We previously demonstrated that the tamoxifen-inducible form of Cre recombinase, CreER T2
, when expressed under neural-specific regulatory elements of the Nestin
gene, enables temporal control of recombination specifically in adult NPCs (Chen et al., 2009
). By crossing the Nestin-CreERT2
mice to the R26
reporter mice (Soriano, 1999
), and inducing Cre-mediated recombination with tamoxifen, hippocampal NPCs and their progeny could be labeled and lineage traced (; R26 Nestin
mice, tamoxifen induction at 2 months of age). It is well documented that chronic antidepressants increase hippocampal proliferation, differentiation and maturation of newly born neurons (Li et al., 2008
; Wang et al., 2008
). We confirmed that chronic treatment of imipramine (20 μ
g/g), or fluoxetine (18 μ
g/g) significantly increased the number of X-Gal-positive cells in the dentate gyrus of R26 Nestin
mice, by 54.8% and 63.0%, respectively. These findings were consistent with prior observations (Encinas et al., 2006
), and demonstrate that the Nestin-CreERT2
transgene can facilitate the monitoring of this population of NPCs.
Figure 1 Nestin-CreERT2 labels adult hippocampal NPCs. A, X-Gal staining on DG sections of control and R26Nestin mice at 2.5 months. Vehicle or tamoxifen was given at 2 months. Scale bar, 100 μm. B, Representative confocal images of the DG immunostained (more ...)
To effectively study neurogenesis after Nf1
deletion in SGZ progenitors, high-efficiency Cre-mediated recombination must be achieved. Therefore, to determine the efficiency of the Nestin-CreERT2
transgene in mediating recombination, we used a Cre-dependent toxin-based cell-ablation approach, that allows identification of any NPCs in the SGZ that evade recombination because they would survive. A mouse line harboring a silent allele of the diphtheria toxin subunit A (DTA) gene (Brockschnieder et al., 2006
), that becomes active upon Cre-mediated recombination, was crossed with Nestin-CreERT2
mice. Without tamoxifen, the DTAflox;Nestin-CreERT2
mice were indistinguishable from their littermate controls in brain weight and hippocampal structural morphology (data not shown). When treated with vehicle (control group) or tamoxifen (DTA Nestin
group) at the age of 2 months and analyzed 2 weeks later, the DTA Nestin
mice had almost no detectable Nestin-expressing cells in the SGZ (<5% compared with controls, ) or SVZ (data not shown). This was echoed by near complete loss of proliferating cells (Ki67-positive), and immature neurons (Dcx-positive) at 3.5 months of age (); however, existing neurons and glia appeared unaffected by tamoxifen induction (data not shown). These results are consistent with a previous report demonstrating efficient ablation of Dcx-positive cells in the adult dentate gyrus upon tamoxifen induction in mice carrying the Nestin-CreERT2
and NSE-DTA transgenes (Imayoshi et al., 2008
). Our results confirm that the Nestin-CreERT2
line used in the present study is highly efficient in targeting the vast majority of NPCs in the SGZ.
Nf1 deletion increases adult hippocampal neurogenesis
We next targeted Nf1 in adult NPCs using mice harboring two conditional (flox) alleles of Nf1 and the Nestin-CreERT2 transgene, treated with tamoxifen at 2 months of age (NF1 Nestin mice). The NF1 Nestin mice appeared healthy and were grossly indistinguishable from control mice (data not shown). Recombination of the flox alleles in NF1Nestin mice was confirmed in DG-derived NPCs by PCR (). To visualize the extent of Cre-mediated recombination, we incorporated the R26 reporter allele into the NF1 Nestin mice and examined X-Gal-positive NPCs and their progeny (R26;NF1 Nestin mice; ). Compared with age-matched R26 Nestin mice, R26;NF1 Nestin mice displayed similar numbers and distribution of X-Gal-positive cells at 2.5 and 3 months (; data not shown). Additional analysis, including immunostaining for Ki67, Dcx, BrdU, and NeuroD demonstrated a similar number of proliferating cells and newly born neurons in the SGZ (Saline groups; see Fig. 4F–I; data not shown). Thus, 1 month after ablation of Nf1 in adult NPCs, basal hippocampal neurogenesis was not significantly affected.
Figure 2 Ablation of Nf1 in adult NPCs in vivo. A, PCR demonstrating Nestin-CreERT2-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 (more ...)
