Figure 2. The anxiolytic agonist SNC80 activates ERK1/2 in vitro and in the limbic and striatal regions of the brain.

(A) Time course analysis of the phosphorylation of ERK1/2, p38, and JNK in CHO cells stably expressing δOR and β-arrestin 2 and stimulated with 10 μM SNC80. Data are from N = 3 experiments; a representative blot is shown, right. (B) Time course analysis of the phosphorylation of ERK1/2 in NG108-15 cells that endogenously express δOR and were stimulated with 10 μM SNC80. Data are from N = 3 experiments; a representative blot is shown, right. (C) Immunofluorescence staining for ERK (green), phosphorylated ERK (red), and nuclear DNA marker DAPI (blue) in limbic and striatal regions of WT mouse brain tissues that were perfused 10 min after i.p. administration with saline or SNC80 (20 mg/kg). 4x-Magnification images (left; scale bars, 1000 μm) were used to stitch together the whole brain-slice images shown. Enlarged images (right; scale bars, 100 μm) are 20x magnification that correspond to the regions marked (white boxes) in the 4x images. (D) Representative images of brain sections with micropunctures of five specified brain regions for the Western blot analysis. (E to I) Western blot analysis of the activation of ERK1/2 at 10 and 30 min after systemic administration of saline (Con) or SNC80 in five brain regions from WT mice (N = 3 to 13 mice; see table S2). Representative images below the respective bar graph. See table S2 for one-way and two-way ANOVA and post-hoc multiple comparisons.