Study of the calcium dynamics and molecular mechanisms in the dendrite of cerebellar Purkinje cell

小脑浦肯野细胞树突钙动力学及分子机制研究

• 批准号: 18300104
• 负责人: INOUE Takafumi
• 金额: $ 10.98万
• 依托单位: Waseda University (2007)The University of Tokyo (2006)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18300104/
• 关键词: cerebellar Purkinie cell; IP3 receptor; dendrite; spine; membrane protein; endoplasmic reticulum; cerebellar granule cell; FRET; プルキンエ細胞; 細胞内カルシウム動態; 電気生理; イメージング; 脳切片

We investigated dynamics and functions of IP3 receptor (IP3R) in the neuronal dendrite. Since IP3R is known to play a pivotal role in synaptic plasticity in the postsynapse, changes in the trafficking of IP3R into/out of dendritic spines would affect the mechanism underlying synaptic plasticity. We compared lateral diffusion efficacy of IP3R with another ER membrane protein, SERCA, in spines of cerebellar Purkinje cells by expressing genes of the ER proteins fused with fluorescent proteins by means of gene gun gene delivery technique and two photon microsoopy. We found that the diffusion efficacy of IP3R was significantly lower than that of SERCA, suggesting a mechanism which enriches IP3R proteins in spines due to their high affinity to the actin cytoskeleton as has been shown in the dendrites of hippocampal pyramidal cells by us previously. In another approach to the IP3R function in the cerebellar cortex, we found that IP3R plays a role in formation of the dendrite of Purkinje cell. It was unexpected that IP3R expressed in the cerebellar granule cells was the key player instead of the highly expressed IP3Rs in the Purkinje. This result shows another important role of IP3R in the cerebellar cortex. We also succeeded to create an IP3-indicator by engineering the IP3-binding part of IP3R with fluorescent proteins, which clearly showed IP3 oscillation during calcium oscillation in the COS-7 cell line. This novel indicator will be a potent tool investigate IP3 dynamics in neuronal dendrites where IP3 and IP3R play important roles in synaptic plasticity.

我们研究了神经元树突中 IP3 受体 (IP3R) 的动力学和功能。由于已知 IP3R 在突触后突触可塑性中发挥关键作用，因此 IP3R 进出树突棘的运输变化将影响突触可塑性的机制。我们通过基因枪基因递送技术和两个光子显微技术表达与荧光蛋白融合的 ER 蛋白基因，比较了 IP3R 与另一种 ER 膜蛋白 SERCA 在小脑浦肯野细胞棘中的横向扩散功效。我们发现IP3R的扩散效率显着低于SERCA，这表明IP3R蛋白由于与肌动蛋白细胞骨架的高亲和力而在棘中富集，正如我们之前在海马锥体细胞的树突中所显示的那样。在研究小脑皮质中 IP3R 功能的另一种方法中，我们发现 IP3R 在浦肯野细胞树突的形成中发挥作用。出乎意料的是，小脑颗粒细胞中表达的IP3R而不是浦肯野细胞中高表达的IP3R才是关键参与者。这一结果显示了IP3R在小脑皮质中的另一个重要作用。我们还通过用荧光蛋白改造 IP3R 的 IP3 结合部分，成功创建了 IP3 指示剂，该指示剂清楚地显示了 COS-7 细胞系中钙振荡期间的 IP3 振荡。这一新颖的指标将成为研究神经元树突中 IP3 动态的有效工具，其中 IP3 和 IP3R 在突触可塑性中发挥重要作用。

项目成果

平行線維-プルキンエ細胞間シナプスにおける短期増強

平行纤维浦肯野细胞突触的短期增强

• 发表时间: 2006
• 作者: 後藤 純一;井上 貴文;来馬 明規;御子柴 克彦
• 通讯作者: 御子柴 克彦

Cytosolic inositol 1,4,5-trisphosphate dynamics during intracellular calcium oscillations in living cells.

• DOI: 10.1083/jcb.200512141
• 发表时间: 2006-06-05
• 期刊: JOURNAL OF CELL BIOLOGY
• 影响因子: 7.8
• 作者: Matsu-ura, Toru;Michikawa, Takayuki;Inoue, Takafumi;Miyawaki, Atsushi;Yoshida, Manabu;Mikoshiba, Katsuhiko
• 通讯作者: Mikoshiba, Katsuhiko

A genetically encoded fluorescent inositol 1,4,5-trisphosphate sensor based on the inositol 1,4,5-trisp hosphate receptor

基于肌醇 1,4,5-三磷酸受体的基因编码荧光肌醇 1,4,5-三磷酸传感器

• 发表时间: 2006
• 作者: Takayuki Michikawa;Toru Matsu-ura;Takafumi Inoue;Atsushi Miyawaki;Manabu Yoshida;Katsuhiko Mikoshiba
• 通讯作者: Katsuhiko Mikoshiba

Inositol 1,4,5-trisphosphate receptor type-1 in granule cells, not in Purkinje cells, regulates the dendritic morphology of Purkinje cells through brain-derived neurotrophic factor production

• DOI: 10.1523/jneurosci.3269-06.2006
• 发表时间: 2006-10-18
• 期刊: JOURNAL OF NEUROSCIENCE
• 影响因子: 5.3
• 作者: Hisatsune, Chihiro;Kuroda, Yukiko;Mikoshiba, Katsuhiko
• 通讯作者: Mikoshiba, Katsuhiko

Improvements of affinity,dynamic range and reaction rate of fluorescent indicators for inositol 1,4,5-trisphosphate (IP3) by mutating of IP3-binding domain

通过 IP3 结合域突变提高肌醇 1,4,5-三磷酸 (IP3) 荧光指示剂的亲和力、动态范围和反应速率

• 发表时间: 2007
• 作者: Toru Matsu-ura;Takayuki Michikawa;Miwako Iwai;Sachiko Ishida;Takafumi Inoue;Katsuhiko Mikoshiba
• 通讯作者: Katsuhiko Mikoshiba