Psychopharmacology (2011) 214:249–260 At 9 a.m. on PND 4, cages were cleaned by replacing some of the sawdust, after which the dam was returned to the nest. Litters from the CON group were culled and partially cleaned at the moment of disturbance on PND 3 (11 a.m.). Cages were placed back into the breeding room and left undisturbed until weaning, with the exception of PND 14, when some of the sawdust was replaced. On PND 21, pups were weaned and housed in groups of four same-sex littermates for experiments I, II, III and IV, or in pairs for experiment V. Only results obtained with the female offspring were analysed for the present study. Experiment I. Baseline and stress-induced corticosterone levels To investigate effects of early life stress on baseline corticosterone levels, 8- to 13-week-old CON (n =30) and MD (n =23) rats were sacrificed by rapid decapitation between 9 and 10 a.m. Trunk blood was collected in EDTA-covered tubes, centrifuged (5,000 rpm for 20 min) and blood plasma was stored at −20°C until further processing. To investigate the effects of early life stress on stress-induced corticosterone levels, we induced stress by (1) exposing animals to an adverse novel situation and (2) by exposing them to swimming in a water maze setup. Both these stressors bear relevance to the behavioural tasks in experiment IV. In detail, stress- induced corticosterone levels were measured in a different cohort of CON (n = 8) and MD (n = 9) rats of 14–16 weeks of age by means of the following procedure. In the morning (between 9 and 10 a.m.), rats were brought to a novel room where a baseline blood sample was collected by tail bleeding from a small incision made halfway along the tail (Fluttert et al. 2000). Blood was collected in an EDTA-covered capillary tube. Thirty minutes later, rats were brought to the same room to collect a second sample by means of tail bleeding, which was taken as the stress-induced blood sample. Blood was processed and stored as described above. Two weeks later, plasma corticosterone levels were determined 1 h after swim stress in the same animals in order to gain more information about the speed of recovery from a stressor. For this reason, rats were placed into a water maze (22°C, 150 cm in diameter, no platform present) for 2 min between 9 and 10 a.m. After this, rats were taken out of the water and placed back into their home cage. One hour after swim stress, rats were decapitated and trunk blood was collected and processed as described above. Plasma corticosterone concentrations from all three experiments were measured in duplicate using a commer- cially available radioimmunoassay kit (MP Biochemicals, Amsterdam, The Netherlands). 251 Experiment II. Adult neurogenesis and granule cell morphology In order to determine lasting effects of early MD on the different stages of the adult neurogenesis process, eight CON and eight MD females (from four CON and four MD litters) were injected with bromodeoxyuridine (BrdU; Sigma-Aldrich, 200 mg/kg body weight, intra- peritoneally) on PND 51 and sacrificed 18 days later. At that day, animals were anaesthetised in the morning by an injection of pentobarbital sodium salt (Nembutal, 1 mg/kg bodyweight; A.U.V. Cuijk, The Netherlands) and perfused transcardially with saline followed by 4% paraformaldehyde in phosphate buffer (PB; 0.1 M; pH 7.4). To prevent pressure artefacts, brains were post-fixed overnight in the skull at 4°C after which they were carefully removed, washed and cryoprotected by 20% sucrose in phosphate-buffered saline. Frozen sec- tions (30 μm thick) were cut using a sliding microtome and collected in PB with azide. Different stages of neurogenesis were studied as described previously (Mayer et al. 2006; Oomen et al. 2007). Immunohistochemistry for BrdU (monoclonal murine anti- BrdU; Roche Diagnostics, The Netherlands, 1:2,000) was used to primarily assess newborn cell survival. The marker Ki-67, a cell cycle-related protein identifying all cells actively engaged in the cell cycle (polyclonal rabbit α-Ki- 67; Novocastra, Newcastle, UK, 1:2,000), was used to assess cell proliferation. In addition, the number of young, differentiating neurons was identified with an antibody against the microtubule-associated protein doublecortin (DCX; polyclonal goat α-DCX, Santa Cruz, 1:800). Ampli- fication was performed with a biotinylated secondary antibody (sheep anti-mouse (1:200; GE Healthcare), goat anti-rabbit (1:200; Vector) and donkey anti-goat (1:500; Jackson), respectively) and avidin–biotin complex (ABC kit; Elite Vectastain, Brunschwig Chemie, Amsterdam, The Netherlands, 1:1,000) in combination with tyramide (1:500; 0.01% H2O2 kindly provided by Dr. I. Huitinga, Netherlands Institute for Neuroscience). Subsequent chromogen develop- ment was done with diaminobenzidine (20 mg/100 ml TB, 0.01% H2O2). All histological quantification procedures described below were performed in a stereological approach in a one-in-ten series of coronal sections taken along the entire rostrocaudal axis in a total of nine sections per animal. The total number of DG granule neurons and DCX-positive cells were quantified by systematic random sampling performed with the StereoInvestigator system (Microbright- field, Germany). The optical fractionator settings were as follows: for DCX cell count, grid size 140 ×80, counting frame 50 × 50, which resulted in 300–500 markers per animal; for total granule cell count, grid size 150 × 150,