Technology, Boston, MA) or DAPI (dihydrochloride salt in water, 14.3 mM, Invitrogen). The percentage of DRAQ5- or DCX- positive cells that colocalized with YFP immunofluorescence in regions of interest was determined by counting of cells in confocal images. Optical Z-plane sectioning (1 mm steps) was used to ensure that colocalized signals belonged to the same cells. The method used for analyses of TAM-induced recombination of the fc2 locus by PCR is provided in Materials and Methods S1. Diazepam Treatment DZP (Sigma Aldrich, St. Louis, MO) was administered p.o. as a suspension in 0.3% tween 80 in saline for a dosage of 1 mg/kg/ day for treatment during P10-16 or 2 mg/kg/2 days for treatments during P14-28, P22-28, P29-35, and P50-56. Control mice were gavaged identically with vehicle alone. Drug and vehicle treated mice were briefly transferred to a new holding cage as was done for TAM treatment. The behavioral effects of DZP administered at 1 mg/kg/day from P10-16 were comparable to those of DZP administered at 2 mg/kg every other day during P14-28. Therefore, for practical reasons all other DZP treatments used a dose of 2 mg/kg every two days. Doses of 1–2 mg/kg DZP have acute anxiolytic but no sedative effects [7,38]. Notably, higher sedative doses of DZP (5–10 mg/kg) were avoided as they affect locomotion of mice in adulthood [39]. Behavioral Assessment All experiments were performed with female mice, the gender that in humans is more vulnerable to anxiety and mood disorders [40,41]. The mice were group housed 6–12 animals per large cage (56640620 cm) in a female only room with a 12 h:12 h light-dark cycle (light from midnight to noon), separated by genotype if applicable, starting in the fourth postnatal week. We found that these conditions suppress estrus cycling thereby alleviating concerns with estrus-dependent behavioral variation (unpublished observation). Behavioral testing of TAM-treated mice was done under red light between 8 and 11 weeks of age, one test per week, at least 72 h after the last cage change, and starting 1 h after onset of the dark phase of the light-dark cycle. Testing of DZP-treated mice was done under the same conditions between 8 and 15 weeks of age, no sooner than four weeks after the last DZP injection. Before testing, the test equipment was routinely exposed to same sex trial mice to saturate the maze with mouse odor. The test apparatus was wiped clean of excrement after each mouse. Testing routinely started with an Open Field Test (OFT), followed by EPMT [42] and a FST [43] with the experimenter blinded to experimental conditions. The FST of P29-35 DZP-treated mice was followed by a Tail Suspension Test (TST) [44]. No OFTs were done for P28/29 TAM-treated and P14-28 and P22-28 DZP-treated mice. The OFT was used to assess possible alterations in baseline locomotion that might affect EPMT and FST measures. The mice were placed into the corner of a novel open field arena (50650 cm), and the total distance travelled over 15 min was recorded with an Ethovision system (Noldus Information Tech- nology, Inc., Leesburg, VA). Given that the OFT was performed under red light it cannot be used to assess neophobia. The EPMT [7] was used to assess anxiety-like aversion to explore a novel elevated open space as opposed to an enclosed space. The test arena consisted of an elevated crossbar (30 cm length per arm X 5 cm wide X 40 cm tall) with two walled (20 cm, clear Plexiglas) and two open arms. The edges of the open arms were surrounded by a 4 mm wide and 2 mm high trim to prevent mice from falling off the maze. The mice were placed onto the center square of the maze and videotaped for 5 min. The % closed arm entries, the % Development of Anxious-Depression-Related Behavior time spent on open arms (% open arm duration) and the number of closed arm entries were recorded. The FST [45] was used to assess behavioral immobility of mice in response to exposure to an inescapable stressful situation. Reduced immobility in this test has predictive validity for antidepressant drug action [46]. Conversely, increased immobility in this test has been proposed as an index of behavioral despair [46] (however, see Discussion) and is correlated with depressive-like behavioral, endocrine and pharmacological characteristics in c2+/2 mice [9]. The mice were lowered into a plastic beaker 19 cm in diameter and 27 cm deep and filled to a height of 18 cm with 24–26uC water and video recorded for 6 min. We recorded the real time spent swimming until the first floating episode and the cumulative time spent immobile during the final 4 min, using a 5 s interval sampling method. The TST [44,47] was used to confirm FST results of P29-35 DZP-treated mice. It assesses escape behavior in an inescapable stressful situation and has predictive validity for antidepressant drug action similar to the FST. The mice were suspended by their tails from a stainless steel rod (affixed with adhesive tape) that was positioned 30 cm above the floor of an apparatus (50650645 cm) consisting of two 25 cm wide compartments separated by an opaque PVC board. Two mice were videotaped side-by-side and the latency to assume an immobile posture and the total immobility time during the entire 6 min trial were recorded. Analyses of the Survival and Differentiation of Adult-born Hippocampal Neurons Female P13/14 TAM-treated CAGGCre-ERTM X fc2/+ mice and littermate controls were injected with the DNA synthesis marker 5-bromo-29deoxyuridine (BrdU, 3675 mg/kg, adminis- tered i.p. in 2 h intervals as a solution of 10 mg/ml BrdU in 0.9% NaCl) at P63 and transcardially perfused at P91 with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS, pH 7.4) postfixed for 12 h in the same solution, and equilibrated in 30% sucrose for at least one day. Free-floating sections cut coronally (50 mm) by Vibratome (Vibratome, St. Louis, MO) were permeabilized with 1% Triton X-100 in PBS, incubated in 2N HCl for 30 min at 37uC, and washed for 5 min in 0.1 M sodium borate (Na2B4O7) at room temperature followed by four times 5 min in PBS. They were stained with a rat antiserum against BrdU (1:500; Accurate Chemical, Westbury, NY) and a mono- clonal antibody for neuronal-specific nuclear protein (NeuN)(1:1000; Chemicon, Temecula, CA), followed by appro- priate Cy3- and Alexa 488-coupled secondary antibodies (1:500, Jackson ImmunoResearch, West Grove, PA). The number of BrdU and BrdU/NeuN positive neurons in the subgranule cell layer of confocal images across sections of the entire bilateral hippocampus were counted as described [8]. Optical sectioning was used to verify colocalization of BrdU and NeuN. The methods for analyses of production, migration and survival of embryo- derived neocortical and hippocampal neurons are provided in Materials and Methods S1. Statistical Analyses Statistical analyses were conducted using Minitab15 (Minitab Inc., State College, PA). Simple comparisons of two group means of behavioral tests were done by two-sample two-tailed t-tests. The latency to immobility data of FSTs and TSTs were all log transformed to satisfy the homogeneous variance assumption. One-way analyses of variance (ANOVAs) were used for compar- ison of behavioral data of multiple genotypes, followed by post hoc analyses of group means by Dunnett’s tests. In the case of large experimental groups of mice that had to be split into two subgroups for testing over two days, the two subgroups were PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e47441