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.

将全细胞斑块夹技术应用于形成胆碱能突触的培养大鼠上颈神经节细胞。在某些实验中，通过Fura-2荧光的比例记录，测量了细胞内游离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。在研究的一半细胞中，高K^+处理导致振幅MEPSC和乙酰胆碱（ACH）诱导的电流的逐渐增强，该电流持续了15-60分钟。在烟碱激动剂施用30-60秒后，在研究的一半细胞中看到的增强也发生。 BAPTA的细胞内应用降低了MEPSC和烟碱反应的增强幅度，并降低了ACH产生的[Ca^<2+>] _ i的升高。当用于测试响应的ACH浓度较高时，ACH诱导的电流的增强很小。一种钙调蛋白依赖性蛋白激酶II（CAMKII）的特定抑制剂KN-62，而不是非活性类似物KN-04，它阻止了MEPSC的增强。结果表明，通过烟碱ACH受体通道输入的MEPSC中期增强的机制导致CAMKII的激活，从而磷酸化了烟碱ACH受体通道本身或相邻的相关蛋白质，并增强了烟碱ACH受体对ACH的敏感性。