腺相关病毒AAV2/8-CaMKIIα-ChR2-mCherry的组织学及通道功能鉴定  

Histological features and channel functions of adeno-associated virus AAV2/8-Ca MKIIα-ChR2-m Cherry in glutamatergic neurons

作　　者：刘树雷[1,2] 孙琳[3] 吴广延[3] 隋建峰[3] 曹文富[1] 

机构地区：[1]重庆医科大学中医药学院中医药教研室,重庆400016 [2]解放军第324医院皮肤科,重庆00020 [3]第三军医大学基础医学部教学实验中心,重庆400038

出　　处：《第三军医大学学报》2015年第13期1297-1301,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金预研项目(NSFYY201108);重庆市基础与前沿基金(cstc2014jcyj A1027);重庆市卫生局基金(2013-2-301);博士后基金(2014M5625592)~~

摘　　要：目的对腺相关病毒AAV2/8-Ca MKIIα-ChR2-m Cherry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-Ca MKIIα-ChR2-m Cherry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（m Cherry）在谷氨酸能神经元（特异启动子Ca MKIIα）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 m W/mm2,5 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;P〈0.001）。结论腺相关病毒AAV2/8-Ca MKIIα-ChR2-m Cherry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-Ca MKIIα-ChR2-m Cherry expressed in medial prefrontal glutamatergic neurons of rats. Methods Adeno-associated AAV 2 /8-Ca MKIIα-ChR2-m Cherry 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 m Cherry in the glutamate neurons. Electrophysiological characteristics of the channel protein 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 m W / mm2,5 ms,5 ~ 80 Hz） stably,and the activations were significant to low frequencies（ 5,10 and 20 Hz） than to high frequencies（ P〈0. 001）. Conclusion Adeno-associated virus AAV2 /8-Ca MKIIα-ChR2-m Cherry successfully leads the expression of channel protein ChR2 in the cell membrane of glutamatergic neurons, and the cells show frequency-selective to light stimulation.

关 键 词：光遗传学 腺相关病毒 ChR2 

分 类 号：R-332[医药卫生] R373