Defects in adult hippocampal neurogenesis have been proposed as a possible cellular substrate for anxiety- and depression-related behavior [17], although several studies suggest that blocking neurogenesis is insufficient to affect performance in corresponding behavioral paradigms [18,19,20,21] (for review see [22,23,24]). GABAergic transmission via c2 subunit-containing GABAARs plays a key role in regulating cell fate decisions in adult quiescent stem cell niches [25] and in the dendritic maturation and synaptic integration of adult-born neurons [26,27] (reviewed in [28,29]). In particular, anxious-depression-related behavior of c2 subunit- deficient mice with global or embryonically induced GABAAR deficits show a marked reduction in the survival of adult-born hippocampal neurons, whereas hippocampal neurogenesis is unaffected in behaviorally normal mice with a developmentally delayed GABAAR deficit [8]. These experiments suggested that the manifestation of anxiety- and depression-related behavior in GABAAR-deficient mice might involve deficits in adult hippo- campal neurogenesis (reviewed in [3]). Differences in the rate of activity-dependent maturation of neural circuits are thought to underlie brain function-specific critical periods, i.e. developmental periods during which a certain disturbance has a significantly greater impact than the same event later in life [30]. A gradual shift from GABAergic neural excitation of immature neurons in the developing brain to mainly inhibitory effects of GABA on mature neurons in adulthood is thought to underlie intrinsic activity-dependent control of neural circuit formation and thereby to provide a neurochemical basis for critical developmental periods of brain disorders [29,31,32]. As a case in point, the critical period underlying ocular dominance plasticity of the visual cortex is sensitive to both intra-cortical genetic reductions in GABA synthesis and pharmacological potentiation of GABAAR function with the BZ site agonist diazepam (DZP) [33]. Based on these data it was proposed that the potential for functional plasticity of neural circuits is retained until a certain level of circuit maturity, defined by a GABAergic inhibitory threshold, is attained [32,34]. Here we present the results from two independent studies designed to define critical developmental periods during which perturbations of intrinsic GABAergic neural activity in mice result in lasting behavioral changes in the Elevated Plus Maze (EPMT) and Forced Swim Test (FST), respectively. In addition, we extend our studies addressing the role of hippocampal neurogenesis in regulating such behavior. First, we employed tamoxifen-inducible heterozygous knockout of the c2 subunit gene at different time points of postnatal brain development, using mice carrying a single copy of a floxed c2 subunit gene locus (fc2/+, [8]) and the CAGGCre-ERTM locus [35] encoding a ubiquitously expressed tamoxifen-inducible Cre-estrogen receptor fusion protein. These experiments narrowed the developmental period underlying altered EPMT and FST behavior to between the beginning of the third and end of fifth postnatal week. In addition, and contrary to previous global or embryonic reductions of the c2 gene dosage in immature neurons [8], these experiments showed that postnatal GABAAR deficit-induced anxiety- and depression-related behav- ioral changes can occur independently of reduced hippocampal neurogenesis. Second, we used pharmacological potentiation of GABAARs with DZP to transiently but more abruptly perturb brain development during distinct postnatal temporal windows predicted to underlie normal anxiety- and depression-related behavior in adulthood. Our experiments show that genetic impairment and pharmacologic potentiation of GABAergic trans- mission between the second and fifth postnatal week of mice have comparable lasting and detrimental consequences on anxiety- and depression-related behavior in adulthood. Moreover, the DZP Development of Anxious-Depression-Related Behavior treatment experiments identify two distinct developmental critical periods that selectively underlie behavior in the EPMT and FST, respectively. Materials and Methods Animals All mice used for this study were backcrossed onto the 129X1/ SvJ genetic background (.5 generations, previously named 129SvJ) and produced in our own breeding colony with food and water available ad libitum, on a 12 h:12 h light-dark cycle. GABAAR c2 subunit global knockout (KO; c22/2 or c2+/2) [6,7] and floxed heterozygous c2 (fc2/+) mice [36] were generated in- house and genotyped as previously described [36]. CAGGCre- ERTM mice [also known as Tg(CAG-cre/Esr1*)5Amc/J] [35] were purchased from Jackson Laboratory (Bar Harbor, ME). ROSA26-YFP (R26Y) mice used as Cre reporters [37] were provided by Dr. A. J. Eisch (University of Texas Southwestern Medical Center). All mutant and control animals compared were produced as littermates. Accordingly, c22/2, c2+/2 and wild-type (WT) mice used to analyze cortical neurogenesis were produced by mating of c2+/2 mice with c2+/2 or WT mice. CAGGCre-ERTM X fc2/+, fc2/+, CAGGCre-ERTM and WT mice were produced by mating hemizygous CAGGCre-ERTM-transgenic mice with fc2/+ mice. All mice were weaned between P20 and P22. The ages of mice given in postnatal days (P) refer to the exact age of all mice in a group at the time of treatment. The ages of mice indicated in weeks refer to the age of mice pooled from multiple litters in number of weeks 63 days, at the time of testing. All animal experiments were performed in accordance with NIH guidelines and approved by the Institutional Animal Care and Use Committee (protocols # 29296, 32425) of the Pennsylvania State University. Tamoxifen Treatment and Quantitation of Tamoxifen- induced Recombination Efficacy Cre-mediated recombination of floxed target genes was induced by a total of two injections of tamoxifen (TAM, LP Biomedicals LLC, Solon, OH), one day apart. The daily dosage was 180 mg/ kg per day (i. p., 30 mg/ml) and the drug was emulsified in ethanol:sunflower seed oil (1:9). As part of the drug treatment procedure the pups were temporarily transferred to a new cage (for maximally 5 min) until all mice of a litter had been treated. For P13/14 TAM and vehicle treatment this procedure included temporarily separating the pups for maximally 5 minutes from their mother. Analyses of Tamoxifen Induced Recombination Efficiency CAGGCre-ERTM X R26Y mice were injected with tamoxifen at P13/14 or P27/28 as described above and anesthetized and trans-cardially perfused with 4% paraformaldehyde in phosphate buffered saline (PBS) at six weeks of age. The brains were postfixed for 16 h in the same fixative, rinsed in PBS three times and stored in PBS containing 0.05% sodium azide at 4uC until all brains were ready for sectioning. Coronal sections (50 mm) were cut with a Vibratome (Vibratome, St. Louis, MO) using a brain matrix (Electron microscopy sciences, Hatfiled, PA) for reproducible positioning of brains and processed for immunofluorescent staining using rabbit anti-green fluorescence protein [GFP (1:1000), recognizing YFP, Invitrogen, Carlsbad, CA] antiserum and goat anti-doublecortin (DCX, 1:250) antibody (Santa Cruz, Santa Cruz, CA), developed with anti-rabbit Alexa 488 and anti- goat Cy3 secondary antibodies (1:500. Jackson ImmunoResearch), and rinsed in nuclear stain DRAQ5 (1:1000, Cell Signaling PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e47441