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)).