Behavior in the last 4 min of the test was manually scored using the Ethovision program, and defined as either swimming (movement) or immobility (the absence of movement

except that necessary to keep afloat). Thirty minutes after the forced swim test, a second blood sample was taken to measure the corticosterone level post-swim stress. Corticosterone assay Approximately 50 μL of whole blood was collected exactly 24 h prior to forced swim testing (baseline or pre-stress measure) and again 30 min after the first forced swim trial (post-stress measure) from the tail vein by nicking the tail without restraint Inhibitors,research,lifescience,medical of the mouse. Blood collection was completed within 120 sec after removing each mouse from its cage. Blood was collected into potassium-EDTA microvette CB 300 tubes (Sarstedt, Nümbrecht, Germany). Plasma corticosterone levels were determined in duplicate

from 20 μL of plasma using commercially available enzyme immunoassay kits (Assay Designs, Ann Arbor, Maine); sensitivity 30 pg/mL. Epigenetic analysis Sample preparation As the male mice produced the most robust Inhibitors,research,lifescience,medical behavioral changes following early life stress, we chose to focus on them for the epigenetic part of the study. Inhibitors,research,lifescience,medical Mice were killed by cervical dislocation and the hippocampus, a key area of the brain involved in behaviors, such as anxiety, aggression and learning and memory (Fernandes et al. 2004), was dissected from 14- to 15-week-old male C57BL/6J control (n = 10), C57BL/6J separated (n = 8), DBA/2J control (n = 12) and DBA/2J separated (n = 6) behaviorally naïve mice (total n = 36). DNA was extracted Inhibitors,research,lifescience,medical from the tissue using the Qiagen AllPrep DNA/RNA kit (Crawley, UK), using the manufacturer’s standard protocol. All DNA samples were quantified and quality tested. DNA methylation analysis Genomic DNA (400 ng) was treated with sodium bisulfite using the EZ-96 DNA Methylation Kit (Zymo Research, Irvine, California) following the manufacturer’s standard Inhibitors,research,lifescience,medical protocol. DNA methylation was quantitatively assessed using the Sequenom EpiTYPER

system (Sequenom Inc., San Diego, California) as described previously (Ehrich et al. 2005). Bisulfite-PCR amplicons were designed to span CpG sites in promoter regions of Nr3c1, Avp, and Nr4a1. Primer sets, locations, and PCR conditions for each region are presented in Table 1. Positive controls, including both artificially Ketanserin methylated and artificially unmethylated samples were included in all experimental procedures to ensure unambiguous PCR amplification of selleck kinase inhibitor bisulfite-treated samples. Each sample was processed in duplicate to reduce technical variance, with the correlation between technical duplicates being 0.95 across all assays. The data presented are the average of duplicate runs. Data generated from the EpiTYPER software were filtered using stringent quality control parameters, and CpG units with low call rates and/or individuals with a high number of missing CpG units were removed.