[1]刘树雷,孙琳,吴广延,等.腺相关病毒AAV2/8-CaMKIIα-ChR2-mCherry的组织学及通道功能鉴定[J].第三军医大学学报,2015,37(13):1297-1301.
 Liu Shulei,Sun Lin,Wu Guangyan,et al.Histological features and channel functions of adeno-associated virus AAV2/8-CaMKIIα-ChR2-mCherry in glutamatergic neurons[J].J Third Mil Med Univ,2015,37(13):1297-1301.
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
37卷
期数:
2015年第13期
页码:
1297-1301
栏目:
论著
出版日期:
2015-07-15

文章信息/Info

Title:
Histological features and channel functions of adeno-associated virus AAV2/8-CaMKIIα-ChR2-mCherry in glutamatergic neurons
作者:
刘树雷孙琳吴广延隋建峰曹文富
重庆医科大学中医药学院中医药教研室;第三军医大学基础医学部教学实验中心;解放军第324医院皮肤科
Author(s):
Liu Shulei Sun Lin Wu Guangyan Sui Jianfeng Cao Wenfu

Department of Traditional Chinese Herbs, School of Traditional Chinese Medicine, Chongqing Medical University, Chongqing, 400016;  Experimental Center of Basic Medicine, College of Basic Medical Sciences, Third Military Medical University, Chongqing, 400038;Department of Dermatology, No. 324 Hospital of PLA, Chongqing, 400020, China

关键词:
光遗传学腺相关病毒ChR2
Keywords:
optogenetics adeno-associated virus channelrhodopsin-2 mCherry
分类号:
R-332; R373; R394
文献标志码:
A
摘要:

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/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-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。

Abstract:

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 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 mW/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-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.

参考文献/References:

[1]Deisseroth K. Optogenetics[J]. Nat Methods, 2011, 8(1): 26-29.
[2]Zhang F, Gradinaru V, Adamantidis A R,et al. Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures[J]. Nat Protoc, 2010, 5(3): 439-456.
[3]Robinson M J, Warlow S M, Berridge K C. Optogenetic excitation of central amygdala amplifies and narrows incentive motivation to pursue one reward above another[J]. J Neurosci, 2014, 34(50): 16567-16580.
[4]Wang X, Pinol R A, Byrne P,et al. Optogenetic stimulation of locus ceruleus neurons augments inhibitory transmission to parasympathetic cardiac vagal neurons via activation of brainstem alpha1 and beta1 receptors[J]. J Neurosci, 2014, 34(18): 6182-6189.
[5]van-Bergeijk P, Adrian M, Hoogenraad C C,et al. Optogenetic control of organelle transport and positioning[J]. Nature, 2015, 518(7537): 111-114.
[6]Benson D L, Isackson P J, Gall C M,et al. Contrasting patterns in the localization of glutamic acid decarboxylase and Ca2+/calmodulin protein kinase gene expression in the rat central nervous system[J]. Neuroscience, 1992, 46(4): 825-849.
[7]Hamilton L S, Sohl-Dickstein J, Huth A G,et al. Optogenetic activation of an inhibitory network enhances feedforward functional connectivity in auditory cortex[J]. Neuron, 2013, 80(4): 1066-1076.
[8]Lin J Y, Sann S B, Zhou K,et al. Optogenetic inhibition of synaptic release with chromophore-assisted light inactivation (CALI)[J]. Neuron, 2013, 79(2): 241-253.
[9]Van-Dort C J, Zachs D P, Kenny J D,et al. Optogenetic activation of cholinergic neurons in the PPT or LDT induces REM sleep[J]. Proc Natl Acad Sci USA, 2015, 112(2): 584-589.
[10]Wu M C, Chu L A, Hsiao P Y,et al. Optogenetic control of selective neural activity in multiple freely moving Drosophila adults[J]. Proc Natl Acad Sci U S A, 2014, 111(14): 5367-5372.
[11]Goshen I. The optogenetic revolution in memory research[J]. Trends Neurosci, 2014, 37(9): 511-522.
[12]Iyer S M, Delp S L. Neuroscience. Optogenetic regeneration[J]. Science, 2014, 344(6179): 44-45.
[13]Yizhar O, Fenno L E, Davidson T J,et al. Optogenetics in neural systems[J]. Neuron, 2011, 71(1): 9-34.
[14]Creed M, Pascoli V J, Luscher C. Addiction therapy. Refining deep brain stimulation to emulate optogenetic treatment of synaptic pathology[J]. Science, 2015, 347(6222): 659-664.
[15]Stefanik M T, Kupchik Y M, Brown R M,et al. Optogenetic evidence that pallidal projections, not nigral projections, from the nucleus accumbens core are necessary for reinstating cocaine seeking[J]. J Neurosci, 2013, 33(34): 13654-13662.

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更新日期/Last Update: 2015-07-07