Neurofibromin Modulates Adult Hippocampal Neurogenesis and Behavioral Effects of Antidepressants

Ablation of Nf1 in adult NPCs in vivo. A, PCR demonstrating Nestin-CreER^T2-mediated recombination of the flox Nf1 alleles in DG-derived adult NPCs. Mice were given tamoxifen at 2 months of age. NPCs were derived from DG at 3 months. B, Diagram of tamoxifen induction regimen. Mice were induced with tamoxifen at 2 months of age and killed at 3 months or 8 months of age. C, R26 reporter revealed enhanced long-term neurogenesis in the DG of NF1^Nestin mice. Mice were given tamoxifen at 2 months and examined at 3 or 8 months of age. Images show representative X-Gal staining. Bottom show higher-magnification view of the boxed areas. TAM, tamoxifen. Scale bar, 100 μm. D, Coimmunostaining for β-Gal (green), Dcx (red), and NeuN (blue) at 3 months (1 month post-tamoxifen). Note that most recombined cells (β-Gal⁺) did not express NeuN, but colocalized with the immature neuronal marker (Dcx). Scale bar, 50 μm. E, In the DG of 8-month-old mice, adult-generated cells (β-Gal⁺) predominantly assumed neuronal fate (NeuN⁺). Images show representative double immunostaining for β-Gal (green) and NeuN (red, top) or GFAP (red, bottom). Scale bars, 100 μm. F, Quantification of β-Gal and NeuN double immunostaining revealed significant increase in the proportion of new-born neurons (β-Gal⁺NeuN⁺) in all NeuN⁺ granule neurons in NF1^Nestin mice. ANOVA (GLM) revealed significant effects of time (F_(2,25) = 101.6, p < 0.0001), genotype (F_(1,25) = 21.49, p < 0.0001), and the interaction of the two (F_(1,25) = 4.621, p < 0.0196). N = 4–6 for each genotype and age. G, H, Adult-born neurons (β-Gal⁺) in the DG of NF1^Nestin mice exhibited increased migration into the middle and outer portions of the granular layers. GL, granular layer. N = 6 for each. Results are mean + SEM. *p < 0.05, **p < 0.01.

Nf1 ablation leads to ERK activation in NPCs and immature neurons. A, At 3 months of age, Nf1 ablation increased the number of phospho-ERK-immunopositive cell bodies in the SGZ. Bottom shows higher-magnification view of the boxed areas. Insets in the top show phospho-ERK-immunopositive mossy fiber pathway. Arrows indicate immunoreactivity. Scale bars, 100 μm. B–E, Coimmunostaining demonstrated that phospho-ERK-positive cells colocalized with Dcx (B), Nestin (D), and Sox2 (E), but not NeuN (C). P, Phosphorylated. Arrows indicate cells with lack of colocalization. Arrowheads indicate colocalization. Scale bars, 30 μm (left) and 5 μm (right) for Nestin (D), 20 μm for all else. F, At 8 months of age, NF1^Nestin mice had dramatically more phospho-ERK-positive cell bodies in the SGZ. Scale bars, 100 μm. G, H, Long-term ablation of Nf1 in adult NPCs led to ERK activation in Dcx⁺ new neurons (G), but not NeuN⁺ mature neurons (H). P, Phosphorylated. Scale bars: G, 15 μm; H, 20 μm. I, Nf1 ablation in adult NPCs did not lead to activation of AKT in vivo. Enhanced phospho-AKT immunoreactivity was observed in the DG granular and molecular layers of Pten conditional knock-out (cKO) mice, but not in control or NF1^Nestin mice. Scale bar, 100 μm.

