GFAP-Tk heterozygous mice (GFAP-Tk^+/−; Bush et al., 1998) allow us to transiently reduce neurogenesis. These mice express herpes thymidine kinase (Tk) under control of the stem and non-stem astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Neural progenitor cells can be eliminated in GFAP-Tk^+/− mice by treatment with VGCV (Roche), which is phosphorylated by Tk leading to abortive replication in mitotically active cells. Since non-stem astrocytes are seldom dividing, they are spared during gancyclovir treatment (Garcia et al., 2004). Nestin-CreERT2 heterozygous R26R-Stop-EYFP heterozygous mice (Dranovsky et al., 2011) allow us to visualize cumulative levels of neurogenesis across time. Nestin-CreERT2 mice express a Tamoxifen-inducible Cre in stem cells. When tamoxifen is administered it binds to the estrogen receptor, which then translocates Cre to the nucleus allowing it to excise the stop sequence resulting in expression of EYFP in nestin stem cells. Thus nestin stem cells and all of their resulting neuronal and astrocytic progeny express the EYFP protein indelibly.

Female GFAP-Tk heterozygous Nestin-CreERT2 heterozygous R26R-Stop-EYFP homozygous mice were mated with R26R-Stop-EYFP homozygous males, all on C57BL/6J 129S6 mixed background. Male pups were genotyped using previously reported PCR reactions (Bush et al., 1998; Dranovsky et al., 2011), weaned at P21, and housed 3–5 per cage with mixed genotypes. Half the mice were GFAP-Tk heterozygous (GFAP-Tk^+/− ) and half were negative for the gene (GFAP-Tk^−/−), in addition half of each group were Nestin-CreERT2 heterozygous and all were R26R-Stop-EYFP homozygous. Mice were given ad libitum access to food and water under a 12:12 h light:dark cycle in a temperature-controlled (72°F) colony. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the New York State Psychiatric Institute Animal Care and Use Committee.

ChowG containing VGCV (165 mg/kg) was administered to mice at P28-P35 in the adolescent group (GFAP-Tk^−/− n = 25, GFAP-Tk^+/− n = 27) and at P56-P63 (GFAP-Tk^−/− n = 21, GFAP-Tk^+/− n = 29) in the adult group.

Thymidine analogs bromodeoxyuridine (BrdU), 5-chloro-2′-deoxyuridine (CldU) and iododeoxyuridine (IdU) incorporate into the DNA of dividing cells and serve as markers of DNA replication. BrdU was used to label DNA replication at a single time point. Discrete antibodies bind to CldU and IdU (Vega and Peterson, 2005) so the two markers were used to visualize DNA replication at two different historical time points in the same animal.

To assess if VGCV reduced cell proliferation after 7 days of treatment from P28-P35, a cohort of mice was injected with BrdU (50 mg/kg, dissolved in 0.1 M PBS, Roche) intraperitoneally on P35 and P36 and mice were sacrificed on P36. To assess if cell proliferation was restored 7 days following cessation of VGCV treatment, a separate cohort of mice treated with VGCV from P28-P35 was injected with the same dose of BrdU on P42 and P43 and sacrificed on P43.

To assess VGCV mediated reduction in neurogenesis in adolescent and adult mice used in behavioral experiments, CldU (42.5 mg/kg dissolved in 0.1 M PBS, Sigma) was injected after 7 days of VGCV treatment intraperitoneally, on P35 and P36 in the adolescent group and on P63 and P64 in the adult group. To assess if cell proliferation was restored 7 days following cessation of VGCV treatment, mice were injected with IdU (57.5 mg/kg dissolved in 0.1 M PBS with 2% 0.2 M NaOH; MP Biochemicals) intraperitonially on P42 and P43 in the adolescent group and on P70 and P71 in the adult group. Finally to assess if cumulative levels of neurogenesis were normalized following VGCV treatment, mice were treated with 2.5 mg tamoxifen (Sigma) suspended in 1:1 honey water via gavage on P42 for the adolescent group and P70 for the adult group. All mice were sacrificed after behavioral experiments on P130.

