CREATION OF NEW CONCEPT AND ITS MECHANISM IN SYNAPTIC PLASTICITY AT CHOLINERGIC SYNAPSES

胆碱能突触突触可塑性新概念的创立及其机制

• 批准号: 08680892
• 负责人: SHIRASAKI Tetsuya
• 金额: $ 1.54万
• 依托单位: KANSAI MEDICAL UNIVERSITY (1997)佐賀医科大学 (1996)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08680892/
• 关键词: PLASTICITY; PERIPHERAL NEURONS; NICOTINIC ACETYLCHOLINE; MINIATURE EXCITATORY POSTSYNAPTIC CURRENTS; PATCH CLUMP; INTRACELLULAR Ca^<2+>; CALMODULIN DEPENDENT PROTEIN KINASE

A whole cell patch clamp technique was applied to cultured rat superior cervical ganglion cells which form cholinergic synapses each other. In some experiments, intracellular free Ca^<2+> concentration ([Ca^<2+>]_i) was measured by the ratiometric recording of fura-2 fluorescence. Raising the external K^+ concentration ([K^+]_o) to 40 mM by a quick solution exchanger swiftly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs). In a half of the cells studied, a high K^+ treatment caused a gradual enhancement of the amplitude mEPSCs and acetylcholine (ACh)-induced currents which lasted for 15-60 min after returning to the normal [K^+]_o. The potentiation, seen in a half of cells studied also occurred after the conditioning application of nicotinic agonist for 30-60 sec. Intracellular application of BAPTA reduced the magnitude of the potentiation of mEPSCs and nicotinic response as well as a rise in [Ca^<2+>]_i produced by ACh. The potentiation of ACh-induced currents was small when the concentration of ACh used for test response was high. A specific inhibitor of calmodulin dependent protein kinase II (CaMKII), KN-62, but not an inactive analogue, KN-04, blocked the potentiation of mEPSCs. The results suggest as the mechanism of the middle-term potentiation of mEPSCs that Ca^<2+> entered through nicotinic ACh receptor channel caused the activation of CaMKII that phosphorylated the nicotinic ACh receptor channel itself or neighboring related protein (s) and enhanced the sensitivity of nicotinic ACh receptor to ACh.

将全细胞膜片钳技术应用于培养的大鼠颈上神经节细胞，这些细胞彼此形成胆碱能突触。在一些实验中，通过fura-2荧光的比率记录来测量细胞内游离Ca 2+ 浓度([Ca 2+ ]_i)。通过快速溶液交换器将外部 K^+ 浓度 ([K^+]_o) 提高到 40 mM，迅速增加了微型兴奋性突触后电流 (mEPSC) 的频率。在一半的研究细胞中，高 K^+ 处理导致 mEPSC 和乙酰胆碱 (ACh) 诱导电流的幅度逐渐增强，在恢复到正常 [K^+]_o 后持续 15-60 分钟。在所研究的一半细胞中观察到，在烟碱激动剂调理应用 30-60 秒后也出现了增强作用。 BAPTA的细胞内应用降低了mEPSC和烟碱反应的增强幅度以及ACh产生的[Ca^2+]_i的增加。当用于测试响应的ACh浓度较高时，ACh诱导电流的增强较小。钙调蛋白依赖性蛋白激酶 II (CaMKII) 的特异性抑制剂 KN-62（而非非活性类似物 KN-04）可阻断 mEPSC 的增强。结果表明，Ca^2+通过烟碱型ACh受体通道进入mEPSCs，引起CaMKII的激活，磷酸化烟碱型ACh受体通道本身或邻近的相关蛋白，从而增强mEPSCs的中期增强机制。烟碱型乙酰胆碱受体对乙酰胆碱的敏感性。