Molecular cytobiological study on hippocampal LTP and LTD

本研究的目的是用分子细胞生物学方法阐明动物学习记忆的机制。当给予突触电高频刺激(HFS)时，突触活动的增强可持续数小时或更长时间。这种现象被称为长时程增强(LTP)，被认为是动物学习和记忆的模型。阐明LTP的作用机制将为理解脑的高活性提供线索。最近的研究表明，蛋白磷酸化，尤其是钙调素/钙调蛋白依赖性蛋白激酶II(CaM Kinase II)参与了海马区LTP的诱导和维持。为了探讨LTP在海马区的分子机制，我们以髓鞘碱性蛋白为底物，通过凝胶内实验分析了LTP诱导LTP过程中LTP对CaM激酶II和丝裂原活化蛋白激酶(MAP)的激活作用。MAPK在LTP诱导过程中被激活。HFS后3min出现激活高峰，10 mm内迅速降至原始水平。预先在海马片上注射MAP激酶抑制剂PD098059，LTP的诱导和激活均被抑制。加入30um的PD098059对LTP和CaM激酶II的激活均无影响，但对MAP的激活有抑制作用。这些结果表明，LTP的诱导与CaM激酶II的激活有关，而与MAP的激活无关。2)在LTP诱导过程中，Calyculin A敏感的蛋白磷酸酶(PP)的活性被抑制，包括PP1和PP2。放射自显影和免疫印迹分析表明，PP2A调节亚基的磷酸化伴随着PP活性的降低。

The purposes of the study is to elucidate the mechanisms of learning and memory of animals on the basis of molecular cytobiological methodology. When electrical high frequency stimulation (HFS) is given to synapses, potentiation of synaptic activities lasts for several hours or longer. This phenomenon is called long-term potentiation(LTP) and considered to be a model for learning and memory of animals. The elucidation of the mechanisms of LTP will give a clue to understand the higher activity of the brain. It has been recently reported that protein phosphorylatiorr especially Ca^2/calmodulin-dependent protein kinaseII (CaM kinase II) is involved in induction and maintenance of LTP in the hippocampus. In an attempt to examine the molecular mechanisms of LTP in the hippocampus, we analyzed LTP.1)Activation of CaM kinase II and mitogen-activated protein kinase (MAP kinase) was analyzed during LTP induction by the in-gel assay with myelin basic protein as a substrate. MAP kinase was activated during LTP induction. The peak of the activation was observed 3 min after HFS and rapidly decreased to the original level within 10 mm. When 50 muM PD098059, a MAP kinase kinase inhibitor, was previously given to hippocampal slices, LTP induction and activation of both CaM kinase II and MAP kinase were inhibited. However, addition of 30 muM PD098059 to slices did not affect both LTP and activation of CaM kinase II but did inhibit activation of MAP kinase. These results indication that LTP induction is correlated with activation of CaM kinase II, but not with activation of MAP kinase.2)Calyculin A-sensitive protein phosphatase (PP) activity, which includes PP1 and PP2 was inhibited during LTP induction. The autoradiography and immunoblot analyses showed that phosphorylation of the regulatory subunit of PP2A is phosphorylated with a concomitant decrease in PP activity.