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Background: Developmental stress has been hypothesised to interact with genetic predisposition to increase the risk of developing substance use disorders. Here we have investigated the effects of maternal separation-induced developmental stress using a behavioural proxy of methamphetamine preference in an animal model of attention-deficit/hyperactivity disorder, the spontaneously hypertensive rat, versus Wistar Kyoto and Sprague-Dawley comparator strains. Results: Analysis of results obtained using a conditioned place preference paradigm revealed a significant strain × stress interaction with maternal separation inducing preference for the methamphetamine-associated compartment in spontaneously hypertensive rats. Maternal separation increased behavioural sensitization to the locomotor-stimulatory effects of methamphetamine in both spontaneously hypertensive and Sprague-Dawley strains but not in Wistar Kyoto rats. Conclusions: Our findings indicate that developmental stress in a genetic rat model of attention-deficit/hyperactivity disorder may foster a vulnerability to the development of substance use disorders.
SHR (Charles River Laboratories, Wilmington, MA, USA), WKY (Harlan Laboratories, Bicester, UK) and SD (Charles River Laboratories, Wilmington, MA, USA) rats were obtained from strains maintained at the University of Cape Town Research Animal Facility. The decision to source WKY from Harlan, UK, rather than Charles River Laboratories, was based on research suggesting that they are the most appropriate behavioural and genetic control [24]. Rats had ad libitum access to water and standard rat chow and were housed in clear Perspex cages with wood chip bedding in a facility maintained at 21–23 °C with a 12/12 h light/dark cycle (lights on at 06h00). All experiments were authorised by the University of Cape Town Faculty of Health Sciences Animal Ethics Committee under application 011/047 and conformed to local and international standards set out for the care and use of animals for scientific purposes [28, 29]. The MS paradigm was performed as previously described [30]. Briefly, male and female rats were pair bred in the University of Cape Town Human Biology satellite animal facility and the day of birth of the resulting litters was designated as postnatal day 0 (P0). On P2, the dam was removed from the cage and the number and sex of the pups was determined. In order to maintain uniformity of care, litters were culled to 8 pups with males preferentially selected for. However, a minimum of 2 female pups were retained in each litter to control for possible altered maternal behaviour and subsequent anxiety in offspring due to varying litter gender composition [31, 32]. Dams of non-separated (nMS) litters were subsequently returned to the home cage and remained with the litter in the animal facility until weaning. Conversely, on P2 MS litters were removed from the dam to a separate room maintained at 31–33 °C with infrared heating lamps. Three hours later the litters were returned to the animal facility and the dam returned to the home cage. This separation paradigm occurred between 09h00 and 13h00 over 13 days from P2 to P14. Cleaning of cages and the initial handling of pups on P2 was consistent across MS and nMS groups to ensure that potential differences would be due to the effect of the separation paradigm. On P21 litters were weaned and male rats we co-housed (2–4 rats/cage) for the remainder of the project. No more than 2 rats from any one litter were assigned to an experimental group so as to avoid potential confounding litter effects. The CPP paradigm was performed over the course of 7 days (P54–P60) in male adolescent rats, thereby corresponding with the most common age of onset for SUDs in humans [33]. This compressed protocol consisted of 3 preconditioning, 3 conditioning and 1 probe trial day. Briefly, a square black Perspex box (43 cm length × 50 cm height) was equally divided by a central partition to produce one chamber with a grid floor and thin vertical white stripes on the walls and a second chamber with unadorned walls and a smooth floor. Rats were allowed to freely explore the apparatus for 30 min during preconditioning, which was performed over the course of 3 days to compensate for the increased exploratory drive and preference for novelty in SHR as well as potential anxiety in WKY [34]. The compartment in which rats spent the most time on the 3rd day of testing was designated as the preferred compartment. The conditioning period was composed of 2 × 1-h trials per day with a vehicle (0.9 % saline administered via intraperitoneal injection at 1 ml/kg volume) injection paired with the preferred compartment, and a methamphetamine (Sigma-Aldrich, St Louis, MO, USA) injection (1.5 mg/kg in 0.9 % saline administered via intraperitoneal injection at 1 ml/kg) paired with the non-preferred compartment. These 2 trials were separated by at least 3 h to allow sufficient time for memory formation with the non-drug pairing conducted first to prevent the association of potential withdrawal effects with the subsequent trial [27]. The selected dose of methamphetamine (1.5 mg/kg calculated as a free base) was based on 3 factors: successful CPP in SHR following conditioning with a 1.25 mg/kg dose; the failure to find an effect of MS on place preference in SD rats administered a 1.0 mg/kg dose; and the need to avoid potential neurotoxic side effects associated with a higher dose, which might reduce locomotor activity due to depressive effects [35–38]. As caudate putamen methamphetamine concentrations peak between 30 and 60 min post intraperitoneal injection, rats were injected 10 min prior to the onset of conditioning trials to ensure that peak cerebral concentrations of methamphetamine were reached within the 1 h conditioning trial [39]. On the final day of testing, P60, rats were exposed to a 30 min probe trial during which they were allowed to freely explore the apparatus. Behaviour was recorded using a Soni Handicam DCR-SX 83E and time spent in each compartment as well as locomotor activity were analysed using Noldus Ethovision XT 7.0 (Noldus Information Technology, Wageningen, Netherlands). This experimental design produced 6 final groups: nMS SHR (n = 13), MS SHR (n = 11), nMS WKY (n = 10), MS WKY (n = 13), nMS SD (n = 13) and MS SD (n = 10). All data were tested for normal distribution using a Shapiro–Wilk W test. Baseline activity data over the course of the 3 preconditioning days was analysed to check for strain and stress effects. The time spent highly mobile (defined as the period of time during which the area detected as the animal changes by at least 60 % per second) was non-parametrically distributed and therefore tested for potential strain × stress effects using a Kruskal–Wallis test with multiple comparisons of mean ranks with Bonferroni adjustment as a post hoc test. To check for differences in the initial strength of compartment preference, the duration spent in the non-preferred compartment on the third day of preconditioning was subjected to a factorial ANOVA with strain and stress as categorical predictors. Significant differences were further investigated using a Tukey post hoc test. Methamphetamine preference scores were calculated by subtracting the time spent in the non-preferred compartment on the third day of preconditioning from the time spent in the same compartment during the probe trial. Therefore a positive value, i.e. increased time spent in the non-preferred compartment following methamphetamine conditioning, was taken as an indication of increased preference for the drug-paired compartment. Preference scores were normally distributed and thus analysed using a factorial ANOVA with strain and stress as categorical factors. Significant differences between groups were probed using a Tukey post hoc test. To determine which groups displayed behavioural sensitisation to methamphetamine, the total distance covered and the time spent highly mobile on the first and third days of conditioning were compared. As these data were non-parametrically distributed, they were analysed with a Wilcoxon Matched Pairs Test. To check for strain × stress effects on sensitisation, we subjected the mobility data to an aligned rank transform for nonparametric factorial analyses [40]. This preprocessing allows common ANOVA procedures to be used to investigate interaction effects in repeated measures non-parametrically distributed data. All statistical analyses were performed using Statistica 13 (Statsoft, Dell Software, Tulsa, OK, USA) and an α value of 0.05 was used to determine significance. Graphs were generated using GraphPad Prism 6.0 (GraphPad, La Jolla, CA, USA).
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