All behavioral experiments were performed by female scientists and took place during the light cycle between 9 AM and 2 PM. Behavioral studies were conducted in adulthood beginning at P90 in the following order: sucrose preference test, social affiliation, forced swim test, stress induced corticosterone, and social defeat. All experiments were performed and scored with experimenters blind to genotype.

An 8-day sucrose preference protocol was performed as previously described (Roybal et al., 2007) with modifications. Mice were group housed and deprived of water in the homecage for 8 days starting 12 h before the beginning of the experiment. Once every 24 h mice were presented with two identical bottles with ball bearing sipper tubes. To avoid a side bias bottle position was alternated daily. The weight of bottles was recorded daily to assess the amount of solution consumed. On days 1 and 2, mice were presented with two identical bottles filled with water (water/water) for 2 h and 1 h respectively. On days 3 and 4, both bottles contained 4% sucrose solution dissolved in the drinking water (sucrose/sucrose) for 1 h and 30 min respectively. On days 5–8, one bottle was filled with water and the other was filled with 4% sucrose solution for 30 min each day (water/sucrose). Preference on each day was calculated as: (weight bottle 1/ (weight bottle 1 + weight bottle 2) ×100). Preference was averaged for each condition (water/water, sucrose/sucrose or water/sucrose).

Social affiliation was performed as described with modifications (Nadler et al., 2004). Mice were placed in an empty three chambered apparatus for a 5 min habituation period under 250 lux overhead light. Mice were shuttled to the center chamber, doors were closed, an empty wire cup was placed in the left chamber and a wire cup containing a conspecific mouse was placed in the right chamber. The doors were opened and mice were free to explore the three chambers for 10 min while being recorded by a video camera. An experienced researcher scored the duration sniffing the empty and social cup. The placement of empty cup and cup with conspecific mouse were counterbalanced across animals.

The forced swim test (FST) was performed as previously described (Cryan et al., 2002). For two consecutive days, mice were placed in a transparent 3 L plastic beaker containing 2.2 L of 25°C water for 6 min. Swimming activity was recorded automatically for 6 min and activity in the last 4 min was analyzed. Water was changed between subjects.

Mice were placed in a clean novel cage for 30 min under 250 lux overhead light and blood was immediately drawn from the submandibular vein. Cortiocosterone levels were assessed by ELISA (Enzo Life Sciences).

The social defeat paradigm was performed as described (Golden et al., 2011) with modifications. Briefly, homecages were divided in half by a transparent perforated plastic divider (Animal Care Systems) and CD1 aggressor mice (Charles River Laboratories, Wilmington, MA) were housed on the right side of the divider and not moved for the 10 day duration of the experiment. For a social defeat session experimental mice were placed on the right side of a homecage with a CD1 for 10 min, then moved to the left empty side opposite the aggressor for 24 h. Once a day for 10 days mice experienced a social defeat session with a new CD1 resident aggressor. Control mice also lived in cages divided by a transparent perforated plastic divider and were moved to a new cage every day. On the last day of defeat or control conditions mice were placed into individual cages.

Twenty four hours following placement into individual cages social interaction was assessed as described with modifications (Golden et al., 2011) in a brightly lit room (250 lux). Mice were placed in a transparent Plexiglas open field (60 × 60 cm) opposite an empty upside-down wire cup and were allowed to explore for 180 s. Mice were removed from the open field for 30 s while an unfamiliar CD1 mouse was placed under a new wire cup. Experimental mice were then returned to the open field and allowed to explore for another 180 s. Sessions were recorded by video and an experienced researcher scored the duration mice sniffed the cup in both conditions and number sniff epochs. The social sociability score was calculated as the (amount of time spent sniffing the CD1 cup)/(amount of time spent sniffing the empty cup).

Mice were anesthetized with a mixture of 150 mg/kg ketamine and 10 mg/kg xylazine and perfused transcardially with ice cold 0.9% saline followed by 4% paraformaldehyde (PFA). Brains were removed and stored in 4% PFA for 24 h, then switched to 30% sucrose solution for 48 h for cryoprotection. Brains were sectioned coronally on a cryostat at 35 μm, collected in serial sections of six and stored in PBS with 0.02% sodium azide.

