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.

应用全细胞膜片钳技术观察了培养的大鼠上级颈神经节细胞之间形成胆碱能突触的过程。在某些实验中，细胞内游离Ca^2+浓度（[Ca^2+] i）是通过比率记录Fura-2荧光来测量的。通过快速溶液交换器将外部K^+浓度（[K^+]_o）提高到40 mM，可迅速增加微小兴奋性突触后电流（mEPSC）的频率。在所研究的一半细胞中，高K^+处理导致mEPSCs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 min。在所研究的一半细胞中观察到的增强作用也发生在烟碱激动剂调理应用30-60秒后。细胞内应用BAPTA降低了mEPSC和尼古丁反应增强的幅度，以及ACh产生的[Ca^<2+>]_i的升高。当ACh浓度较高时，ACh诱导电流的增强作用较小。钙调蛋白依赖性蛋白激酶II（CaMKII）的特异性抑制剂KN-62，但不是无活性的类似物KN-04，阻断了mEPSC的增强作用。结果提示，mEPSCs的中时程增强机制可能是Ca^2+通过烟碱型ACh受体通道进入，激活CaMKII，使烟碱型ACh受体通道本身或邻近相关蛋白磷酸化，增强烟碱型ACh受体对ACh的敏感性。