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

将全细胞膜片钳技术应用于培养的大鼠颈上神经节细胞之间形成胆碱能突触。在一些实验中，用Fura-2荧光比率记录法测定细胞内游离钙离子浓度([Ca^&lt；2+&gt；]i)。用快速溶液交换器将外源性K+浓度([K+]o)增加到40 mM，可迅速增加微小兴奋性突触后电流(MEPSCs)的频率。在所研究的一半细胞中，高K~(++)处理引起mEPSCs和乙酰胆碱(ACh)诱发电流的幅度逐渐增强，在恢复到正常的[K~(++)]_o后持续15-60分钟，在被研究的一半细胞中，这种增强也发生在施加尼古丁激动剂30-60秒之后。细胞内应用BAPTA可降低ACh对mEPSCs的增强作用和烟碱反应，并使[Ca~(2+)&gt；]_i升高。当用于测试反应的ACh浓度较高时，ACh诱发电流的增强作用较小。钙调素依赖蛋白激酶II(CaMKII)的特异性抑制剂KN-62，但不是非活性类似物KN-04，可阻断mEPSCs的增强。结果提示，通过烟碱型ACh受体通道进入mEPSCs，引起CaMKII激活，使烟碱型ACh受体通道本身或邻近相关蛋白(S)磷酸化，从而增强烟碱型ACh受体对ACh的敏感性，这可能是MEPSCs中期增强效应的机制。

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.