利用GCaMP6f蛋白对小鼠伏隔核神经元活动的钙离子成像

基础医学与临床 ›› 2016, Vol. 36 ›› Issue (6): 763-766.

利用GCaMP6f蛋白对小鼠伏隔核神经元活动的钙离子成像

刘泽玥¹,许琪²

1. 中国医学科学院基础医学研究所北京协和医学院基础学院
2. 北京协和医学院基础学院

收稿日期:2016-03-17 修回日期:2016-04-20 出版日期:2016-06-05 发布日期:2016-05-27
通讯作者: 许琪 E-mail:xuqi@pumc.edu.cn
基金资助:
973计划

Calcium imaging of the activity of mice neurons in the nucleus accumbens using GCaMP6f

Liu Ze-Yue¹,

Received:2016-03-17 Revised:2016-04-20 Online:2016-06-05 Published:2016-05-27

摘要/Abstract

摘要： 目的在小鼠伏隔核表达GCaMP6f蛋白，并在束缚状态下，应用钙成像技术实时监测小鼠伏隔核内活化的神经元。方法首先，包装并纯化aav-Syn1-GCaMP6f-P2A-Tomato病毒，并通过感染原代神经元验证目的蛋白的表达和功能。然后，通过脑立体定位技术注射aav-Syn1-GCaMP6f-P2A-Tomato病毒于小鼠伏隔核脑区，动物恢复21d后，插入光纤，在其清醒状态下，给予束缚刺激，用钙成像技术实时记录伏隔核神经元的活化。结果表达GCaMP6f的原代神经元在给予谷氨酸刺激后，神经元被活化，Ca2+内流而产生绿色荧光；通过在体实验，给予小鼠束缚刺激，可在视野内检测到因伏隔核神经元活化而发出的绿色荧光信号；鼠脑切片同样可检测到位于胞质的绿色荧光信号与位于细胞核的病毒红色荧光标记共定位于相同细胞。结论 aav-Syn1-GCaMP6f-P2A-Tomato 腺相关病毒可成功用于在体活化神经元的实时监测。通过在小鼠伏隔核定点注射该病毒，给予束缚刺激，应用钙成像技术可实时记录到小鼠伏隔核的活化神经元。

关键词: 关键词：钙成像, 腺相关病毒, 伏隔核, GCaMP6f

Abstract: Objective Expressing GCaMP6f in nucleus accumbens of mice using brain stereotaxic injection，examined neuron activity of restrained mice by calcium imaging in real time. Methods To get recombination virus, cultured 293T cell, then packed and purified aav-Syn1-GCaMP6f-P2A- Tomato virus. After being confirmed the recombination virus worked by infecting primary neurons, the virus was injected into nucleus accumbens by stereotaxic injection. And after the animals recovered, the mice were implanted optical fiber in brains, and restrained, then recorded neuronal activity by calcium imaging in the nucleus accumbens in time. Results After stimulated by glutamic acid, the neurons expressed GCaMP6f protein released green fluorescence because of the calcium influx. Brain slices showed that the green fluorescence produced by GCaMP6f and the red fluorescence produced by a carried marker protein in virus could co-localize in the same neuron. Conclusions The adeno-associated virus aav- Syn1-GCaMP6f-P2A-Tomato could be successfully used to real timely monitor activated neurons employing calcium imaging in vivo.

Key words: Key words: calcium imaging, adeno-associated virus, nucleus accumbens, GCaMP6f

中图分类号:

R338.2+6

刘泽玥许琪. 利用GCaMP6f蛋白对小鼠伏隔核神经元活动的钙离子成像[J]. 基础医学与临床, 2016, 36(6): 763-766.

Liu Ze-Yue. Calcium imaging of the activity of mice neurons in the nucleus accumbens using GCaMP6f[J]. Basic & Clinical Medicine, 2016, 36(6): 763-766.