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Ca^<2+>的条件下，破伤风诱导的Mg^<2+>积累增强了MEPP频率。我们观察了Ca^<2+>对蛙神经肌肉接点微型终板电位（MEPP）频率破伤风和破伤风后增强的影响。在egta螯合不含Ca^<2+>的Mg^<2+>外源性溶液中，100 Hz强电50秒可使MEPP频率增强约30倍。Ca^<2+>-显像显示破伤风期间突触前末端[Ca^<2+>]_i未增加。外源Mg^<2+>浓度从5 mM降低到2 mM，使破伤风引起的MEPP频率增强的峰值降低约一半。ω- concontoxin GVIA阻断n型Ca^<2+>通道，使增强峰值降低到25%左右。Mg^<2+>-显像显示破伤风期间终末[Mg^<2+>]_i增加约1.5倍，并以约1 min的时间常数恢复到静息水平。ω-螺帽毒素GVIA处理后，其n [Mg^<2+>]_i减少到1 / 3左右。这些结果表明，通过n型Ca^<2+>通道在突触前终末积聚Mg^<2+>是破伤风诱导MEPP频繁增强的主要原因。一种不依赖于Ca^<2+>和Mg^<2+>的新型递质释放增强我们研究了上述突触前激活是否也影响终板电位（EPPs）。神经-肌肉预备液循环使用无Ca^<2+>的林格氏液。0.25Hz刺激诱发epp，同时向观察突触局部喷入0.9mM Ca^<2+>林格氏液。破伤风前3分钟停止泡芙，破伤风期间细胞外无Ca^<2+>。破伤风后，EPP振幅增加到5倍，并以130s左右的时间常数呈指数衰减。影像学显示破伤风期间突触前末端[Ca^<2+>]_i未升高，但[Mg^<2+>]_i升高。在不同的细胞外浓度Mf^<2+>（2、5和10 mM）下，[Mg^<2+>]_i峰值升高，但这种可塑性没有改变。因此，我们得出结论，这种可塑性既不取决于[Ca^<2+>]_i，也不取决于[Mg^<2+>]_i。酪蛋白激酶2 （CK2）抑制剂DRB将这种可塑性降低到60%。CK2一定是造成这种可塑性的部分原因。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