新型递质释放增强作用是否也影响终板电位。神经肌肉制剂用无钙林格液循环。用0.25赫兹刺激诱发EPPS，同时向观察到的突触局部喷洒0.9 mM钙离子林格液。破伤风前3分钟停止喷雾，因此破伤风时细胞外无钙。破伤风后，EPP波幅增加到5倍，并呈指数衰减，时间常数约为130s。成像技术显示，强直性脊柱炎时，突触前终末的[Ca^&lt；2+&gt；]_i没有升高，但[mg^&lt；2+&gt；]_i升高。在不同胞外浓度(2，5和10 mM)的MF^&lt；2+&gt；作用下，[mg^&lt；2+&gt；]_i峰增大，但这种可塑性没有改变。因此，我们得出结论，这种可塑性既不依赖于[Ca^&lt；2+&gt；]_i，也不依赖于[Mg^&lt；2+&gt；]_i，酪蛋白激酶2(CK2)抑制剂DRB使这种可塑性降低到60%。CK2一定是这种可塑性的部分原因。较少

1.Tetanus-induced Mg^<2+> accumulation enhances MEPP frequency under Ca^<2+>-free conditionsEffects of Ca^<2+> on tetanic and post-tetanic enhancement of miniature end-plate potential (MEPP) frequency were examined at the frog neuromuscular junction. About thirty times enhancement of MEPP frequency was induced by 100 Hz tetanus for 50 sec in an EGTA-chelated Ca^<2+>-free Mg^<2+> containing external solution. Ca^<2+>-imaging indicated that there was no increase in [Ca^<2+>]_i at the presynaptic terminals during tetanus. Decreasing external concentration of Mg^<2+> from 5 mM to 2 mM reduced peak value of teh tetanus-induced enhancement of MEPP frequency to about a half. Blocking of N-type Ca^<2+> channels with ω-conotoxin GVIA reduced the peak value of the enhancement to about 25%. Mg^<2+>-imaging indicated that [Mg^<2+>]_i in the terminals increased to about 1.5 times during tetanus and it returned to the resting level with a time constant of about 1 min. This tetanus-induced increase i … More n [Mg^<2+>]_i was reduced to about one-third after treated with ω-conotoxin GVIA. These results suggest that Mg^<2+> accumulation in the presynaptic terminals through N-type Ca^<2+> channels is the main cause of the tetanus-induced enhancement of MEPP frequently.2.A new type enhancement of transmitter release not depended on Ca^<2+> and Mg^<2+>We examined whether above presynaptic activation also affected endplate potentials (EPPs). The nerve-muscle preparation was circulated with Ca^<2+>-free Ringer's solution. EPPs were evoked by 0.25Hz stimulation, simultaneously 0.9mM Ca^<2+> Ringer's solution was puffed locally to the observed synapse. Puff was stopped 3 minutes before tetanus, therefore the extracellular condition was Ca^<2+>-free during tetanus. After tetanus, EPP amplitude increased to 5 times and this enhancement decayed exponentially with a time constant of about 130s. Imaging techniques indicated that [Ca^<2+>]_i did not increased but [Mg^<2+>]_i increased in presynaptic terminals during tetanus. Under various extracellular concentrations of Mf^<2+> (2,5 and 10 mM), [Mg^<2+>]_i peak increased, but this plasticity did not change. Therefore, we concluded that this plasticity depended on neither [Ca^<2+>]_i nor [Mg^<2+>]_i. Casein kinase 2 (CK2) inhibitor, DRB, reduced this plasticity to 60%. CK2 must be partially responsible for this plasticity. Less