duration, F(2, 41) = 1.64, p = 0.206, ANOVAs] (Figure 2c). The data indicate that P13/14 bot not P27/28 TAM treatment of CAGGCre-ERTM X fc2/+ mice results in anxiety-like behavior in adulthood. Next we compared TAM-treated CAGGCre-ERTM, fc2/+ and CAGGCre-ERTM X fc2/+ mice in the FST. Reduced immobility in this test is predictive of antidepressant drug activity [43,45], while increased immobility in c2+/2 mice is associated with anhedonia-like reductions in sucrose consumption, hypercortiso- lism and increased behavioral sensitivity to diazepam, fluoxetine and desipramine indicative of an anxious-depressive state [7,9]. P13/14 TAM-treated mice showed significant genotype effects for latency to immobility [F(2,46) = 10.47, p,0.001] and immobility duration [F(2,46) = 3.68, p,0.05, ANOVAs]. Posthoc tests confirmed that CAGGCre-ERTM X fc2/+ mice showed a reduced latency to immobility compared to fc2/+ and CAGGCre-ERTM controls [CAGGCre-ERTM X fc2/+ vs. fc2/+, q (46, 3) = 3.80, p,0.01; CAGGCre-ERTM X fc2/+ vs. CAGGCre-ERTM, q (46, 3) = 4.23, p,0.001, n = 14–20, Dunnett’s tests] (Figure 2e). The immobility duration of P13/14 TAM-treated CAGGCre-ERTM X fc2/+ mice was also increased but only when compared to CAGGCre-ERTM mice [q (46, 3) = 22.55, p,0.05, n = 14–16] and not when compared to fc2/+ controls (p.0.05, Dunnett’s tests). Importantly, the aforementioned lox P site-independent effect of TAM treatment on immobility duration of CAGGCre- ERTM mice (Figure 1b) indicates that CAGGCre-ERTM but not fc2/+ mice serve as the appropriate control for the parameter immobility duration. Similar to the EPMT, the behavior of P27/ 28 TAM-treated CAGGCre-ERTM X fc2/+ mice in the FST was indistinguishable from that of identically treated fc2/+ and CAGGCre-ERTM littermate controls [latency, F(2,42) = 0.38, p = 0.689; immobility, F(2,42) = 2.37, p = 0.106, ANOVAs] (Figure 2e). Altered behavior of P13/14 TAM-treated CAGGCre-ERTM X fc2/+ mice in the EPMT and FST was not due to a change in locomotion as indicated by the absence of genotype effects on closed arm entries in the EPMT [F(2,31) = 0.22, p = 0.8, ANOVA] (Figure 2a) and distance traveled during a 15 min OFT [F(2,25) = 0.44, p = 0.65, ANOVA] (Figure 2f). Thus, TAM-induced inactivation of the fc2 gene of CAGGCre-ERTM X fc2/+ mice at P13/14 but not P27/28 resulted in anxiety- and depression-related behavioral changes in the EPMT and FST similar to those previously described for mice with germ line or embryonically-induced GABAAR deficits [8,9]. Altered Behavior of P13/14 TAM-treated CAGGCre-ERTM X fc2/+ Mice does not Involve Deficits in the Survival of Adult-born Hippocampal Neurons We previously showed that the manifestation of heightened aversion to open arms in the EPMT and increased immobility in the FST of three different c2-subunit-deficient mouse lines (c2+/2, Emx1Cre X fc2/+, CaMKIICre2834 X fc2/+) was correlated with reduced survival of adult-born hippocampal neurons [8]. Here we assessed whether the same correlation between behavior and adult hippocampal neurogenesis could be extended to mice with GABAAR deficits induced at P13/14. CAGGCre-ERTM X fc2/+ mice and CAGGCre-ERTM and fc2/+ littermate controls were treated with TAM at P13/14, followed by metabolic labeling of replicating neural progenitor cells with BrdU at P63 and immunofluorescence analyses of brains at P91, allowing for four weeks of maturation of BrdU-labeled neurons. Interestingly, the numbers of BrdU-positive cells and BrdU positive hippocampal granule cells co-labeled with the mature neural marker NeuN in P13/14 TAM-treated CAGGCre-ERTM X fc2/+ mice were indistinguishable from corresponding cell counts in identically Development of Anxious-Depression-Related Behavior treated CAGGCre-ERTM and