At 1 month after tamoxifen induction (3 months of age), few β-Gal-positive cells expressed a mature neuronal marker (NeuN), but rather expressed markers of undifferentiated NPCs and immature neurons (). Six months post-tamoxifen induction (8 months of age), the X-Gal-positive cell population expanded from the SGZ into the inner granular layer, indicative of progressive neuronal differentiation (; see for diagram). By comparison, the R26;NF1 Nestin mice had a higher number of X-Gal-positive cells, with more robust migration into the inner and outer portions of the granular layer (). Consistent with the notion that newly generated cells in the adult DG preferentially assume neuronal fate, we found a majority of β-Gal-positive cells coexpressing NeuN but not GFAP (). In concert with the X-Gal staining (), we found more β-Gal-positive neurons in R26;NF1 Nestin mice at 8 and 11 months of age (; data not shown). Quantitative analysis revealed that a higher percentage of β-Gal-positive neurons was found in the middle and outer granular layers in the 8-month-old R26;NF1 Nestin mice (). Further analysis of β-Gal costaining revealed that the proportion of cells expressing NeuN or GFAP among all β-Gal-positive cells was unaffected by genotype (data not shown), suggesting relatively normal lineage commitment of newborn cells from adult NPCs in the absence of Nf1. These results demonstrate that ablation of Nf1 in adult NPCs increases neurogenesis in the DG.
Activation of ERK signaling in Nf1−/− NPCs and immature neurons
As described above, Nf1
loss in adult hippocampal NPCs beginning at 2 months of age resulted in an obvious increase in neurogenesis by 8 months of age (). It is known that the ERK and PI3K signaling cascades can be genetically activated by loss of the Nf1
gene (Klesse and Parada, 1998
). We therefore examined the levels of phosphorylated ERK and AKT in the hippocampus. At 3 months of age, control and NF1 Nestin
mice exhibited phospho-ERK immunostaining in the axons of dentate granule neurons along the mossy fiber pathway (, insets in top panels), and occasionally in pyramidal neurons of the CA1 regions (data not shown). In the SGZ, however, we observed phospho-ERK-positive cell bodies in NF1 Nestin
mice but not in littermate controls (; number of positive cells per section: 0.33 ± 0.16 in control mice; 3.44 ± 1.00 in NF1Nestin
mice). Many of the phospho-ERK-positive SGZ cells colocalized with the immature neuronal marker Dcx (), but not mature neuronal marker NeuN (). In addition, some phospho-ERK-positive cells colabeled with Nestin () or Sox2, markers for adult NPCs (). We note that not all NPCs or immature neurons identified with the cell type-specific markers displayed elevated phospho-ERK. Quantitative analyses revealed that 36.67 ± 4.41% of Dcx-positive cells expressed phospho-ERK, suggesting that ablation of Nf1
led to partial activation of ERK signaling in NF1 Nestin
mice at this relatively early stage (1 month post-tamoxifen).
Figure 3 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 (more ...)
We next examined the status of ERK signaling in 8-month-old NF1Nestin mice. In the SGZ, we detected a significant increase in the number of phospho-ERK-positive cells compared with controls and younger NF1 Nestin counterparts (; number of positive cells per section: 0.26 ± 0.16 in control mice; 30.86 ± 9.82 in NF1 Nestin mice; n = 6 for each; p < 0.05). Similar to 3-month-old NF1 Nestin mice, coimmunostaining revealed that the phospho-ERK-positive cells expressed markers of immature neurons such as doublecortin (), but not markers of mature neurons, such as NeuN (). In addition, a high level of ERK activity was seen in almost all Dcx-positive cells (98.33 ± 1.67%), indicating increased penetrance of ERK activation in 8-month-old NF1 Nestin mice compared with their 3-month-old counterparts. Together, these findings demonstrate that ablation of Nf1 in adult NPCs causes progressive activation of the ERK pathway.
We also examined phospho-AKT but observed no evidence of activity in mice of either genotype or age, although intense staining was seen within the SGZ, granular layer and molecular layer in brain tissues from Pten
conditional knock-out mice, in which activation of the AKT pathway is expected in Pten−/−
cells (Zhou et al., 2009
) (). Thus, Nf1
ablation in adult NPCs leads to cell-autonomous activation of ERK but not AKT.
Loss of Nf1 facilitates behavioral and neurogenic response to antidepressants
Hippocampal neurogenesis has been implicated in the behavioral effects of chronic antidepressants (Santarelli et al., 2003
; Airan et al., 2007
; Li et al., 2008
; Wang et al., 2008
; David et al., 2009
). Consistent with this, the DTANestin
mice, which are incapable of undergoing neurogenesis (), showed muted behavioral response to chronic fluoxetine (18 μ
g/g) or imipramine (20 μ
g/g) in the NSFT and the Tail-Suspension Test (), thus reflecting a lack of change in anxiety- and depression-like behaviors. These data provided genetic confirmation that acute depletion of adult NPCs in mice abrogates behavioral response to chronic antidepressants in the NSFT and TST.
Figure 4 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. DTANestin mice showed (more ...)