For immunostaining, tissue was washed with PBS prior to incubation in 2 N HCl for 30 min at 37°C followed by 10 min in 0.1 M boric acid. Tissue was washed again in PBS, blocked with 10% normal donkey serum and incubated in primary antibody for 48 h at 4°C. The following primary antibodies were used: rat anti-BrdU (1:500, Accurate) for CldU, mouse anti-BrdU (1:1000, Becton Dickinson) for IdU, chicken anti-GFP (1:500, Santa Cruz) and Hoechst which served as a counterstain. All fluorescent secondary antibodies were obtained from Jackson ImmunoResearch and diluted 1:200 in PBS.

Tissue was imaged at 20× on a fluorescent microscope (Olympus IX83) and CldU, IdU and GFP positive cells were identified and counted in the SGZ of the DG in a series of six coronal sections that traversed the septotemporal axis of the hippocampus.

All statistics were calculated by GraphPad Prism. All data are presented as the means ± SEM and significance was set at p < 0.05. Immunostaining, plasma corticosterone levels and social interaction post-defeat results were analyzed by t-test. Forced swim test was analyzed by two-way analysis of variance (ANOVA) with day and genotype as between subject variables. Sucrose preference test was analyzed by two-way ANOVA with day and genotype as between subject variables. Social affiliation was analyzed by two-way ANOVA with cup and genotype as between subject variables. ANOVAs that yielded statistically significant main effects were followed with Bonferroni post hoc tests.

To observe the immediate effects of VGCV on cell division, a cohort of mice was administered VGCV from P28-P35, injected with BrdU on P35 and P36 and sacrificed on P36 (Figure 1A, Proliferation 1). A separate cohort of mice was administered VGCV from P28-P35 injected with BrdU on P42 and P43 and sacrificed on P43 (Figure 1B, Proliferation 2). After 7 days of VGCV treatment, the number of BrdU+ cells was reduced by 85% in GFAP-Tk^+/− mice (Figure 2A; t₍₃₎ = 4.916, p = 0.016) but no differences in BrdU+ cells were detected 7 days following cessation of VGCV by t-test (Figure 2B). Therefore, cellular proliferation was dramatically reduced by 1 week VGCV treatment and returned to baseline within 1 week of discontinuing VGCV.

Mice used for behavioral experiments were treated with VGCV from P28-P35 in the adolescent group (Figure 1C, Adolescent) and P56-P63 in the adult group (Figure 1D, Adult). We used two thymidine analogs CldU and IdU, which can be identified by separate antibodies, to observe cells that divided when each of the compounds was administered (Figure 3G). We also used tamoxifen to label the cumulative neural stem cell lineage in Nestin-CreERT2 heterozygous R26R-Stop-EYFP homozygous mice (Figure 3H). In the adolescent cohort, the number of cells dividing at the end of VGCV treatment that survived until P130 was reduced by 60% in GFAP-Tk^+/− mice compared to GFAP-Tk^−/− littermate control mice (Figure 3A; t₍₂₉₎ = 3.258, p = 0.0029). In adult mice the number of cells dividing at the end of VGCV treatment that survived until P130 was reduced by 75% in GFAP-Tk^+/− mice compared to GFAP-Tk^−/− littermate control mice (Figure 3D; t₍₂₆₎ = 5.125, p < 0.0001). No difference in the number of IdU+ cells was detected between GFAP-Tk^+/− and GFAP-Tk^−/− mice in either adolescent or adult group by t-test (Figures 3B,F). In addition we observed that GFAP-Tk^−/− mice appeared to have a higher levels of neurogenesis at P35-P36 compared to P42-P43 and P63-P64 (Figures 3A,D). This reflects a well-documented age-related decline in neurogenesis (Ansorg et al., 2012) and may be further accentuated by the use of different labels (CldU and IdU) at the different time points. Neurogenesis remained constant in GFAP-Tk^−/− mice when assessed at P42-43, P63-64 and P70-P71 (Figures 3B,D,E). Finally no difference was observed in the number of GFP cells in GFAP-Tk^−/− and GFAP-Tk^+/− mice in adolescent and adult cohorts by t-test (Figures 3C,F) and as expected GFAP-Tk^−/− mice had a larger accumulation of cells in the nestin lineage when recombination was initiated at P42 compared to P70 (Figures 3C,F).

