GABAergic basket cells controls the density of initial perisomatic synaptic innervation of pyramidal cells, while GABA release in addition controls the subsequent pruning of GABAergic synapses [74]. It is conceivable therefore that GABAergic innervation is disturbed by the manipulations of GABAergic transmission used in our study. Moreover, GABA-potentiating anesthetics including BZs given to two-week-old mice have been reported to transiently increase dendritic spine formation of principal cells in the prefrontal cortex [75,76]. These observations are consistent with electrophysiological evidence showing that GABA-mediated de- polarization of immature neurons promotes the developmental functional maturation of glutamatergic synapses [77]. The identification of GABAAR function-dependent critical periods regulating anxiety- and emotion-related behavior in mice may help elucidate the developmental substrate of anxious depression in patients. Based on a large number of structural and functional parameters, the limbic and cortical brain areas of a ten day-old mouse (29 days post conception) correspond to those of a human fetus at the 143rd and 197th day of gestation, respectively [48]. Based on the critical developmental period regulating ocular dominance plasticity, the visual cortex of a 23– 33 day-old mouse corresponds to that of an approximately six- year-old human [33,78]. Thus, based on the mouse data presented here the critical periods for anxiety- and depression-related behavior in humans is predicted to span the time from mid- gestation (anxiogenic effects of DZP) to school age (depressive-like effects of DZP). Future experiments will need to address whether DZP-regulated critical periods for anxiety- and emotional behavior can be mapped onto different neuroanatomical sub- strates, whether they are reflected in permanent changes in structure and function at the cellular level, and whether any such changes are reversible by antidepressant drug treatment. Supporting Information Figure S1 Unaltered proliferation, migration and survival of c22/2 and c2+/2 embryo-derived neocortical neurons. The density of neurons labeled with BrdU at different embryonic time points (E12.5 and 15.5) and accumulating in different embryonic and postnatal brain structures was determined using immunoflu- orescent staining of brain sections for BrdU or BrdU and NeuN and analyses by confocal microscopy. Timed pregnant females (c2+/2 x c2+/2 matings) were injected with BrdU at gestational day E12.5 (a) or E15.5 (b–d) and the brains harvested at E18.5 (a, b) or P21 (c, d). The density of BrdU-labeled neurons (cells/ 62500 mm2) that had migrated to the cortical subplate by E18.5 (a) and layer II/III (b) of c2+/2 and c22/2 vs. c2+/+ embryos was independent of genotype. Similarly, the density of E15.5 derived BrdU-positive cells or BrdU/NeuN double positive neurons that had accumulated in the neocortex (C) or hippocampal CA1, CA3 and dentate gyrus (DG) regions (d) of 3-week-old c2+/2 vs. c2+/+ mice was unaffected by genotype (c). Note that c22/2 mice exhibit a perinatal lethal phenotype that precluded their analyses in (c). Data indicate means 6 SEM. n = 5/genotype, p.0.05 for all comparisons [Kruskal-Wallis (a, b) and Mann-Whitney (c, d)]. (DOCX) References 1. Murphy JM, Horton NJ, Laird NM, Monson RR, Sobol AM, et al. (2004) Anxiety and depression: a 40-year perspective on relationships regarding prevalence, distribution, and comorbidity. Acta Psychiatr Scand 109: 355–375. 2. Gamez W, Watson D, Doebbeling BN (2007) Abnormal personality and the mood and anxiety disorders: Implications for structural models of anxiety and depression. J Anxiety Disord 21: 526–539. Development of Anxious-Depression-Related Behavior Figure S2 Characterization of tamoxifen induced, CAGGCre- ERTM-mediated recombination. Tamoxifen was injected into CAGGCre-ERTM X R26Y mice on P13 and P14 or P27 and P28 to induce recombination at the start of the third or fifth postnatal week, respectively and harvested at 6 weeks of age. a. Representative micrographs of sections through the dentate gyrus of CAGGCre-ERTM X R26Y mice treated with tamoxifen at the ages indicated. Scale bar, 50 mm. b. Quantitation of YFP positive cells in the dentate gyrus as a percentage of cells visualized by staining with the nuclear stain DRAQ5 (n = 4–6, p.0.05, Mann- Whitney). (DOCX) Figure S3 Analyses of TAM-induced recombination of the fc2 locus in CAGGCre-ERTM X fc2/+ mice by PCR of genomic forebrain DNA. Duplicate CAGGCre-ERTM X fc2/+ mice were treated with TAM on P13/14 (lanes 1–4) or P27/28 (lanes 5, 6) and euthanized 24 (lanes1, 2) or 48 h later (lanes 3–6) as indicated. Untreated Emx1Cre X fc2/fc2 [1] and fc2/+ mice (lacking a Cre transgene) were analyzed as positive and negative controls, respectively. The Emx1Cre transgene drives recombination of the fc2 locus in the large majority of cells of the forebrain including glutamatergic neurons and glia but not GABAergic cells [1]. Genomic DNA (125 ng) from forebrain was subject to PCR using primers mapping to sites upstream and downstream of the 59 and 39 loxP site in the fc2 locus, respectively, thereby allowing for simultaneous amplification of both the fc2 locus and the recombined locus, fc2D. Genomic DNAs of Emx1Cre X fc2/ fc2 (lane 7) and CAGGCre-ERTM mice (lane 8) were amplified as positive and negative controls, respectively. The Cre loci of all samples were analyzed in parallel PCR reactions to verify the integrity of all DNA samples and as an internal standard. Note the bands of similar intensity representative of the fc2D locus in DNA isolated 48 vs. 24 h after the second injection of TAM (lanes 1 and 2, vs. lanes 3 and 4) as well as 48 h after TAM injection o P27/28 (lanes 5,6). Abbreviations: c2, region of gabrg2 locus containing exon 8; fc2, corresponding pseudo-WT locus containing lox P sites upstream and downstream of exon 8; fc2D, gabrg2 locus following Cre-mediated recombination and deletion of exon 8. (DOCX) Materials and Methods S1 GABAergic control of critical developmental periods for anxiety- and depression-related behav- ior in mice. (DOCX) Acknowledgments We thank A. J. Eisch for proving R26Y mice and we are grateful to Yao Guo for technical assistance. Author Contributions Conceived and designed the experiments: QS TF NS BL. Performed the experiments: QS TF NS. Analyzed the data: QS TF NS BL. Wrote the paper: QS TF BL. 3. Luscher B, Shen Q, Sahir N (2011) The GABAergic deficit hypothesis of major depressive disorder. Mol Psychiatry 16: 383–406. 4. Olsen RW, Sieghart W (2009) GABA(A) receptors: Subtypes provide diversity of function and pharmacology. Neuropharmacology 56: 141–148. 5. Pritchett DB, Sontheimer H, Shivers BD, Ymer S, Kettenmann H, et al. (1989) Importance of a novel GABAA receptor subunit for benzodiazepine pharma- cology. Nature 338: 582–585. PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e47441