Imaging of presynaptic activity in cultured cerebellar granule neurons

培养的小脑颗粒神经元突触前活动的成像

• 批准号: 23700387
• 负责人: IBATA KEIJI
• 金额: $ 2.75万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2011
• 资助国家: 日本
• 起止时间: 2011 至 2013
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-23700387/
• 关键词: イメージング; 膜輸送; シナプス

To understand the cerebellar neuronal circuit, it is important to measure the presynaptic activities of granule cells when animals are moving or learning. In my research, I developed the reporter proteins for presynaptic activity using luciferase. Because luminescence of luciferase is product of chemical reaction, it does not need excitation light, which may sometimes hurt the cells or make autofluorescence from tissue and cell. Therefore using luminescence should give a better signal to noise ratio. I used Gaussia luciferase, which was pH sensitive. I performed the random mutagenesis to obtain bright luminescence. To visualize presynaptic activity, VAMP2 presynaptic protein was used. As a result, I obtained mutant Gluc (mGluc), which showed about 10 times brighter than wild type Gluc. The VAMP2 mGluc transfected neuron showed strong luminescence after neuronal depolarization, indicated this VAMP2-mGluc presynaptic reporter protein can be used to monitor presynaptic activity.

为了了解小脑神经元回路，测量动物运动或学习时颗粒细胞的突触前活动是很重要的。在我的研究中，我用荧光素酶开发了突触前活动的报告蛋白。由于荧光素酶的发光是化学反应的产物，不需要激发光，激发光有时会伤害细胞或使组织和细胞自发荧光。因此，使用发光应该给出更好的信噪比。我用的是Gaussia荧光素酶，它对pH值敏感。我进行了随机突变以获得明亮的发光。为了可视化突触前活性，使用VAMP 2突触前蛋白。 结果，我获得了突变型Gluc（mGluc），其显示出比野生型Gluc亮约10倍。VAMP 2-mGluc转染的神经元在神经元去极化后显示出强烈的发光，表明该VAMP 2-mGluc突触前报告蛋白可用于监测突触前活动。

项目成果

小脳顆粒細胞の軸索から神経活動依存的に分泌されるCbln1の動態解析

小脑颗粒细胞轴突分泌的 Cbln1 神经元活动依赖性动态分析

• 发表时间: 2013
• 作者: 井端啓二;柚崎通介
• 通讯作者: 柚崎通介

Development of Luminescent Coelenterazine Derivatives Activatable by β-Galactosidase for Monitoring Dual Gene Expression

• DOI: 10.1002/chem.201302002
• 发表时间: 2013-10-25
• 期刊: CHEMISTRY-A EUROPEAN JOURNAL
• 影响因子: 4.3
• 作者: Lindberg, Eric;Mizukami, Shin;Kikuchi, Kazuya
• 通讯作者: Kikuchi, Kazuya

Development of cell-impermeable coelenterazine derivatives

• DOI: 10.1039/c3sc51985f
• 发表时间: 2013-01-01
• 期刊: CHEMICAL SCIENCE
• 影响因子: 8.4
• 作者: Lindberg, Eric;Mizukami, Shin;Kikuchi, Kazuya
• 通讯作者: Kikuchi, Kazuya