We next tested whether acute ablation of Nf1
in adult NPCs would have opposing effects on behavioral response to antidepressant. At 3 months of age, basal level anxiety- and depression-like behaviors as measured by NSFT, TST, and FST were indistinguishable in control and NF1Nestin
mice (Saline groups in ; data not shown). Furthermore, both groups showed significant behavioral improvements in these behavioral tests after chronic fluoxetine or imipramine treatment, compared with saline-treated mice (21 d regimen, data not shown). We next subjected control and NF1 Nestin
mice to a shortened antidepressant treatment paradigm, in which animals were treated with saline, fluoxetine (18 μ
g/g) or imipramine (20 μ
g/g) for 7 d, and tested in the NSFT and TST paradigms on day 8 and 9, respectively (). The NF1Nestin
mice receiving subchronic fluoxetine or imipramine displayed significantly decreased latency to feed in the NSFT compared with those receiving saline (). We found that control mice of all treatment groups performed similarly, confirming previous reports that this duration of antidepressant exposure was not sufficient to produce behavioral change in the NSFT paradigm (Santarelli et al., 2003
; Wang et al., 2008
). Similar findings were obtained with the TST analysis, where NF1Nestin
mice receiving antidepressant for 7 d displayed significantly shorter length of immobility, while the control mice did not respond (). We note that the TST was performed 48 h after the last dose of antidepressant to assess chronic (neurogenesis-dependent) rather than acute (neurogenesis-independent) response.
To examine whether the enhanced behavioral response to antidepressants was coupled with changes in the activity of the DG neurogenic niche, we examined proliferation and new neuron generation. Consistent with previous reports (Malberg et al., 2000
), this subchronic fluoxetine or imipramine treatment did not significantly increase proliferation in control mice (). The NF1Nestin
mice, however, displayed a significant increase in the number of Ki67-positive cells after subchronic antidepressant treatment (). Quantification of phospho-histone H3-positive cells further confirmed this increase in proliferation (data not shown). Furthermore, the NF1Nestin
mice displayed an increased number of Dcx-positive cells, indicating enhanced production of new neurons ().
Complex dendritic morphology in Dcx-positive cells represents greater cellular maturation (Plümpe et al., 2006
; Sahay et al., 2011
), and it has been shown that chronic antidepressants can enhance neurogenesis by stimulating new neuron maturation (Wang et al., 2008
; David et al., 2009
). We analyzed the dendritic morphology of Dcx-positive neurons, and found that subchronic fluoxetine or imipramine increased the number of Dcx-positive cells bearing tertiary dendrites in NF1Nestin
but not control mice, indicating enhanced maturation of the new neuron pool in response to subchronic fluoxetine (). We next evaluated the dendritic morphology of individual Dcx-positive cells with tertiary dendrites, and found that subchronic fluoxetine or imipramine enhanced the complexity of dendritic branching in NF1 Nestin
mice (), while control mice showed no significant changes. Furthermore, analysis of the total length of dendritic branches showed a similarly enhanced response in NF1 Nestin
Figure 5 Subchronic fluoxetine accelerates neuronal maturation in NF1Nestin 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 (more ...)
We next specifically evaluated the effect of subchronic antidepressant on the maturation of already-existing young neurons, by birth-dating cells born 7 d before the onset of saline or fluoxetine treatment with BrdU, and analyzing their maturation state after treatment (). By coimmunostaining for BrdU and NeuN (), we observed that a substantial number of BrdU-positive cells had assumed a mature neuronal pheno-type, and that subchronic fluoxetine treatment increased the percentage of BrdU and NeuN double-positive cells in the DG of NF1Nestin mice (, n = 5 for each genotype and treatment), but not their littermate controls. These results are consistent with the model that fluoxetine can enhance neurogenesis in NF1 Nestin mice by promoting the maturation of already-existing young Nf1−/− neurons. Collectively, these findings demonstrate that ablating Nf1 in adult NPCs enhances sensitivity to subchronic antidepressant-induced production and maturation of new neurons.
