Fig. 2.

The overexpression of GSK-3β alters the establishment and maturation of inhibitory synaptic contacts made onto newborn dentate granule cells (DGCs). A, B Density (number/µm) of Gephyrin⁺ clusters in 1st (A) and higher branching (B) order dendrites of newborn dentate granule cells of distinct ages (1, 2, 3, 4, and 8 weeks post-infection) in wild-type (WT) and GSK-3β-overexpressing (OE) mice. C Representative image of an 8-week-old newborn dentate granule cell transduced with a Gephyrin:GFP-encoding retrovirus in a GSK-3β-OE mouse. D Colocalization between Bassoon⁺ presynaptic area and Gephyrin⁺ clusters in 4-week-old newborn dentate granule cells of WT and GSK-3β-OE mice. E Percentage of GSK-3β-OE 1-week-old newborn dentate granule cells surrounded by Parvalbumin (PV)⁺ interneurons. F Representative image of a GSK-3β-OE 8-week-old newborn dentate granule cell transduced with a Gephyrin:GFP-encoding retrovirus, and high-power magnification of axonal Gephyrin⁺ clusters surrounded by PV⁺ terminals. G PV⁺ volume surrounding Gephyrin⁺ clusters in the soma of newborn dentate granule cells of distinct ages (1, 2, 3, 4 and 8 weeks post-infection) in WT and GSK-3β-OE mice. H, I Lectin (H) and Aggrecan (I) fluorescence intensity in perineuronal nets (PNNs) that surround PV⁺ interneurons in the dentate gyrus. J, K Representative images of Lectin, Aggrecan, and PV staining in the dentate gyrus of WT (J) and GSK-3β-OE (K) mice. In (C, F, J, and K), Z-projection images are shown. In A, B and G, a two-way ANOVA, followed by a Tukey post hoc test, was used. In (D, H), a Mann–Whitney U test was applied. A Chi-square test was used to analyze the data shown in (E). In (I), a Student t-test was applied. In A, a minimum of 30 dendritic segments of 1st branching order per cell age, obtained from 4–5 animals per genotype, were analyzed. In (B), a minimum of 30 dendritic segments of each order (namely 1st, 2nd, 3rd, 4th, and 5th) per cell age, obtained from 4–5 animals per genotype, were analyzed. In D and E, a minimum of 50 newborn dentate granule cells, obtained from 4–5 animals per genotype, were analyzed. In G, at least 30 somatic Gephyrin⁺ clusters per cell age, obtained from 4–5 animals per genotype, were analyzed. In (H, I), 40–50 stacks of dentate gyrus images, obtained from 8–10 mice per genotype, were analyzed. Graphs represent mean values ± SEM. ML: Molecular layer. GCL: Granule cell layer. H: Hilus. White scale bar: 25 μm. Orange scale bar: 10 μm. Magenta scale bar: 5 µm. Red scale bar: 40 μm. Light blue triangles: Gephyrin⁺ clusters. Orange triangles: PV⁺ interneurons. ⁺ 0.09 > p ≥ 0.05; * 0.05 > p ≥ 0.01; ** 0.01 > p ≥ 0.001; and *** 0.001 > p ≥ 0.0001