Inhibition of IKKβ Reduces Ethanol Consumption in C57BL/6J Mice

Effect of IKKβ inhibitors on ethanol (EtOH) intake and preference after 3 h in a limited access two-bottle choice drinking-in-the-dark test in C57BL/6J mice. A–C, 50 mg/kg TPCA-1 vs saline treated (n = 6 per group). D–F, 100 mg/kg sulfasalazine vs saline treated (n = 8 per group). A, D, 15% ethanol consumption (g/kg/3 h). B, E, Preference for ethanol. C, F, Total fluid intake (g/kg/3 h). Day 2 in each panel shows the averages of 2 d of saline injections for each group ± SEM. Remaining time points are the 2 d drinking averages in the presence of saline or drug ± SEM. Significant main effect of drug treatment is shown by the p value (two-way ANOVA with repeated measures). Significant post hoc effect of each drug compared with the corresponding saline group is indicated by the symbols above each time point (Bonferroni test for multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001).

IKKβ protein knockdown (3 and 8 weeks post-injection) in NAc of Ikkb^F/F mice. A fluorescent light microscope image of a representative stain from the 3 week post-injection time point in NAc is shown. A, Anti-IKKβ fluorescently labeled antibody. B, Anti-EGFP fluorescently labeled antibody. C, Overlay of A and B (“IKKβ–” represents transduced cells without IKKβ, and “IKKβ+” represents transduced cells with IKKβ). D, Knockdown of IKKβ (LV-EGFP-Cre) measured by IKKβ-positive cells colocalized with EGFP-positive cells relative to their time-matched control (LV-EGFP-Empty). The mean ± SEM of eight fields of view (20×) per mouse for four mice are shown (n = 4 for each group: 3 weeks after LV-EGFP-Cre, 3 weeks after LV-EGFP-Empty, 8 weeks after LV-EGFP-Cre, 8 weeks after LV-EGFP-Empty). Student’s t test: **p < 0.01, ***p < 0.001.

Effect of IKKβ knockdown in NAc on ethanol (EtOH) intake and preference during the 24 h two-bottle choice test in Ikkb^F/F mice. A, Ethanol consumption (g/kg/24 h). B, Preference for ethanol. C, Total fluid intake (g/kg/24 h). Each point is the average of 2 d of drinking ± SEM. Significant main effect of treatment is shown by the p value (two-way ANOVA with repeated measures). Significant post hoc effect of LV-EGFP-Cre compared with LV-EGFP-Empty treatment is indicated by symbols above each time point (Bonferroni test for multiple comparisons *p < 0.05, **p < 0.01). n = 32 animals injected with LV-Cre-EGFP; n = 20 injected with LV-Cre-Empty.

Effect of IKKβ knockdown in CeA on ethanol (EtOH) intake and preference during the 24 h two-bottle choice test in Ikkb^F/F mice. A, Ethanol consumption (g/kg/24 h). B, Preference for ethanol. C, Total fluid intake (g/kg/24 h). Significant main effect of treatment is shown by the p value (two-way ANOVA with repeated measures). Significant post hoc effect of LV-EGFP-Cre compared with LV-EGFP-Empty treatment is indicated by *p < 0.05 (Bonferroni test for multiple comparisons). n = 20 injected with LV-EGFP-Cre; n = 10 injected with LV-EGFP-Empty.

Lentiviral-mediated knockdown of IKKβ in the NAc and CeA had no effect on saccharin preference or total fluid intake in the 24 h two-bottle choice test in Ikkb^F/F mice. A, B, The effect of IKKβ knockdown in NAc (n = 32, LV-EGFP-Cre; n = 20, LV-EGFP-Empty) is shown in A (preference for saccharin) and B (total fluid intake (g/kg/24 h). C, D, The effect of IKKβ knockdown in CeA (n = 20, LV-EGFP-Cre; n = 10, LV-EGFP-Empty) is shown in C (preference for saccharin) and D (total fluid intake (g/kg/24 h). Each point is the average of 2 d of drinking ± SEM.

Injection target verification of lentiviral-mediated IKKβ knockdown in the NAc and CeA. A, C, Composite microscope images of a coronal section in the NAc (A) or CeA (C) of a representative lentiviral injection using fluorescent microscopy (on left) to show EGFP marker signal (green) and bright-field (on right) to demonstrate neuroanatomy. B, D, Coronal brain atlas figures of the injection sites with blue circles indicating the NAc (B) or CeA (D), and the green ovals illustrating the typical lentiviral injection location and spread.

IKKβ protein levels and mRNA expression of IKKβ, TNF-α, and IL-6 at the injection site upon completion of behavioral studies. A, IKKβ protein levels in NAc and CeA (A; n = 5 per group: NAc LV-EGFP-Cre, NAc LV-EGFP-Empty, CeA LV-EGFP-Cre, and CeA LV-EGFP-Empty). B–D, mRNA levels of IKKβ (B), TNF-α (C), and IL-6 (D) in the NAc (n = 10, LV-EGFP-Cre; n = 5, LV-EGFP-Empty) and CeA (n = 5, LV-EGFP-Cre; n = 5, LV-EGFP-Empty). Values are shown relative to LV-EGFP-Empty-treated mice. IKKβ protein levels were analyzed using immunohistochemistry. IKKβ mRNA levels at the target site in the NAc and CeA were assessed by quantitative RT-PCR and normalized relative to GADPH. **p < 0.01, ***p < 0.001 determined by Student’s t test. All data are shown as the mean ± SEM.

Cell type-specific localization of IKKβ in the NAc and CeA. A–I, Representative fluorescent light microscope images illustrating cell type-specific antibodies in the first columns [anti-NeuN for neurons (A); anti-GFAP for astrocytes (D); anti-IBA1 for microglia (G)], anti-IKKβ stains in the second columns (B, E, H), and overlay of the first two in the third columns (C, F, I). Arrows illustrate cells showing colocalization of anti-IKKβ and cell type-specific stains.