Figure 4
From: Chondroitin Sulfate Is Required for Onset and Offset of Critical Period Plasticity in Visual Cortex
Inhibitory maturation requires aggrecan-dependent accumulation of Otx2 within PV-cells. (A) Co-immunoprecipitation (IP) of aggrecan and Otx2. Homogenates of V1 are treated with (+) or without (−) ChABC digestion (Ch’ase). Lanes from the left, input (1/40), supernatant (sup), IP with anti-aggrecan, anti-Otx2 antibody, normal IgG, soluble recombinant Otx2. The full-length blots are shown in a Supplementary Information. (B) Co-localization of Otx2 (red) with aggrecan (green) is observed in the binocular zone of the adult V1 with immunostaining using nonpermeabilizing conditions for labeling of extracellularly localized Otx223,24 (top, WT; bottom, KO; right, merged images with DAPI). The scale bars represent 10 μm. (C) Reduction in Otx2 protein in juvenile (P28–30) T1 KO mice (top, specific bands for Otx2; bottom, relative volume of protein, six mice, p = 0.02, t-test). The full-length blots are shown in a Supplementary Information. (D) Decreased number of positive cells for Otx2 and PV in juvenile (P28–30) T1 KO mice, but no difference in the number of Calretinin (CR)-positive cells in supragranular layers of the binocular zone (600 × 350 μm; 4–6 mice on P28–30, WT 34.6 ± 1.1 versus KO 18.1 ± 2.9, p = 0.0007 for Otx2, WT 32.6 ± 1.46 versus KO 18.67 ± 2.29, p < 0.0001 for PV, WT 23.9 ± 1.6 versus KO 24.7 ± 1.5, p = 0.72 for CR, t-test). (E) Quantitative analysis of intensity within somata for Otx2, PV, and CR (600 × 350 μm; p < 0.0001 for Otx2 and PV, p > 0.05 for CR, t-test). Reduced intensity of GAD65 in supragranular layers of the binocular zone (400 × 350 μm; 3–4 mice, p = 0.038, t-test). (F–M) Reduction in Otx2 (F,G), PV (H,I), and GAD65 (L,M) staining in supragranular layers of the binocular zone in T1 KO (bottom) compared to WT (top) mice at P28–30. Notably, CR staining is not altered (J,K). The scale bars represent 100 μm (F–K) and 20 μm (L,M).
