Role of IP_3 in LTD in cerebellar purkinje cells

IP_3 在小脑浦肯野细胞 LTD 中的作用

基本信息

批准号：
13470019
负责人：
HIRASE Kenzo
金额：
$ 9.15万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470019/
关键词：
cerebellum Purkinje cells Inositol trisphosphate calcium ryanodine Inositol phospholipids

项目摘要

The aim of the present research project was to clarify the role of inositol lA5-trisphosphate (IP_3) in the induction oflong tem depression, especially focusing on the spatio-temporal aspects of IP_3 signaling. To achieve this end, we have developed novel technologies that enable us to visualize the intracellular dynamics of IP_3 in cerebellar Purkinje neurons. As an IP_3 imaging probe, we employed GFP-PHD which is a fusion protein consisting of GFP and pleckstrin homology domain of phospholipase C δ1. We first proved that IP_3 can be visualized in cultured Purkinje neurons which is transduced with GFP-PHD. Furthermore, by directly injecting Sindbis virus vector containing cDNA encoding GFP-PHD into the mouse brain, we successfully introduced GFP-PHD into Purkinje neurons in cerebellar slice. The transduction efficiency of GFP-PHD was found to be high after dissection of cerebellar slices. Using these novel techniques, we found that AMPA receptor activation induces intracellular accumu … More lation of IP_3 in cultured Purkinje neuron. AMPA receptor-mediated IP_3 production was not a mere epiphenomenon, because AMPA receptor activation through climbing fiber stimulation also elicited IP_3 production in Purkinje neuron in cerebellar slice preparation. Our findings are thus important in an understanding of the mechanism of LTD induction.The aim of the present research project was to clarify the role of inositol lA5-trisphosphate (IP_3) in the induction oflong tem depression, especially focusing on the spatio-temporal aspects of IP_3 signaling. To achieve this end, we have developed novel technologies that enable us to visualize the intracellular dynamics of IP_3 in cerebellar Purkinje neurons. As an IP_3 imaging probe, we employed GFP-PHD which is a fusion protein consisting of GFP and pleckstrin homology domain of phospholipase C δ1. We first proved that IP_3 can be visualized in cultured Purkinje neurons which is transduced with GFP-PHD. Furthermore, by directly injecting Sindbis virus vector containing cDNA encoding GFP-PHD into the mouse brain, we successfully introduced GFP-PHD into Purkinje neurons in cerebellar slice. The transduction efficiency of GFP-PHD was found to be high after dissection of cerebellar slices. Using these novel techniques, we found that AMPA receptor activation induces intracellular accumulation of IP_3 in cultured Purkinje neuron. AMPA receptor-mediated IP_3 production was not a mere epiphenomenon, because AMPA receptor activation through climbing fiber stimulation also elicited IP_3 production in Purkinje neuron in cerebellar slice preparation. Our findings are thus important in an understanding of the mechanism of LTD induction. Less

本研究项目的目的是阐明肌醇LA5-三磷酸（IP_3）在诱导长TEM抑郁症中的作用，尤其是专注于IP_3信号传导的时空方面。为了实现这一目标，我们开发了新型技术，使我们能够可视化小脑Purkinje神经元中IP_3的细胞内动力学。作为IP_3成像探针，我们采用了GFP-PHD，它是由磷脂酶Cδ1的GFP和Pleckstrin同源域组成的融合蛋白。我们首先证明了IP_3可以在用GFP-PHD翻译的培养的Purkinje神经元中可视化。此外，通过将含有编码GFP-PHD的cDNA的sindbis病毒载体直接注射到小鼠切片中的Purkinje神经元中，我们成功地将GFP-PHD引入了小脑切片。小脑切片解剖后发现GFP-PHD的转移效率很高。使用这些新型技术，我们发现AMPA受体激活细胞内积累……培养的Purkinje神经元中IP_3的更多细节。 AMPA受体介导的IP_3的产生不仅仅是Epiphenomenon，因为通过攀爬纤维刺激的AMPA受体激活也引起了小脑切片制剂中Purkinje神经元中的IP_3产生。因此，我们的发现在理解LTD诱导机制方面很重要。本研究项目的目的是阐明肌醇LA5-三磷酸（IP_3）在诱导长TEM抑郁症中的作用，尤其是专注于IP_3信号传导的空间周期方面。为了实现这一目标，我们开发了新型技术，使我们能够可视化小脑Purkinje神经元中IP_3的细胞内动力学。作为IP_3成像探针，我们采用了GFP-PHD，它是由磷脂酶Cδ1的GFP和Pleckstrin同源域组成的融合蛋白。我们首先证明了IP_3可以在用GFP-PHD翻译的培养的Purkinje神经元中可视化。此外，通过将含有编码GFP-PHD的cDNA的sindbis病毒载体直接注射到小鼠切片中的Purkinje神经元中，我们成功地将GFP-PHD引入了小脑切片。小脑切片解剖后发现GFP-PHD的转移效率很高。使用这些新型技术，我们发现IP_3在培养的Purkinje神经元中IP_3的细胞内积累。 AMPA受体介导的IP_3的产生不仅仅是Epiphenomenon，因为通过攀爬纤维刺激的AMPA受体激活也引起了小脑切片制剂中Purkinje神经元中的IP_3产生。因此，我们的发现对于理解LTD诱导机制很重要。较少的