Nf1 ablation in adult NPCs enables rapid behavioral and neurogenic response to subchronic antidepressants. A, In the NSFT, chronic fluoxetine or imipramine shortened the latency to feed in control mice, indicating lowered anxiety. DTA^Nestin mice showed no behavioral changes after treatments. N = 9–10 for each. ANOVA (GLM) found significant effects of genotype (F_(1,52) = 14.07, p = 0.0004), and the interaction of genotype and treatment (F_(2,52) = 3.214, p = 0.0483). Sal, Saline; Flx, fluoxetine; Imi, imipramine. B, The total length of immobility in the TST reflects the level of depression-like behavior and could be reduced by chronic fluoxetine or imipramine in control mice, but not in DTA^Nestin mice. N = 9–10 for each. ANOVA (GLM) found significant effects of antidepressant treatment (F_(2,52) = 4.603, p = 0.0144), genotype (F_(1,52) = 28.81, p < 0.0001), and the interaction of the two (F_(2,52) = 6.103, p = 0.0042). C, Diagram of subchronic antidepressant treatment regimen. Mice were treated with vehicle or tamoxifen at 2 months of age and exposed to saline or antidepressants at 3 months of age for 7 d. TAM, Tamoxifen; Sal, saline; AD, antidepressant. D, E, Subchronic antidepressants reduced the latency to feed in the NSFT (D) and the length of immobility in the TST (E) in the NF1^Nestin mice, while control mice did not display significant behavioral responses. NSFT: F_(2,51) = 3.715, p = 0.0312 for treatment; F_(1,51) = 5.896, p = 0.0187 for genotype. TST: F_(1,51) = 12.76, p = 0.0008 for genotype. F, G, Subchronic fluoxetine or imipramine increased the number of Ki67-positive (green) proliferating cells in the DG of NF1^Nestin but not control mice. ANOVA (GLM) revealed significant effects of treatment (F_(2,52) = 6.279, p = 0.0036), genotype (F_(1,52) = 29.74, p < 0.0001), and the interaction of the two (F_(2,52) = 4.730, p = 0.0130). Scale bar, 100 μm. H, I, The numbers of Dcx-positive (red) newborn neurons in the DG of NF1^Nestin but not control mice increased after subchronic fluoxetine or imipramine treatment. Significant effect of genotype (F_(1,52) = 22.69, p < 0.0001) was found. Scale bar, 100 μm. Results are mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Subchronic fluoxetine accelerates neuronal maturation in NF1^Nestin mice. A, Immunostaining for Dcx (red) revealed the dendritic morphology of immature neurons. B, Subchronic fluoxetine or imipramine increased the percentage of Dcx-positive neurons that harbored tertiary dendrites. ANOVA revealed significant effect of treatment (F_(1,16) = 5.693, p = 0.0297). Sal, Saline; Flx, fluoxetine; Imi, imipramine. C, Tracing of representative Dcx-positive cells in the DG of saline- or fluoxetine-treated control and NF1^Nestin mice. D, E, Subchronic fluoxetine or imipramine increased the number of dendritic intersections (D) and dendritic length (E) of Dcx-positive cells in NF1^Nestin but not control mice. ANOVA post hoc test of antidepressant-to-saline comparisons found significant difference in NF1^Nestin mice. F–H, BrdU pulse-chase was used to label cells born 7 d before saline or fluoxetine treatment (F). Coimmunostaining for NeuN (red) and BrdU (green) was used to identify 14-d-old mature neurons (G) born 7 d before the onset of fluoxetine or saline treatment. Subchronic fluoxetine increased the percentage of all BrdU⁺ cells that expressed NeuN in NF1^Nestin but not control mice (H). ANOVA revealed significant effect of treatment (F_(1,16) = 9.394, p = 0.0074), and genotype (F_(1,16) = 7.821, p = 0.0129). Results are mean + SEM. *p < 0.05, **p < 0.01.

Eight-month-old NF1^Nestin mice show enhanced basal proliferation and neurogenesis. A, B, Immunostaining for Ki67 (green) and Dcx (red) revealed a higher level of proliferation and neurogenesis in the DG of NF1^Nestin mice compared with control mice. NeuN, blue. Scale bar, 50 μm. C, D, Quantitative analyses of the dendritic morphology of Dcx+ immature neurons showed that NF1^Nestin mice had an increased number of dendritic intersections (C) and longer length (D) compared with controls. E, Diagram of BrdU pulse-chase analysis. Mice were induced with tamoxifen at 2 months of age and injected with BrdU at 8 months of age to label cells that were born at 8 months and were present 28 days later. Results are mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001.