Put together in both adolescent and adult groups VGCV treatment in GFAP-Tk^+/− mice reduced markers of cell division after 7 days of treatment and markers of cell division were restored 7 days following VGCV cessation. Moreover we detected no differences in the numbers of cells resulting from lineage tracing of nestin stem cells in both adolescent (Figure 3C) and adult (Figure 3F) groups. We therefore transiently reduced neurogenesis for 1–2 weeks specifically during adolescence and adulthood.

To test if reductions in neurogenesis during adolescence and adulthood influence depression-related behavior, mice were tested in three behavioral measures related to depression and antidepressant response. The Porsolt forced swim test is sensitive to antidepressant treatment and is thought to measure passive stress coping or behavioral despair in rodents (Porsolt et al., 1977). We previously showed that baseline FST behavior is altered in mice genetically altered to model depression (Richardson-Jones et al., 2010). However, a reduction in neurogenesis in adolescence or adulthood did not influence behavior in this task in adult mice. No differences between groups were detected in immobile duration on day 1 or day 2 of the forced swim test in adolescent (Figure 4A) or adult (Figure 4B) groups by two-way ANOVA. The sucrose preference test is also sensitive to antidepressant treatment and assesses the hedonic drive in adult mice. Reduction in neurogenesis during adolescence or in adulthood did not influence hedonic state in adult mice. When mice with reductions in adolescent and adult neurogenesis were presented with either two identical water bottles (water/water) or two identical bottles containing 4% sucrose (sucrose/sucrose), they consumed fluid equally from the two bottles (Figures 4C,D). However when mice were presented with one bottle of water and one bottle containing 4% sucrose (water/sucrose), both GFAP-Tk^−/− and GFAP-Tk^+/− mice from adolescent and adult cohorts preferred the bottle containing sucrose. No differences in preference to consume sucrose were detected between GFAP-Tk^−/− and GFAP-Tk^+/− mice in either the adolescent or adult cohort by two-way ANOVA. Mice from all groups consumed approximately 75–80% sucrose solution. To assess social affiliation mice had an opportunity to explore an empty wire cup or a cup containing a stranger conspecific mouse. We did not detect an effect of reducing neurogenesis in adolescent or adult groups as all mice showed a strong preference to explore a conspecifc mouse compared to an empty cup (Figures 4E,F; main effect of cup F_(1,36) = 46.38, p < 0.0001, main effect of cup F_(1,28) = 50.71, p < 0.0001). Finally exposure to a novel cage activates the HPA axis above baseline resulting in an increase of corticosterone into the bloodstream (Schloesser et al., 2009). Plasma levels of corticosterone were measured in mice following exposure to a novel cage for 30 min. We did not detect differences in corticosterone levels in adult mice with neurogenesis reduced during adolescence or adulthood by t-test (Figures 5A,B).

To assess responses to chronic stress mice underwent a chronic social defeat paradigm. Control groups naïve to social defeat were also used in each case. During the social defeat paradigm we observed similar levels of aggression in the CD1 mice across the 10 min defeat sessions so each experimental mouse received comparable treatment. In the adolescent cohort, GFAP-Tk^−/− and GFAP-Tk^+/− mice in the naïve group preferred to explore a wire cup containing a CD1 mouse compared to an empty cup thus had sociability scores >1 (Figure 6A). GFAP-Tk^−/− mice exposed to social defeat did not show a preference to explore a wire cup containing a CD1 mouse thus exhibiting significantly reduced sociability scores compared to the naïve group (Figure 6A; t₍₁₃₎ = 2.412, p = 0.031). However, similar to the naïve group, GFAP-Tk^+/− mice exposed to social defeat preferred to explore the CD1 mouse (Figure 6A), demonstrating a resilient phenotype. In the adult group, both GFAP-Tk^−/− and GFAP-Tk^+/− mice in the defeat condition showed reduced sociability scores compared to the control condition (Figure 6B; t₍₁₃₎ = 2.206, p = 0.046, t₍₂₀₎ = 3.460, p = 0.0025) indicating a susceptible phenotype as expected.