Molecular cytobiological study on hippocampal LTP and LTD

本研究的目的是从分子细胞生物学的角度阐明动物学习记忆的机制。当电高频刺激（HFS）给予突触时，突触活动的增强持续几个小时或更长时间。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。对LTP机制的阐明将为理解脑的高级活动提供线索。近年来有研究表明，蛋白磷酸化酶特别是钙调蛋白依赖性蛋白激酶II（CaM kinase II）参与海马LTP的诱导和维持。为了研究海马LTP的分子机制，我们分析了LTP。1）通过以髓鞘碱性蛋白为底物的凝胶内分析，分析了LTP诱导过程中CaM激酶II和丝裂原活化蛋白激酶（MAP激酶）的活化。MAP激酶在LTP诱导过程中被激活。HFS后3 min出现激活高峰，10 min内迅速下降至原始水平。预先给予50 μ M的MAP激酶抑制剂PD098059，可抑制LTP诱导和CaM激酶II和MAP激酶的激活。然而，加入30 μ M的PD098059的切片不影响LTP和激活的CaM激酶II，但抑制激活的MAP激酶。这些结果表明LTP的诱导与CaM激酶II的激活有关，而与MAP激酶的激活无关。2）在LTP诱导过程中，Calyculin A敏感的蛋白磷酸酶（PP）活性（包括PP 1和PP 2）受到抑制。放射自显影和免疫印迹分析表明，磷酸化的调节亚基的PP2A磷酸化与PP活性的伴随减少。

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.