Methods Animals Serotonin transporter knockout rats (Slc6a41Hubr) were generated by ENU-induced mutagenesis (Smits et al., 2006). Experimental animals were derived from crossing 3 month old heterozygous 5-HTT knockout (5-HTT+/-) rats that were outcrossed for at least eight generations with commercial (Harlan, Ter Horst, The Netherlands) wild-type Wistar rats. The pregnant dams were housed in standard polypropylene cages (40 x 20 x 18 cm) with sawdust bedding and ad libitum access to water and rodent chow (Ssniff Spezialdiäten, Soest, Germany) in a temperature (21 ± 1 °C) and humidity-controlled room (45-60% relative humidity), with a 12 : 12 h light : dark cycle (lights on at 07.00 a.m.). The dams were inspected daily for delivery at 5.00 p.m. and day of birth was designated as postnatal day (PND) 0. At PND1, two paper towels (22,5 x 24,5 cm) were supplied to the dam for nest construction. Further, the litters were culled to a maximum of 10 pups, with gender ratios in favor of a male majority to maximally 7 : 3, and litters were randomly allocated to one of the following rearing conditions (from PND 2 to 14): maternal separation for 180 min (MS180) or a control treatment with immediate reunion of dam and pups (MS0). MS180 was started daily between 08.30 and 09.00 a.m., and consisted of the following procedure: The dam was removed from the home cage and placed into an identical cage until the end of the separation period. Pups were then removed from the nest as complete litters and placed into a cage (24 x 15 x 14 cm) with only sawdust bedding, and then transferred to an adjacent room. There, the cages were placed on heat pads, which were set to maintain a bedding temperature of 31-33 °C for PND 2-7 and at 29-31 °C at PND8-14. At the end of the separation period, litters were returned to their home cage by first rolling them in the home cage bedding material and then placing them in the nests. This was followed by reunion with the dams. During PND 0-22, half of the bedding material of the home cages was refreshed every week. At PND 22, the pups were weaned and housed in groups of 2-3 littermates of the same sex, under the same conditions as mentioned above. Only the male offspring were used for subsequent experimental testing. All experiments were approved by the Committee for Animal Experiments of the Radboud University Nijmegen, The Netherlands, and all efforts were made to minimize animal suffering and to reduce the number of animals used. Inescapable shock stress When the male rats reached adulthood, they were subject to inescapable shock stress in a shuttle box (model ENV-010MD, Med Associates, St. Albans, VT, USA) located within a sound-attenuating cubicle. The box was equipped with eight infrared beams to detect the position of the animal, and separated into two identical chambers by an automated door that opened vertically. The grid floor of the apparatus was connected to a scrambled shock generator (model ENV-412, Med Associates), which enabled the variation of potential differences between the bars of the grid floor in order to prevent animals from avoiding the foot shocks. For two consecutive days, between 08.30 and 12.00 a.m., rats received 50 inescapable, unpredictable 0.6 mA foot shocks. After a 5 min (day 1) or 2.5 min (day 2) habituation period, the IS session started with varying interval durations (10-18 s) and shock durations (6-14 s), amounting to a total session duration of 25 or 22.5 min, respectively. During the intervals, the door separating the chambers was raised, allowing the rats to move freely across the shuttle box. Escape testing Exactly 24 h after the start of the second exposure to IS, the rats were assessed for their ability to escape shocks in the same apparatus. The difference with the IS sessions was that the door was now raised 1 s after the onset of the shock exposure and that rats could escape the shock by moving to the opposite end of the shuttle box. The rats were tested for 30 consecutive trials with a fixed interval duration (25 s) and a maximum shock duration of 15 s. The latency of the rats to escape the shocks was automatically determined by use of the infrared beams. If the rat failed to escape, the maximum 15 s was registered as the escape latency score. Statistics The results are presented as the mean and the standard error of the mean (SEM) for all experimental groups. Mean escape latencies were analyzed with factorial analysis of variance (ANOVA), and if a significant main effect (“genotype”, “treatment”) or interaction (“genotype x treatment”) was found, appropriate a posteriori tests were performed (oneway ANOVA and independent samples t-test). If doubt about the normality of the sample distributions existed, bootstrapping was applied to test the robustness of the parametric tests. Statistical significance was set at p < 0.05. All statistical tests were carried out using SPSS (version 20, IBM corporation, Armonk, NY, USA)).