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

递质释放及其调节机制的研究：突触前神经末梢离子动力学和释放过程的成像

• 批准号: 14580671
• 负责人: SUZUKI Naoya
• 金额: $ 2.11万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580671/
• 关键词: Presynaptic terminal; Neuro-muscular junction; Magnesium sensitive dye; Calcium ion; Magnesium ion; spontaneous release; Short-term plasticity; Casein kinase; マグネシウムイオン感受性蛍光色素; ミオシン軽鎖リン酸化酵素

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

1.在无Ca^<2+>条件下，破伤风诱导的Mg^<2 +>蓄积增强MEPP频率在蛙神经肌肉接头处，观察了Ca^<2+>对强直和强直后微终板电位（MEPP）频率增强的影响。在含有EGTA螯合的无Ca ^2+的Mg ^2+溶液中，100 Hz强直刺激50秒可使MEPP频率增加约30倍。Ca^<2+>显像显示强直时突触前终末[Ca^<2+>] i无明显增加。将外源性Mg^<2+>浓度从5 mM降低到2 mM时，破伤风诱导的MEPP频率增强的峰值降低了约一半。用ω-芋螺毒素GVIA阻断N-型Ca^<2+>通道后，增强的峰值降低到约25%。Mg^<2+>显像显示，在破伤风时，末梢[Mg^<2+>] i增加约1.5倍，并在约1分钟后恢复到静息水平，这种破伤风引起的增加是由于[Mg^<2+>] i的增加而引起的。 ...更多信息 用ω-芋螺毒素GVIA处理后，n [Mg^<2+>]_i下降到约1/3。以上结果提示，Mg^<2+>通过N型Ca^<2+>通道在突触前末梢的积聚是强直刺激引起MEPP频繁增强的主要原因。2.一种不依赖于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（endplate potentials，EPPs）。神经-肌肉制备物用不含Ca^<2+>的林格氏液循环。用0.25Hz刺激诱发EPPs，同时用0.9mM Ca^<2+>林格氏液局部刺激所观察到的突触。在破伤风前3分钟停止抽吸，因此在破伤风期间细胞外条件是无Ca^<2+>的。破伤风后EPP振幅增加5倍，呈指数衰减，时间常数约为130 s。影像学技术显示，强直时突触前终末[Ca ^2+] i不增加，[Mg^2+] i增加。在不同细胞外浓度的Mf^<2+>（2、5和10 mM）下，[Mg^<2+>]_i峰值增加，但这种可塑性没有改变。因此，我们认为这种塑性既不依赖于[Ca^<2+>] i，也不依赖于[Mg^<2+>] i。酪蛋白激酶2（CK 2）抑制剂DRB将这种可塑性降低至60%。CK 2必须部分负责这种可塑性。少

项目成果

鈴木直哉: "蛍光測定 第5章 共焦点顕微鏡法"学会出版センター(印刷中). (2003)

Naoya Suzuki：《荧光测量第 5 章共焦显微镜》学会出版中心（正在出版）（2003 年）。

蛍光測定(第6章 共焦点顕微鏡法)

荧光测量（第 6 章共焦显微镜）

• 发表时间: 2005
• 作者: Tateishi Y.;et al.;鈴木 直哉
• 通讯作者: 鈴木 直哉

鈴木 直哉: "蛍光測定 第5章 共焦点顕微鏡法"学会出版センター(印刷中). (2004)

Naoya Suzuki：《荧光测量第 5 章共焦显微镜》学会出版中心（正在出版）（2004 年）。

Naoya Suzuki: "Ca^<2+>dynamics in presynaptic terminals and control of transmitter release by Ca^<2+>buffering"Recent Research Developments in Molecular & Cellular Biology. (印刷中). (2003)

Naoya Suzuki：“突触前末梢的 Ca^<2+> 动力学和 Ca^<2+> 缓冲对递质释放的控制”，分子与细胞生物学的最新研究进展（2003 年）。