treated as blocks with the blocking effect treated as a random factor. Group means of cell counts of morphological analyses were compared by Mann-Whitney tests (for two groups) or Kruskal- Wallis tests (three groups). The latter were followed up by pairwise comparisons using Mann-Whitney tests. Results Unaltered Neocortical Development of c2+/2 Mice Previous analyses of mice with developmentally distinct conditional hemizygous deletions of the c2 subunit locus indicated that the anxious-depressive-like phenotype of these mice had a developmental origin between embryonic day 10 (E10, corresponding to the start of cortical neurogenesis [48]) and approximately the fifth postnatal week in the forebrain [8]. However, the densities of E12.5 and E15.5-derived neocortical neurons that had migrated to the subplate and layer II/III of the neocortex, respectively of c2+/2 and c22/2 mice were in- distinguishable from values of WT littermates (Figure S1a–c). Similarly, the densities of E15.5-derived neurons surviving to P21 in the CA1 and CA3 region and dentate gyrus of the hippocampal formation were unaltered in c2+/2 vs. WT littermates (Figure S1d). The data suggested that previously described behavioral alterations of c2+/2 mice had most likely a postnatal develop- mental origin. Characterization of TAM-mediated Recombination Patterns Given the evidence that embryonic corticogenesis was un- affected even in c22/2 mice we from here onwards focused on delineating critical periods of c2+/2 mice during postnatal development. We employed a TAM-inducible global KO strategy, making use of mice that are heterozygous for a gabrg2 locus containing lox P sites (fc2/+) [36] and a CAGGCre-ERTM transgenic line that expresses a TAM-inducible Cre recombinase (Cre-ERTM) [35]. Comparing the behavioral consequences of Cre- mediated recombination induced by TAM at different develop- mental time points requires that the recombination efficiency is independent of the age at which recombination is induced. We used Cre-mediated recombination of the R26Y locus as a proxy for recombination of the fc2 locus. Double transgenic offspring of CAGGCre-ERTM X R26Y matings were injected with TAM on two sequential days (P13/14 or P27/28, respectively) and the brains analyzed at six weeks of age for recombination in the dentate gyrus, CA1 region of the hippocampus and frontal cortex, i. e. primary brain regions implicated in anxious and depression- related behavior of global c2+/2 mice (Discussion). Immunostain- ing of the dentate gyrus granule cell layer revealed similar recombination efficiency at P13/14 and P27/28, independent of the time point of TAM injection (YFP-positive cells as a fraction of total number of cells visualized with DRAQ5 or DAPI nuclear stains: P13/14, 33.1610.6%; P27/28, 35.864.4%, p.0.05, n = 4–6 mice, Figure S2). The average recombination efficiency of P13/14 and P27/28 TAM-treated mice was homogenous across brain regions as indicated by similar recombination efficiencies in the hippocampal CA1 region (38.462.6%) and frontal cortex (31.568.6%, p.0.05, n = 3–6 mice, Mann-Whitney tests). Furthermore, analyses of genomic DNA of TAM-treated CAGGCre-ERTM X fc2/+ mice by PCR indicated that conditions identical to those used to induce recombination of the R26Y locus of CAGGCre-ERTM X R26Y mice reliably resulted in re- combination of the fc2 locus within 24–48 h of TAM treatment (Figure S3). Development of Anxious-Depression-Related Behavior Behavioral Characterization of TAM-treated Control Mice We showed previously that anxiety- and emotion-related behavior of pseudo-WT fc2/+ mice was indistinguishable from WT controls [8], in agreement with normal expression of c2- containing GABAARs in fc2/fc2 mice [36]. Recombination of floxed target genes of CAGGCre-ERTM-transgenic mice relies on TAM-induced translocation of Cre protein to the nucleus. High levels of nuclear Cre activity can lead to unspecific recombination of genomic DNA at cryptic loxP sites with Cre-dependent long- term toxic consequences [49]. To address whether TAM-induced unspecific Cre-activity or TAM alone might affect baseline behavior of TAM-treated CAGGCre-ERTM mice, TAM was injected into CAGGCre-ERTM, fc2/+ and WT littermates at either P13/14 or P27/28 followed by behavioral testing between 8 and 10 weeks of age. P13/14- and P27/28-TAM-treated mice were indistinguishable in any of the test parameters; therefore the data for the two treatment ages were combined. TAM-treated CAGGCre-ERTM, fc2/+ and WT mice analyzed in the EPMT (Figure 1a) and FST (Figure 1b) were indistinguishable from each other with respect to % open arm entries [F(2,31) = 0.2, p = 0.82] and % open arm duration [F(2,31) = 0.37, p = 0.70, n = 7–14] in the EPMT, as well as latency to immobility in the FST [F(2,43) = 2.2, p = 0.12, n = 14–16, ANOVAs). However, there was a significant genotype effect on FST immobility duration [F(2, 43) = 4.32, p = 0.02]. Posthoc analyses confirmed that the immo- bility duration of TAM-treated CAGGCre-ERTM mice was reduced compared to that of identically treated fc2/+ [q (43, 3) = 2.715, p,0.05, n = 14–16] and WT mice [q (43, 3) = 2.49, p,0.05, n = 14–16, Dunnett’s test] (Figure 1b). This Cre- transgene-mediated effect was independent of a floxed locus and therefore likely due to unspecific Cre-mediated recombination of cryptic loxP sites [50]. Importantly, the direction of this change in immobility duration observed in TAM-treated CAGGCre-ERTM vs. TAM-treated fc2/+ mice was the inverse of the change described below for P13/14 TAM-treated CAGGCre-ERTM X fc2/+ vs. CAGGCre-ERTM mice (Figure 2), which is consistent with an unspecific mechanism entirely different from GABAAR deficit-induced increases in immobility. Heterozygosity of the c2 Subunit Gene Induced at P13/ 14 but not P27/28 Results in Heightened Anxiety- and Depression-related Behavior in Adulthood We first analyzed the consequences of c2 subunit gene deletion induced at P13/14 or P27/P28 on anxiety-related behavior in the EPMT in adulthood. CAGGCre-ERTM X fc2/+ mice and CAGGCre-ERTM and fc2/+ littermate controls were treated with TAM either at P13/14 or P27/P28 and subjected to behavioral testing between 8 and 10 weeks of age (Figure 2a). In the EPMT, P13/14 TAM-treated mice showed a significant genotype effect for the parameter % open arm entries [F(2,67) = 4.03, p = 0.022, ANOVA] and a corresponding strong trend for % open arm duration [F(2,27) = 2.82, p = 0.077, ANOVA]. Posthoc analyses confirmed that CAGGCre-ERTM X fc2/+ mice visited the open arms less often than fc2/+ and CAGGCre-ERTM littermates and that they spent less time on the open arms than fc2/+ controls (% open arm entries, CAGGCre-ERTM X fc2/+ vs. fc2/+, q (67, 3) = 2.64, p,0.05, CAGGCre-ERTM X fc2/+ vs. CAGGCre- ERTM, q (67, 3) = 2.37, p,0.05, n = 19–32; % open arm duration, CAGGCre-ERTM X fc2/+ vs. fc2/+, q (27, 3) = 2.33, p = 0.05, n = 9–13, Dunnett’s tests) (Figure 2b). By contrast, P27/28 TAM- treated CAGGCre-ERTM X fc2/+ mice tested analogously were not different from fc2/+ and CAGGCre-ERTM littermate controls [% open arm entries, F(2, 41) = 0.61, p = 0.548; % open arm PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e47441