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

• 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

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 IA5-三磷酸 (IP_3) 在诱导长期抑郁中的作用，特别关注 IP_3 信号传导的时空方面。为了实现这一目标，我们开发了新技术，使我们能够可视化小脑浦肯野神经元中 IP_3 的细胞内动态。作为IP_3成像探针，我们使用GFP-PHD，它是由GFP和磷脂酶Cδ1的pleckstrin同源结构域组成的融合蛋白。我们首先证明IP_3可以在用GFP-PHD转导的培养的浦肯野神经元中可视化。此外，通过将含有编码GFP-PHD的cDNA的辛德比斯病毒载体直接注射到小鼠大脑中，我们成功地将GFP-PHD引入到小脑切片的浦肯野神经元中。解剖小脑切片后发现 GFP-PHD 的转导效率很高。使用这些新技术，我们发现 AMPA 受体激活会诱导培养的浦肯野神经元中 IP_3 的细胞内积累。 AMPA 受体介导的 IP_3 产生不仅仅是一种附带现象，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备中浦肯野神经元中 IP_3 的产生。因此，我们的研究结果对于理解 LTD 诱导机制非常重要。本研究项目的目的是阐明肌醇 IA5-三磷酸 (IP_3) 在诱导长期抑郁中的作用，特别关注 IP_3 信号传导的时空方面。为了实现这一目标，我们开发了新技术，使我们能够可视化小脑浦肯野神经元中 IP_3 的细胞内动态。作为IP_3成像探针，我们使用GFP-PHD，它是由GFP和磷脂酶Cδ1的pleckstrin同源结构域组成的融合蛋白。我们首先证明IP_3可以在用GFP-PHD转导的培养的浦肯野神经元中可视化。此外，通过将含有编码GFP-PHD的cDNA的辛德比斯病毒载体直接注射到小鼠大脑中，我们成功地将GFP-PHD引入到小脑切片的浦肯野神经元中。解剖小脑切片后发现 GFP-PHD 的转导效率很高。利用这些新技术，我们发现 AMPA 受体激活会诱导培养的浦肯野神经元中 IP_3 的细胞内积累。 AMPA 受体介导的 IP_3 产生不仅仅是一种附带现象，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备中浦肯野神经元中 IP_3 的产生。因此，我们的发现对于理解 LTD 诱导机制非常重要。较少的

项目成果

森 泰男: "Transient Receptor Potential 1 Regulates Capacitative Ca^<2+> Entry and Ca^<2+> Release from Endoplasmic Reticulum in B lymphocytes"Jouural of Experimental Medicine. 195巻. 673-681 (2002)

Yasuo Mori：“瞬时受体电位1调节B淋巴细胞内质网的电容性Ca ^ 2+ 进入和Ca ^ 2+ 释放”实验医学杂志195。673-681（2002）。

Eto, K.: "Phosphatidylinositd 3-kinase suppresses glucose-stimulated insulin secretion by affecting post-cytosolic [Ca^<2+>] elevation signals"Diabetes. 51. 87-97 (2002)

Eto, K.：“磷脂酰肌醇 3-激酶通过影响胞质后 [Ca^2>] 升高信号来抑制葡萄糖刺激的胰岛素分泌”糖尿病。

並木 繁行: "Mapping of heme binding domain in soluble guanylyl cyclase β1 subunit"Biochemical Biophysical Research Communication. 288巻. 798-804 (2001)

Shigeyuki Namiki：“可溶性鸟苷酸环化酶 β1 亚基中血红素结合域的图谱”《生物化学生物物理研究通讯》，第 288 卷，798-804（2001 年）。

Okubo Y.: "Visualization of IP_3 dynamics reveals a novel AMPA receptor-triggered IP_3 production pathway mediated by voltage-dependent Ca^<2+> influx in Purkinje cells"Neuron. 32. 113-122 (2001)

Okubo Y.：“IP_3 动力学的可视化揭示了浦肯野细胞中电压依赖性 Ca^2 流入介导的新型 AMPA 受体触发的 IP_3 产生途径”Neuron。

並木繁之, 他: "Mapping of heme binding domain in soluble guanylyl cyclase β1 subunit"Biochemical and Biophysical Research Communication. 288巻4号. 798-804 (2001)

Shigeyuki Namiki 等人：“可溶性鸟苷酸环化酶 β1 亚基中的血红素结合域的映射”《生物化学和生物物理研究通讯》，第 288 卷，第 4 期，第 798-804 期（2001 年）。