Development of Anxious-Depression-Related Behavior distance traveled of P10-16, P29-35 and P50-56 DZP-treated mice in a 15 min OFT was unaltered compared to controls, showing that locomotion was unaffected. *p,0.05, **p,0.01, ***p,0.001, t-tests. doi:10.1371/journal.pone.0047441.g004 unaffected [7]. Third, a similar anxiety- and depression-related phenotype was also observed following selective reduction of c2 subunit expression selectively in glutamatergic neurons of the telencephalon [8]. Fourth, the manifestation of this behavioral phenotype in different conditional or global c2-deficient strains of mice was associated with deficits in the long-term survival of adult- born hippocampal granule cell neurons [8], which encouraged speculation that deficits in hippocampal neurogenesis might contribute to anxious depressive behavior of c2+/2 mice. Here, we showed that selective knockdown of c2 expression in two-week- old mice results in a comparable anxiety- and depression-related phenotype that did not involve altered production or survival of hippocampal granule cells. By extension, the aforementioned deficits in hippocampal neurogenesis of global c2-deficient mice [8] are likely to represent an epiphenomenon of GABAergic deficit-induced anxiety- and depression-related behavior. These results complement and extend a number of other studies that concluded that reduced hippocampal neurogenesis was insufficient to induce anxious or depressive-like behavior [18,19,20,21]. However, there is also evidence that blocking hippocampal neurogenesis may be sufficient to induce heightened anxiety- [53,54] and depression-related behavior [17]. In a second study, we used potentiation of GABAARs with low concentrations of DZP to more precisely map critical develop- mental periods involved in establishing anxiety- and depression- related behavior. These experiments identified P10-16 and P29-35 as two critical periods that separately and specifically control anxiety-related (EPMT) and immobility (FST) behavior, respec- tively in adulthood. The idea that critical periods may be sensitive to both a reduction and potentiation of GABAergic transmission refutes the intuitive presumption that opposite manipulations must have opposite outcomes. Previous studies of ocular dominance plasticity of the visual cortex suggested that both genetic reduction of GABA synthesis and pharmacological potentiation of GA- BAARs with diazepam perturb sensory input-mediated ocular dominance plasticity [32]. Similarly, we here provide evidence that the developmental substrate for anxiety and depression- related behavior is sensitive to both genetic impairment and pharmacological potentiation of GABAergic transmission through GABAARs. Both types of manipulations relied on altering the function of c2-containing GABAARs and thereby likely targeted similar neuroanatomical substrates. We postulate that both genetic impairment and pharmacological potentiation of GABAARs interfered with neural activity-dependent processes that normally drive the development of neural circuits underlying anxiety and depression-related behavior. Our study is subject to several limitations. First, it relied principally on the EPMT and FST as proxies of anxiety- and depression-related behavior. The EPMT has face and construct validity for generalized anxiety, as well as high predictive validity for anxiolytic drug action [55]. By contrast, the validity of the FST (and the closely related TST) for depressive disorders is largely derived from its predictive validity for antidepressant drug action [55,56], although the test has also been proposed as a measure of depression-related behavioral despair [57]. Therefore, future experiments will need to address whether the critical periods identified for FST immobility extend to other behavioral measures with face and construct validity for specific aspects of depression such as reduced reward sensitivity or increased stress sensitivity [58]. A second limitation is due to the fact that mice treated with TAM or DZP at different ages were part of independent cohorts that were treated and tested independently. Therefore, behavioral alterations attributed to different TAM or DZP treatment ages could be corroborated by differences in baseline behavior due to slight variation in experimental conditions. Future verification will require comparison of groups of animals produced as a single large cohort that were subjected to treatment at different ages and behaviorally tested on the same day. Third, we cannot exclude that drug withdrawal-induced stress and stress-induced increases rather than DZP-induced decreases in neural excitability contributed to the long-term behavioral consequences of DZP treatment. Lastly, the behavioral effects of gene knockout or DZP treatment might be corroborated by interactions with handling-induced stress [59]. The incomplete correspondence of sensitive periods mapped by genetic and pharmacologic methods might in part reflect the fact that the first approach relied on gradually and modestly reducing GABAergic input, thereby allowing for compensatory adaptations of neural excitability, while DZP treatment affects GABAergic input instantaneously and probably more potently. The slower time course and reduced potency of the genetic vs. pharmacologic manipulation may also explain the unaltered behavior of TAM28/ 29-treated CAGGCre-ERTM X fc2/+ mice, which based on results of the DZP-treatment experiment would be predicted to show increased immobility in the FST. The anxiety–related critical period identified here (P10-16) maps to within a larger temporal window (P5-21) previously implicated in the developmental programing of anxiety by analyses of 5-HT1A receptor knockout mice [60]. Similar early programming of anxiety-related behavior is also evident based on developmental effects of fluoxetine on adult behavior of rodents, as well as studies of serotonin transporter (SERT) knockout mice and hypomorphic SERT alleles in humans (reviewed in [61]). This is consistent with rapidly emerging evidence that serotonergic innervation modulates the functional maturation of GABAergic neural circuits [62,63,64,65,66]. The critical period identified as important for immobility behavior in the FST (P29-35) matches a developmental period (P30-35) that is sensitive to social isolation stress in rats [59]. An earlier developmental origin of anxiety vs. emotion-related behavioral traits is corroborated by empirical evidence from human subjects, showing greatest risk for anxiety disorders in childhood, and a maximal risk for depressive disorders in adolescence [15,16,67,68]. However, we caution that critical periods of developmental vulnerability are not necessarily identical with the ages at which these disorders first manifest themselves behaviorally or clinically. Our analyses of corticogenesis failed to detect overt changes in the genesis of embryo-derived neurons in both c2+/2 and c22/2 embryos, thereby likely excluding developmental processes that overtly affect the majority of neocortical neurons of c2+/2 mice. However, these studies do not exclude deficits in numbers or activity-dependent differentiation of specific subtypes of GABAer- gic interneurons that have recently been implicated in MDD [69,70]. Indeed, consistent with a role in controlling postnatal critical windows of vulnerability, GABAergic innervation is a protracted process that is regulated by network activity and known to extend well into the fifth postnatal week of mouse brain development [71,72]. At developmental stages corresponding to the fourth and fifth postnatal week of the mouse, activity also controls the innervation field of cortical GABAergic interneurons [73]. A recent report suggests that neural activity of cortical PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e47441