Loss of neuronal Rac1 alters Gadd45b, BDNF, and TrkB expression. A, Isolation of NPCs and mature neurons from the hippocampus of adult mice. Microdissected DGs were collected form naive mice and mice trained in the DMP task and rapidly dissociated into live cell suspensions that were enriched by Percoll density centrifugation for immature neural progenitors or mature neurons and glia. B, C, Relative BDNF, Gadd45b, and TrkB mRNA levels in enriched cell fractions of DG. Gadd45b expression was significantly upregulated during 7 d of learning in the mature neuron fraction (B) of Rac1 control learners (n > 5 for all groups of mice; ANOVA with post hoc test, **p < 0.01, *p < 0.05). In the NPC enriched fraction (C), TrkB and BDNF expression was increased in Rac1 control (ctrl) learners, but not in Rac1 mutant learners (control naive, n = 8, control learners, n = 5; ANOVA with post hoc test, ^#p < 0.1 trend, **p < 0.01; control learners vs mutant learners, n = 4; ANOVA with post hoc test, ^#p < 0.1 trend, *p < 0.05). D, Gadd45b expression in NeuN⁺ mature dentate granule cells of the adult mouse hippocampus. Shown are sample confocal images of Gadd45b (red), NeuN (green), and Dcx (blue) immunostaining in Rac1 control (left) and Rac1 mutant mice (right) after 7 d of training in the DMP task. Scale bars, 50 μm. E, Quantification of the percentage of Gadd45b-positive pixels within the NeuN⁺ cells in the granular cell layer. Learning significantly increased immunoreactivity for Gadd45b in mature neurons in control learner (control naive vs learners, ANOVA with post hoc test, ***p < 0.001), and to a lesser extent in Rac1 mutant learners (mutant naive vs mutant learners, ANOVA with post hoc test, **p < 0.01). Comparison between learners revealed significant differences between genotypes (control learner vs mutant learner, ANOVA with post hoc test, *p < 0.05). F, Bisulfite sequencing analysis of the methylation status within the BDNF IX regulatory promoter region as described previously (Ma et al., 2009a). No changes in DNA methylation frequency in seven individual CpG sites of BDNF IX were observed in Rac1 control and mutant mice after 7 d of training in the DMP task. G, Immunostaining of phosphorylated TrkB (p-TrkB, red) and the neuronal precursor marker PSA-NCAM (green) in the DG of Rac1 control learner (left) and Rac1 mutant learner (right). Scale bars: 50 μm. H, I, Percentage of p-TrkB⁺ cells among all PSA-NCAM⁺ NPCs (H) and the NeuN⁺ mature neurons (I) within the DG of naive, DMP-trained mice (learners) and runner-learners (Fig. 7A). Learning significantly increased the number of p-TrkB⁺ NPCs (H) in Rac1 control and mutant learners (naive vs learners for both genotypes, ANOVA with post hoc test, **p < 0.01, n = 3 for all groups). Comparison between learners revealed significantly lower p-TrkB⁺ cells numbers in Rac1 mutant learners in NPCs (H) and in NeuN⁺ mature neurons (I) (control learners vs mutant learners, ANOVA with post hoc test, *p < 0.05, n = 3 for all groups). Running before learning had no significant effect on p-TrkB levels and no changes were observed between Rac1 control and mutant mice within the runner-learner groups (control runner-learners vs mutant runner-learners, n = 3 for both groups; ANOVA, p > 0.2).