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刘树雷,孙琳,吴广延,隋建峰,曹文富.腺相关病毒AAV2/8-CaMKIIα-ChR2-mCherry的组织学及通道功能鉴定[J].陆军军医大学学报(原第三军医大学学报),2015,37(13):1297-1301.
Liu Shulei,Sun Lin,Wu Guangyan,Sui Jianfeng,Cao Wenfu.Histological features and channel functions of adeno-associated virus AAV2/8-CaMKIIα-ChR2-mCherry in glutamatergic neurons[J].J Amry Med Univ (J Third Mil Med Univ),2015,37(13):1297-1301.
刘树雷,孙琳,吴广延,隋建峰,曹文富.腺相关病毒AAV2/8-CaMKIIα-ChR2-mCherry的组织学及通道功能鉴定[J].陆军军医大学学报(原第三军医大学学报),2015,37(13):1297-1301. DOI:
Liu Shulei,Sun Lin,Wu Guangyan,Sui Jianfeng,Cao Wenfu.Histological features and channel functions of adeno-associated virus AAV2/8-CaMKIIα-ChR2-mCherry in glutamatergic neurons[J].J Amry Med Univ (J Third Mil Med Univ),2015,37(13):1297-1301. DOI:
目的 对腺相关病毒AAV2/8-CaMKIIα-ChR2-mCherry在大鼠内侧前额叶谷氨酸能神经元中表达的组织学及通道功能予以鉴定。 方法 以立体定位注射技术将腺相关病毒AAV2/8-CaMKIIα-ChR2-mCherry注射到SD雄性大鼠内侧前额叶,当病毒注射3~4周后,采用免疫荧光组织化学技术检测标记蛋白(mCherry)在谷氨酸能神经元(特异启动子CaMKIIα)的表达情况;利用在体细胞外记录技术观测谷氨酸能神经元所表达的通道蛋白(ChR2)对不同光刺激频率的电生理反应特性。 结果 病毒成功注射到内侧前额叶并特异表达通道蛋白于谷氨酸能神经元的细胞膜上,表达成功率为(94.6±0.9)%;表达ChR2通道蛋白的谷氨酸能神经元对蓝光(470 nm, 20 mW/mm
2
,5 ms波宽,5~80 Hz)产生稳定的光激活反应,并且对低频光刺激(5、10、20 Hz)的反应性优于高频光刺激(40 Hz和80 Hz;P0.001)。 结论 腺相关病毒AAV2/8-CaMKIIα-ChR2-mCherry能成功表达在谷氨酸能神经元的细胞膜上,对光刺激有频率选择性。
Objective To identify the histological features and channel functions of adeno-associated AAV2/8-CaMKIIα-ChR2-mCherry expressed in medial prefrontal glutamatergic neurons of rats. Methods Adeno-associated AAV 2/8-CaMKIIα-ChR2-mCherry was injected into the medial prefrontal cortex of SD male rats by using stereotaxic apparatus. In 4 to 6 weeks later
immunofluorescence assay was used to detect the expression of mCherry in the glutamate neurons. Electrophysiological characteristics of the channel prot
ein channelrhodopsin-2 (ChR2) expressed in glutamatergic neurons were observed under light stimulations at different frequencies. Results The viruses were successfully injected into the medial prefrontal cortex of the rats
and the objective channel protein ChR2 was specifically expressed in the cell membrane of glutamatergic neurons
with an expression rate of (94.6±0.9)%. Glutamate neurons with ChR2 expression showed optogenetic stimulation to the blue light (470 nm
20 mW/mm
2
5 ms
5~80 Hz) stably
and the activations were significant to low frequencies (5
10 and 20 Hz) than to high frequencies (P0.001). Conclusion Adeno-associated virus AAV2/8-CaMKIIα-ChR2-mCherry successfully leads the expression of channel protein ChR2 in the cell membrane of glutamatergic neurons
and the cells show frequency-selective to light stimulation.
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