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. 2015 May 15;8(5):6725-34.
eCollection 2015.

Silencing the NR2B gene in rat ACC neurons by lentivirus-delivered shRNA alleviates pain-related aversion

Shou-Gang Guo  1 , Xiu-Hua Lv  2 , Shan-Hui Guan  2 , Hui-Lu Li  3 , Yong Qiao  4 , Hao Feng  4 , Lin Cong  1 , Gong-Ming Wang  3
Affiliations

Silencing the NR2B gene in rat ACC neurons by lentivirus-delivered shRNA alleviates pain-related aversion

Shou-Gang Guo et al. Int J Clin Exp Med. .

Abstract

The N-methyl-D-aspartate (NMDA) receptor NR2B subunit on neurons in the anterior cingulate cortex (ACC) is implicated in the affective response to noxious stimuli. Selectively silencing this NR2B subunit in ACC neurons could therefore alleviate pain-related aversion. However, to date, there is no optimal approach to selectively silence the NR2B gene in ACC neurons. In the present study, we constructed lentiviral vectors and delivered shRNA (NR2B-RNAi-LV) to effectively silence the NR2B gene in ACC neurons. The use of lentivirus resulted in 95% transfection efficiency and 83% silencing of the NR2B gene in ACC neurons. Electrophysiological experiments showed that the total INMDA was similarly reduced by 48% in lentivirus-transfected ACC neurons. The biochemical and functional data demonstrated that lentiviral shRNA delivery produced a high transfection and silencing efficiency in the ACC neurons. SNI rats weighting 220-250 g were randomly divided into three groups: normal saline group (NS), lenti-siRNA/NC (LV-NC) group, and lenti-siRNA/NR2B (LV-NR2B) group, and conditioned place avoidance was conducted. The results indicated that NR2B-RNAi-LV decreased greatly the conditioning scores of F-CPA while NC-GFP-LV has no effects. NR2B mRNA expression in the NR2B-RNAi-LV group was significantly lower than that in the control group and NC-GFP-LV group. This novel approach of silencing the NR2B gene in ACC neuron could potentially be used to alleviate pain-related aversion.

Keywords: ACC; NR2B; RNA interference; gene silencing.

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Figures

Figure 1
Figure 1
Culture and identification of ACC neurons. A. ACC neurons on day 7 of culture in vitro (7 DIV). The clear pale halo was around each neurons. Neuronal processes mutually interconnected into a network. B. Identification of neurons by immunofluorescence staining. More than 95% of cultured neurons were MAP2 positive at 7 DIV. (scale bar: 50 μm).
Figure 2
Figure 2
Design and construction of pFU-GW-iRNA-NR2B. A. The identification of plasmid pFU-GW-iRNA-NR2B by PCR analysis. Lane 1: negative control (ddH2O); Lane 2: negative control (pFU-GW-iRNA); Lane 3: 250-bp DNA ladder; Lane 4: PCR analysis of the recombinant plasmid pFU-GW-iRNA-NR2B. B. Inhibitory effects of the plasmid pFU-GW-iRNA-NR2B on NR2B protein expression were observed by Western blotting. GAPDH protein was used as a control. Lane 1: 293T cells; Lane 2: plasmid expressing NR2B protein; Lane 3: plasmid expressing NR2B protein and the plasmid pFU-GW-iRNA; Lane 4: plasmid expressing NR2B protein and the plasmid pFU-GW-iRNA-NR2B.
Figure 3
Figure 3
Determination of NR2B-RNAi-LV vector transduction efficiency in ACC neurons. GFP expression was observed under a light microscope (A) or fluorescence microscope (B). The increase in GFP expression in ACC neurons peaked when the MOI was 3 (scale bar: 50 μm).
Figure 4
Figure 4
NR2B-RNAi-LV transfection silenced the expression of NR2B mRNA in ACC neurons. These data were analyzed by the relative gene expression (2-ΔΔCt) method. The figures show that NR2B-RNAi-LV decreased significantly NR2B mRNA expression in neurons (# vs. control group, P<0.05) or negative transfection (* vs. NC-GFP-LV group, P<0.05).
Figure 5
Figure 5
The levels of NR2B protein in NR2B-RNAi-LV-transduced cells was significantly lower than that in NC-GFP-LV-transduced or Control cells on 72 h after treatment. CON = ACC neurons uninfected with lentivirus; NC-GFP-LV = ACC neurons infected with control lentivirus; NR2B-RNAi-LV = ACC neurons infected with lentivirus carrying NR2B shRNA. A and B show the protein level measured by Western blotting and band densitometry, respectively. # vs. control group, P<0.05, * vs. NC-GFP-LV group, P<0.05.
Figure 6
Figure 6
Functional evidence of silencing of NR2B gene expression in ACC neurons provided by whole cell patch-clamp recordings. A. Representative traces of the peak NMDA current elicited in control cells by 10 μmol/L NMDA and after silencing of the NR2B gene. The peak NMDA current was significantly decreased in silenced ACC neurons. B. The averaged data from patch-clamp experiments. Knockdown of NR2B protein expression by NR2B-RNAi-LV vectors reduced NMDA responses to 52% of that in control neurons. # vs. control group, P<0.05, * vs. NC-GFP-LV group, P<0.05.
Figure 7
Figure 7
Comparison of the time spent in the formalin-paired compartment among three groups. For NC-GFP-LV group and Control group rats, the time spent in the formalin-paired compartment was 868.30±13.2 s preconditioning vs. 115.83±21.3 s post-conditioning (independent-sample t test, t=7.804, P<0.05, n=10), 967.05±12.2 s preconditioning vs. 135.50±21.3 s post-conditioning (independent-sample t test, t=17.844, P<0.05, n=10) was 868.30±13.2 s preconditioning vs. 115.83±21.3 s post-conditioning (independent-sample t test, t=7.804, P<0.05, n=10), 967.05±12.2 s preconditioning vs. 135.50±21.3 s post-conditioning (Independent-sample T test, t=17.844, P<0.05, n=10). *P<0.05 compared with pre-conditioning day.
Figure 8
Figure 8
Comparison of the conditioning score among three groups. The conditioning score was 752.47±24.6 s and 831.55±12.3. The times spent in the formalin-paired compartment were 1060.78±19.8 s for preconditioning, 1035.74±29.7 s for post-conditioning in NR2B-RNAi-LV group. The conditioning scores were 124.45±23.3 s. The conditioning scores of rats in the Control groups and NC-GFP-LV groups were similar (independent-sample t test, t=0.738, P>0.05, n=10), but reduced significantly in NR2B-RNAi-LV groups (independent-sample t test, t=7.815, P<0.05, n=10; t=9.477, P<0.05, n=10). *P<0.05 compared with CON group, #P<0.05 compared with NC-GFP-LV group.
Figure 9
Figure 9
ACC NR2B mRNA expression among three groups. NR2B mRNA expression in the NR2B-RNAi-LV groups was significantly lower than levels in the control groups and NC-GFP-LV groups (independent-sample t test, t=24.149, P<0.05; independent-sample t test, t=17.976, P<0.05). *P<0.05 compared with CON group, #P<0.05 compared with NC-GFP-LV group.
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