R-project.org). For the behavioral data, a qualitative comparison between sexes was made, and sexes were then analyzed separately as the epigenetic work was conducted exclusively in males. Independent factors were: Strain (G; C57BL/6J and DBA/2J) and Environment (E; MS or control). The data were analyzed with a factorial analysis of variance (ANOVA) to Inhibitors,research,lifescience,medical determine the significance of the main factors (strain and environment as

fixed factors) and any interaction between the main factors. For the epigenetic data, differences in DNA methylation were analyzed by two-tailed unpaired t-test for each CpG unit between groups within a given strain. In all cases, the nominal level of significance was P < 0.05. Results Behavioral changes in response to MS As expected, behavioral differences between sexes and strains were frequently observed in the different tests (see Fig. 1 for a detailed overview of the data), but not elaborated on here Inhibitors,research,lifescience,medical unless in the context of a difference between maternally separated and control animals. Inhibitors,research,lifescience,medical Figure 1 Behavioral tasks. Means (±SEM) for significantly different behavioral measures. (a) Maternally separated males differed from controls in speed (P < 0.05) and distance (P < 0.01) in the non-feeding zone of the homecage task. (b) ... Home cage In the home cage test, significant differences between the maternally separated and control groups

were only seen in male mice in the habituated dark hour (Fig. 1a), when mice are typically most active. Maternally separated males from both strains moved faster (E factor: F[1,34] = 5.4, P < Inhibitors,research,lifescience,medical 0.05) and over shorter distances (E factor: F[1,34] = 7.9, P < 0.01) in the non-feeding zone than controls. Open field Maternally separated mice reacted in a sex-specific way in the open field test (Fig. 1b). The time spent in the center

of the arena was Inhibitors,research,lifescience,medical significantly find more greater in the MS male mice of both strains (E factor: F[1,40] = 4.3, P < 0.05) but significantly lower in the MS female mice from the DBA/2J strain only, demonstrating a genotype by environment interaction Bumetanide in an anxiety phenotype in response to MS (G × E interaction: F[1,36] = 5.1, P < 0.05). Novel object exploration Maternally separated DBA/2J male mice differed from controls in the novel object test, with no differences seen in C57BL/6J males, indicating another genotype by environment interaction in an exploratory phenotype in response to MS. The time spent exploring the novel object was significantly reduced in MS DBA/2J males compared to controls (G × E interaction: F[1,40] = 6.2, P < 0.05, Fig. 1c). There were no differences in exploration in the female mice of either strain. Holeboard and forced swim No significant differences were seen in either the holeboard or the forced swim tasks (Fig. 1d and e).