High time resolution imaging of CaィイD12+ィエD1 dynamics in the presynaptic nerve terminal : Investigation on the mechanisms of transmitter release and short-term plasticity

突触前神经末梢 CaD12+D1 动力学的高时间分辨率成像：递质释放和短期可塑性机制的研究

• 批准号: 10680631
• 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: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680631/
• 关键词: Synapse; Neuro-mescular junction; Calcium sensitive dye; Calcium ion; Confocal microscope; Synaptic terminal; Sunaptic plasticity

1. CaィイD12+ィエD1 dynamics in the presynaptic terminalWe loaded CaィイD12+ィエD1 sensitive dyes into presynaptic nerve-terminals of the frog neuro-muscular junction and measured CaィイD12+ィエD1 during and after nerve stimulation. The free CaィイD12+ィエD1 concentration rose about 1-2 μM during 10 stimulus at 100 Hz in a 1.8mM CaィイD12+ィエD1 ringer solution. After the end of a tetanus, CaィイD12+ィエD1 concentration declined quickly with a decay time constant about 50 ms and about 80 % of rose CaィイD12+ィエD1 was cleared within 200 ms. This indicate that rapid and high capacity CaィイD12+ィエD1 clearance mechanism exist in the presynaptic terminal. CCCP increased the CaィイD12+ィエD1 concentration during tetanus and elongated the time constant of CaィイD12+ィエD1 clearance. This indicates that the uptake of CaィイD12+ィエD1 into mitochondria largely contributes as this CaィイD12+ィエD1 clearance mechanism.2. Visualization of CaィイD12+ィエD1 microdomainWe succeeded to take images of presynaptic CaィイD12+ィエD1 dynamics with a time res … More olution of 2 ms. And we visualized the spatial heterogeneity of CaィイD12+ィエD1 concentration in the terminal during increase transient phase of CaィイD12+ィエD1 after a single nerve stimulation.3. The dependency of the facilitation of transmitter release on presynaptic CaィイD12+ィエD1 dynamicsWe investigated the effect of BAPTA-AM and EGTA-AM on the CaィイD12+ィエD1 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ィイD12+ィエD1 transient and reduced the amplitude of fast-facilitation significantly. The slow kinetic CaィイD12+ィエD1-buffer, EGTA, reduced the amplitude of the rapid CaィイD12+ィエD1 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 slow CaィイD12+ィエD1 transient significantly. These results suggest a direct coupling between CaィイD12+ィエD1 nerve-terminal and the facilitation of transmitter release. Less

1.突触前终末Ca ~（2+）D_1的动态我们将Ca ~（2+）D_1敏感染料负载于蛙神经-肌肉接头突触前神经终末，在神经刺激过程中和刺激后测定Ca ~（2+）D_1。在1.8mMCa β D12 + β D1的林格氏液中，在100 Hz的10次刺激中，游离Ca 2 + β D12 + β D1的浓度升高约1-2 μM。强直后，突触前末梢内Ca ~（2+）D_（12+）D_1浓度迅速下降，衰减时间常数约为50 ms，约80%的Ca ~（2+）D_（12+）D_1在200 ms内被清除，表明突触前末梢存在快速、高容量的Ca ~（2+）D_（12+）D_1清除机制。CCCP可增加破伤风时Ca ~（2+）D_1浓度，延长Ca ~（2+）D_1清除时间常数。这表明线粒体对Ca ~（2+）D12+ Ca ~（2+）D1的摄取在很大程度上有助于Ca ~（2+）D12+ Ca ~（2+）D1的清除机制。突触前Ca ~（2+）D_12 + Ca ~（2+）D_1微区的动态观察 ...更多信息 并观察了单次神经刺激后，在Ca ~（2+）D_12 + Ca ~（2+）D_1增加的瞬间，末梢内Ca ~（2+）D_12 + Ca ~（2+）D_1浓度的空间异质性.突触前Ca ~（2+）D_（12）+Ca ~（2+）D_1动态对递质释放易化的依赖性我们研究了BAPTA-AM和EGTA-AM对突触前末梢Ca ~（2+）D_（12+）D_1动态的影响以及10次100 Hz强直刺激后递质释放的快易化和慢易化。BAPTA可明显阻断快速的Ca ~（2+）D_1 + Ca ~（2+）D_1瞬变，并显著降低快易化的幅度。缓慢动力学的Ca ~（2+）D_12 + Ca ~（2+）D_1-缓冲液（EGTA）降低了快速Ca ~（2+）D_12 + Ca ~（2+）D_1瞬变的幅度，但与应用BAPTA的情况相比没有消除。EGTA对快易化的幅度影响不大，但缩短了其时间常数。EGTA可显著降低慢易化和慢Ca 2 + D12+ D1瞬变。这些结果表明，Ca ~（2+）D_12 + Ca ~（2+）D_1神经末梢与递质释放的易化作用之间存在直接耦合。少

项目成果

Maki Koike: "Regulation of kinetic properties of GluR2 AMPA receptor channels by alternative splicing"J. neurosci.. 20・6. 2166-2174 (2000)

小池真希：“通过选择性剪接调节 GluR2 AMPA 受体通道的动力学特性”J. Neurosci.. 20・6（2000）。

M. Murakami & H. Kijima: "Transduction ion channels directly gated by sugars on the insect taste cell"J.Gen.Physiol.. 115-4. 455-466 (2000)

村上先生

M. Koike, S. Tsukada, K. Tsuzuki, H. Kijima, & S. Ozawa: "Regulation of kinetic properties of GluR2 AMPA receptor channels by alternative splicing"J.Neurosci.. 20-6. 2166-2174 (2000)

M.小池、S.冢田、K.都筑、H.木岛、

Kazuhiko Narita: "A Ca^<2+>-induced Ca^<2+> release mechanism involved in asynchronous exocytosis at frog motor nerve terminals" J.Gen.Physiol.112・11. 593-609 (1998)

Kazuhiko Narita：“青蛙运动神经末梢异步胞吐作用中涉及的 Ca^<2+> 诱导的 Ca^<2+> 释放机制”J.Gen.Physiol.112・11（1998）。

木島 博正: "シナプス前神経末端におけるカルシウムイオン動態" CLINICAL CALCIUM. 8・2. 14-23 (1998)

Hiromasa Kijima：“突触前神经末梢的钙离子动力学”CLINICAL CALCIUM 8・2（1998）。