G1 stimulates mTOR-dependent Arc synthesis in CA3 through BDNF release. Hippocampal slices were pretreated with DMSO/IgG-Fc (Cnt), TrkB-Fc, ANA-12, or rapamycin, and then incubated with G1 or E2 for an additional 60 min or the indicated times. (A) Representative images of Arc immunostaining in CA1 and CA3 under various experimental conditions. Bar, 100 µm. (B) Quantification of density of Arc-positive punctas in CA1 stratum radiatum (CA1sr) and CA3sr. *, P < 0.05; ***, P < 0.001 versus control (n = 3–16, one-way ANOVA). (C) High-magnification images of CA3 from slices double-stained for Arc (red) and PSD95 (green) along with DAPI (blue). Bar, 20 µm. (D and E) Quantification of the density of Arc (D)- and PSD95 (E)-positive punctas in CA3sr. ***, P < 0.001 versus control; #, P < 0.05; ###, P < 0.001 versus G1 (n = 6–29, one-way ANOVA). (F) Percentage of Arc-positive punctas that colocalized with PSD95 (M1 coefficient, n = 11–12). Error bars indicate means ± SEM.
