Development of Anxious-Depression-Related Behavior Figure 3. Quantitation of hippocampal neurogenesis and analyses of recombination in neural progenitors. a) P13/14 TAM-treated CAGGCre-ERTM X fc2/+ mice and littermate controls (CAGGCre-ERTM and fc2/+) were injected with BrdU at P63 and transcardially perfused at P91. The number of BrdU-positive or BrdU/NeuN doubly positive hippocampal granule cells quantitated in serial sections was indistinguishable in TAM treated CAGGCre-ERTM X fc2/+ mice compared to identically treated littermate controls (mixed CAGGCre-ERTM and fc2/+ genotypes). b) Cre-mediated recombination in adult-born hippocampal neurons of P13/14 TAM-treated CAGGCre-ERTM X ROSA26-YFP mice was analyzed at 6 weeks of age. The micrographs show a representative section immunostained for YFP (upper left panel, green), DCX (upper right, red), with merged images showing YFP/DCX-doubly positive cells (lower left) and the outlines of the granule cell layer stained with the nuclear stain, DAPI (lower right). Arrows point to DCX positive immature neurons lacking YFP that therefore were not subject to Cre-mediated recombination. The arrowhead points to a rare DCX/YFP positive immature neuron that was subject to Cre-mediated recombination. Scale bar, 50 mm. doi:10.1371/journal.pone.0047441.g003 treatment selectively during P29-35 but not P10-16, P14-28, P22- 28 or P55-56 resulted in a decreased latency to immobility, as well as increased immobility duration in this test [P29-35: latency to immobility, t(20) = 3.49, p,0.01, immobility duration, t(20) = 2.63, p,0.05; P10-16, 14-28, 22-28 and 55-56: n = 8–13, p.0.05 for all comparisons, t-tests] (Figure 4g–k). Increased immobility of P29-35 DZP-treated WT mice was also evident based on the increased immobility duration in the TST [t(20) = 2.1, p = 0.05, t-test], although the latency to immobility in this test was unaffected (p.0.05) (Figure 4l). Altered behavior in the FST and TST of P29-35 DZP-treated mice was associated with normal baseline locomotion, as indicated by the unaltered number of closed arm entries in the EPMT (Figure 4e) and the unaltered distance traveled in a 15 min OFT (Figure 4n) (p.0.05, n = 10, for both comparisons, t-tests). Locomotion was also unaffected by DZP treatment during P22-28 and P50-56 as evidenced by the unaltered number of closed arm entries in the EPMT for P22-28 and P50-56 DZP-treated mice (Figure 4d, f) and the normal distance traveled in the OFT of P50-56 DZP- treated mice (Figure 4o) (p.0.05 for all three comparisons, n = 8– 10, t-tests). Collectively, the data indicate that anxiety-related behavior in the EPMT and immobility behavior in the FST are regulated independently by developmentally distinct critical periods that are similarly sensitive to treatment with diazepam. Discussion We have presented the combined results of two independent studies assessing postnatal developmental periods during which perturbations of GABAergic transmission via c2-containing GABAARs affect anxiety- and emotion-related behavior in adult- hood. In the first study, irreversible TAM-induced knockdown of the GABAAR c2 subunit gene dosage identified a postnatal two-week period during which induction of a permanent GABAergic deficit led to increased anxiety- and depression-related behavioral measures in adulthood. Conditional TAM-induced recombination of the c2 gene at P13/14 resulted in heightened anxious behavior in the EPMT and FST, similar to behavior previously reported for mice with global or embryonically reduced GABAAR expression [8]. By contrast, delayed TAM-induced inactivation of the c2 locus at P27/28 was without significant behavioral consequences, similar to CaMKIICre2834-mediated delayed knockdown of the c2 gene previously reported [8]. Under the conditions used for TAM-induced knockdown of the c2 gene dosage, Cre-mediated recombination of the R26Y reporter was observed in about 35% of cells (Figure S2). Moreover, TAM-induced, CAGGCre-ERTM- mediated recombination of the fc2 locus was complete after less than 48 hours after the second injection of TAM (Figure S3). However, our attempts to quantitate the reduction of c2 protein expression following TAM-induced recombination were not successful. Based on a recombination efficiency of one of two alleles in 35% of cells one might expect a reduction of c2- containing GABAARs by 17%. However, in global c2+/2 mice c2- containing GABAARs were reduced on average across brain regions by approximately 25% only, i. e. about half of what was expected [7]. Accordingly, we estimate that TAM-treated CAGGCre-ERTM X fc2/+ mice suffered from loss of the c2 subunit in about 8.5% of GABAARs. Due to the known delay of TAM-induced and Cre-mediated recombination of floxed target genes (1–2 days) [35] (Figure S3) and an estimated extra 2–3 days needed for maximal decay of the c2 subunit mRNA and protein, we estimate that the minimal vulnerability window during which reduced GABAAR gene dosage affects adult behavior maps to in- between P14 and P35 (the beginning of the 3rd to the end of the 5th postnatal week) of postnatal mouse development. Additional studies are needed to assess whether the phenotype of P13/14 TAM-treated CAGGCre-ERTM X fc2/+ mice includes depres- sion-related hypercortisolism, pharmacological alterations and anhedonia-like deficits previously reported for global c2+/2 mice [9]. Our previous studies pointed to the hippocampus and cortex as likely substrates for GABAAR deficit-induced anxious-depression- related brain states. First, c2+/2 mice with a global reduction of the c2 gene dosage revealed functional deficits in postsynaptic GABAARs mainly in hippocampus and cingulate, piriform and frontal cortex (225 to 235%), while such deficits were below threshold for reliable quantitation in the amygdala (,13%) [7,8]. Second, anxious-depressive-like behavior of these mice was associated with impaired performance in trace fear conditioning and ambiguous cue fear conditioning [7], which are critically dependent on hippocampal structures [51,52], while hippocam- pus-independent but amygdala-dependent delay conditioning was PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e47441