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

采用全细胞膜片钳技术培养大鼠颈上神经节细胞，这些细胞相互形成胆碱能突触。在一些实验中，通过比值记录fura-2荧光测定细胞内游离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，可以迅速增加微型兴奋性突触后电流（mEPSCs）的频率。在研究的一半细胞中，高K^+处理导致mEPSCs和乙酰胆碱（ACh）诱导电流的振幅逐渐增强，并在恢复正常[K^+]_o后持续15-60分钟。在一半的细胞中观察到的增强也发生在烟碱激动剂调节应用30-60秒后。细胞内应用BAPTA降低了mepsc的增强程度和烟碱反应，以及ACh产生的[Ca^<2+>]_i的增加。当用于测试响应的乙酰胆碱浓度较高时，乙酰胆碱诱导电流的增强作用较小。钙调素依赖性蛋白激酶II （CaMKII）的特异性抑制剂KN-62，而不是无活性类似物KN-04，可以阻断mEPSCs的增强。结果提示，Ca^<2+>通过烟碱ACh受体通道进入mEPSCs，引起CaMKII激活，使烟碱ACh受体通道本身或邻近相关蛋白磷酸化，增强烟碱ACh受体对ACh的敏感性，是mEPSCs中期增强的机制。

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.