Sustained adult NPC activation and antidepressive-like behavior in aged NF1 Nestin mice
To investigate the long-term effects of Nf1 deletion on adult DG NPCs, we examined the level of proliferation, differentiation and neuronal maturation in the SGZ of 8-month-old NF1 Nestin mice in the absence of antidepressant exposure. Immunostaining for Ki67 revealed significantly more dividing cells in the NF1 Nestin mice compared with littermate controls, indicating enhanced basal proliferation (
A; number of positive cells: 1022 ± 69.9 in control mice; 1670 ± 57.6 in NF1Nestin mice; n = 6 for each; p< 0.001). As 3-month-old NF1 Nestin mice showed normal basal proliferation (), additional cohorts of NF1 Nestin mice were analyzed at 2.5 and 5 months of age (2 weeks and 3 months post-tamoxifen, respectively). We found wild-type levels of proliferation in the former, and increased Ki67-positive cells in the latter (; n = 4–5 for each; p < 0.05 at 5 months). Coinciding with the increase in proliferation, we observed significantly more Dcx-positive cells in the SGZ of 8-month-old NF1 Nestin mice, indicating elevated production of immature neurons (; number of positive cells: 3086 ± 356.4 in control mice; 5420 ± 187.5 in NF1 Nestin mice; n = 6 for each; p < 0.001). We next evaluated the dendritic morphology of Dcx-positive immature neurons and found increases in dendritic complexity including a higher percentage of Dcx-positive neurons with tertiary dendrites (11.70 ± 1.76% in control mice; 17.24 ± 0.60% in NF1 Nestin mice; n = 4 for each; p < 0.05), an increased number of dendritic intersections () and longer length () compared with controls. Furthermore, using BrdU pulse-chase, we labeled cells born at 8 months of age (), and found that the DG of NF1Nestin mice harbored significantly more BrdU and NeuN double-positive neurons after 4 weeks (number of BrdU-positive cells: 324.0 ± 19.86 in control mice; 429.6 ± 28.24 in NF1 Nestin mice; p < 0.05; number of BrdU and NeuN double-positive cells: 276.0 ± 17.22 in control mice; 376.8 ± 22.96 in NF1 Nestin mice; p < 0.01; n = 5 for each). Thus, coincident with activation of the ERK signaling pathway, ablation of Nf1 in adult NPCs appears to elicit spontaneous activation of NPC proliferation, increased new neuron production, and enhanced maturation. While these data are intriguing, the mechanism by which this increase in proliferation over time occurs is not completely understood and warrants further investigation.
Figure 6 Eight-month-old NF1Nestin 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 NF1Nestin mice compared with control mice. NeuN, (more ...)
To examine whether genetic activation of adult NPCs produces antidepressive-like behavioral changes, we tested cohorts of 8-month-old mice in the TST paradigm. Compared with littermate control mice, NF1Nestin mice displayed significantly shorter length of immobility suggesting decreased depression-like behavior (160.58 ± 9.81 s for control mice; 128.36 ± 11.91 s for NF1 Nestin mice; p < 0.05; n = 11–12). This was further confirmed in the FST, in which NF1 Nestin mice displayed decreased immobility (106.08 ± 6.65 s for control mice; 77.92 ± 7.96 s for NF1 Nestin mice; p < 0.05; n = 12–13). We used the NSFT to measure anxiety-like behaviors, and found a downward trend in latency to feed in NF1 Nestin mice at 8 months of age (data not shown), and significantly lower latency in a separate cohort of 11-month-old NF1 Nestin mice, suggesting that lack of Nf1 conferred greater resistance to novelty-induced acute anxiety (273.0 ± 16.40 s for control mice; 226.0 ± 12.99 s for NF1 Nestin mice; p < 0.05; n = 10 for each). We further confirmed that at 11 months NF1Nestin mice had higher levels of phospho-ERK expression in the SGZ, and increased NPC proliferation and neurogenesis (data not shown). Together, these findings suggest that enhanced neurogenesis influenced basal anxiety- and depression-like behaviors.
We next assessed whether genetic activation of NPCs can provide antidepressive-like benefits in the CMS paradigm, an established rodent model that reflects some of the core behavioral symptoms of depression (Willner, 2005
). We subjected cohorts of control and NF1 Nestin
mice to mild, unpredictable stress for 5 weeks starting at 8 months of age. Animals of either genotype were further subgrouped to receive saline, fluoxetine (18 μ
g/g) or imipramine (20 μ
g/g) in the last 3 weeks (). At the end of the 5 week regimen, control stressed mice showed declining coat condition and less frequent self-grooming in the splash test compared with age-matched non-stressed mice (data not shown). Chronic exposure to fluoxetine or imipramine reversed these changes (; n
= 6–7 for each genotype, housing condition, and treatment). To the contrary, NF1 Nestin
mice were resistant to CMS, and treatment with either antidepressant did not lead to additional changes (). Furthermore, CMS increased TST immobility in control mice (data not shown), which was reversed by chronic antidepressant (). NF1 Nestin
mice, however were unaffected by CMS in the TST paradigm (). Thus Nf1
deletion mimicked the effects of chronic antidepressants in ameliorating CMS-induced depression-like behaviors. Collectively, our results demonstrate that specific ablation of Nf1
in adult NPCs progressively activated hippocampal neurogenesis, and, by 8 months of age (6 months after Nf1
inactivation), produced antidepressive-like behaviors both at the basal state and under conditions of CMS.
Figure 7 Long-term Nf1 ablation leads to antidepressive-like behaviors. A, Diagram of the 5 week CMS paradigm. AD, antidepressant; Sal, saline. B, C, In control mice, chronic fluoxetine and imipramine reversed CMS-induced deterioration in coat condition (B) and (more ...)