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Type I metabotropic glutamate receptor dependent long-term depression and Homer

I 型代谢型谷氨酸受体依赖性长期抑郁症和荷马

基本信息

批准号：
16500198
负责人：
AKANEYA Yukio
金额：
$ 1.54万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16500198/
关键词：
mGluR1 mGluR5 neuronal activity synaptic plasticity raft dark-rearing RNA interference glutamate receptor 発現制御 RNAi siRNA

项目摘要

To overcome shortcomings of the conventional "knockout" manipulation, I have established RNAi-iduced Gene Silencing by Local Electroporation (RISLE). The method of RISLE is to introduce siRNA into cells of the targeting brain region by local electroporation. The paradigms of electric pulses are the combination of a preceding poring pulse of high-voltage and short-duration and a following driving pulse of low-voltage and long-duration in tandem. I have made the Homer-knockdown rats using this method. It is known that Homer can bind to GluR1 or GluR5. It is shown that I type metabotropic glutamate receptor-dependent long-term depression of visula cortex is impaired in Homer-knockdown rats, suggesting the involvement of the interaction of Homer and I type metabotropic glutamate receptors. I have obtained the result that the expressions of Homer1-3, GluR1 and GluR5 are regulated by neuronal activity. In the present study, I have employed dark-rearing as a in vivo model of neuronal activity, and have investigated whether and how neuronal activity controls expression of the glutamate receptors and the binding proteins in the membrane microdomains. The membrane contains 'raft' which is cholesterol-rich and is suggested to be involved in the intracellular signal transduction. In the current study, I have carried out the sucrose-gradient method to separate biochemically the component of raft and those of others from the tissues of visual cortex of wild- and knockdown rats which are reared normally or at the completely dark-condition. I have checked localization of glutamate receptors, the binding proteins, the protein kinases and the phosphorylated protein kinases. Finally, I have obtained the conclusion that the localization of these proteins at the raft and at extra-raft is regulated in a activity-dependent manner.

为了克服传统的“敲除”操作的缺点，我已经建立了通过局部电穿孔的RNAi诱导的基因沉默（RISLE）。RISLE的方法是通过局部电穿孔将siRNA导入靶向脑区域的细胞中。电脉冲的范例是前一个高电压、短持续时间的穿孔脉冲和后一个低电压、长持续时间的驱动脉冲的串联组合。我用这种方法制作了Homer击倒大鼠。已知Homer可以与GluR 1或GluR 5结合。结果表明，I型代谢型谷氨酸受体依赖的长时程抑制在Homer基因敲低大鼠的视皮层受损，这表明Homer和I型代谢型谷氨酸受体的相互作用。结果表明，Homer 1 -3、GluR 1和GluR 5的表达受神经元活动的调节。在本研究中，我采用暗饲养作为体内模型的神经元活动，并研究了是否以及如何神经元活动控制谷氨酸受体和膜微区中的结合蛋白的表达。膜含有富含胆固醇的“筏”，并被认为参与细胞内信号转导。本研究采用蔗糖梯度法从正常饲养和完全黑暗条件下饲养的野生大鼠和击倒大鼠视皮层组织中分离出筏状成分和其他成分。我已经检查了谷氨酸受体的定位，结合蛋白，蛋白激酶和磷酸化蛋白激酶。最后，我得到的结论是，这些蛋白质在筏和筏外的本地化是以活性依赖的方式进行调节。