fc2/+ controls, indicating unaltered production and survival of adult generated granule cells (BrdU- positive cells, p = 0.53; BrdU/NeuN-doubly positive cells, p = 0.83, n = 5, Mann-Whitney) (Figure 3a). Consistent with these findings, immunostaining of the dentate gyrus of P13/14 or P27/ 28 TAM-treated CAGGCre-ERTM X R26Y mice at six weeks of age for the Cre reporter YFP and the immature neuronal marker doublecortin (DCX) revealed negligible rates of Cre-mediated recombination in adult-born neurons expressing DCX (% DCX- positive neurons colocalized with YFP averaged across both treatment groups: 5.0361.2%, n = 6) (Figure 3b). Thus, height- ened anxiety- and depression-related behavior of P13/14 TAM- treated CAGGCre-ERTM X fc2/+ mice did not involve deficits in the production or survival of adult-born hippocampal neurons. Diazepam Treatment from P10 to P16 has Anxiogenic Effects on WT Mice Analyzed in Adulthood Assuming that gradual loss of the c2 protein following TAM injection occurred with a four to five day delay, we reasoned that reduced function of GABAARs between the end of the second and fifth postnatal week interferes with maturation of neural circuits underlying normal anxiety- and emotion-related behavior in adulthood. However, more precise mapping of critical periods by genetic means was hampered by the ill-defined temporal delay in loss of receptor function following TAM-induced gene deletion. Therefore, in a second study we tested whether the aforemen- tioned results from genetic analyses could be corroborated by a pharmacological approach that is predicted to more abruptly perturb intrinsic neural activity-dependent developmental pro- cesses. Analysis of cortical ocular dominance plasticity indicates that critical developmental periods are not only sensitive to genetic reductions in GABA input but also to pharmacological potenti- ation of GABAARs with low, anxiolytic concentrations of DZP. Therefore, to test whether DZP treatment could be used to delimit critical periods for behavior in the EPMT and FST, WT mice were subjected to one- or two-week treatment with DZP or vehicle starting at different postnatal ages (P10-16, P14-28, P22-28, P29- 35, P50-56), followed by behavioral analyses starting at eight weeks of age at least four weeks after the end of DZP treatment, one test per week (Figure 4a). Treatment with DZP during P10-16 resulted in anxiety-like behavior in the EPMT as indicated by the reduced % open arm entries compared to vehicle-treated controls [t(22) = 4.25, p,0.001, t-test] (Figure 4b). A strong trend in the same direction [t(19) = 1.92, p = 0.07, t-test] was also evident for P14-28 DZP-treated mice, representing a slightly delayed but temporally overlapping treatment window (Figure 4c). By contrast, DZP treatment during P22-28 (n = 8–10), P29-35 (n = 21–22) or P50-56 (n = 8–9) had no effect on behavior in the EPMT (p.0.05 for all three treatment periods, t-tests)(Figure 4d–f). The anxio- genic effects seen in P10-16 and P14-28 DZP-treated mice were not due to alterations in locomotion as indicated by unaltered closed arm entries in the EPMT [DZP(P10-16): p.0.05, n = 11,13; DZP(P14-28): p.0.05, n = 10,12, t-tests] (Figure 4b, c) and unaltered distance traveled of P10-16 DZP-treated mice in a 15 min. OFT (p.0.05, t-test) (Figure 4m). The data indicate that DZP treatment selectively during P10-16 had anxiogenic con- sequences in adulthood. Diazepam Administration from P29 to P35 Increases Immobility Responses in the FST in Adulthood We next asked whether the critical period regulating anxiety- like behavior in the EPMT was the same or could be separated from that regulating immobility in the FST. Interestingly, DZP PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e47441