Figure 4.

Fosb-dependent expression of C5ar1 and C5ar2 in mouse hippocampus. A. Western blotting of Fosb gene products. FOSB, 43 kDa; ΔFOSB, 34-37 kDa; vFOSB, 31 kDa (see Fig. 7A); Δ2ΔFOSB, 25 kDa; GAPDH protein was detected as an internal control. B & C. Expression levels of C5ar1 (B) and C5ar2 (C) mRNAs in wild-type (WT) and Fosb-null (Null) hippocampus, 24 h after saline, or kainate (KA) injection. Relative expression levels were estimated using the expression level in wild-type hippocampus treated with saline as a control. * p = 0.0059, ** p = 0.0089, # p = 0.0128, ## p < 0.0001 (Student’s t-test). Error bars show mean ± SEM. To quantify the expression level of each mRNA, total RNA was reverse-transcribed to cDNA and used for qPCR analysis using appropriate primer sets (Table 1). D. Inducible expression of C5aR1 in hippocampal CA3 region after kainate administration. Coronal sections prepared from wild-type (WT) and Fosb-null (Null) mouse brains were subjected to laser scanning confocal immunofluorescence microscopy with anti-C5aR1 and anti-Iba-1 antibodies. Nuclei were counter-stained with DAPI. Top panels: Merged images of samples 24 h after saline injection. Scale bar = 100 μm. Middle panels: Merged images of samples 24 h after kainate injection. Scale bar = 100 μm. Bottom panels: High magnification images of samples 24 h after kainate injection. Blue, DAPI; green, C5aR1; red, Iba-1. Scale bar = 50 μm. E. Seizure responses after kainate administration. Fosb-null mice exhibited significantly reduced seizure responses to kainate-induced excitotoxicity compared with wild-type mice (Generalized Linear Model, Difference; p < 0.0001, χ²=821.7, df = 39, Genotype; p < 0.0001, time; p < 0.0001, Interaction; p < 0.0001, WT N = 28, Fosb-null N = 10). Error bars show mean ± SEM. F. Survival rate after kainate administration. No Fosb-null mice died within 90 min after kainate administration (Kaplan-Meier method and log-rank test, p = 0.012, χ² = 6.14, df = 1; WT, N = 32; Fosb-null, N = 12).