Subjects

The subjects were 51 inbred male albino Wistar rats bred in our own facility, weighing on average 332±8.6 g at the start of testing. Rats were housed in groups of no more than eight per cage during all phases of the experiment. Food (Young's Stock Feeds Rat and Mouse Breeder cubes, Allied Feeds, Sydney) was freely available to all subjects during all phases of the experiment. Water was freely available to all subjects except during fluoxetine treatment, when 26 subjects had their water replaced by fluoxetine solution (see below). The colony room temperature was controlled at 22°C with a 12 h reverse light cycle (lights on at 20:30 h). All behavioral testing was conducted during the dark cycle. All experimentation was approved by the University of Sydney Animal Ethics Committee, in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Experimental Procedures

Acute drug treatment

(±)3,4-Methylenedioxymethamphetamine hydrochloride was supplied by the Australian Government Analytical Laboratories (Pymble, NSW), and was diluted in 0.9% saline. Acute administration of MDMA involved procedures reported previously (Gurtman et al, 2002; McGregor et al, 2003a, 2003b; Morley et al, 2001). Rats received a 5 mg/kg i.p. injection of MDMA (n=26) or saline (n=25) every hour for 4 h on 2 consecutive days, giving a cumulative dose of 40 mg/kg. This dose regime of MDMA produces long-term behavioral and neurochemical effects (Gurtman et al, 2002; McGregor et al, 2003a, 2003b; Morley et al, 2001).

During MDMA or vehicle administration, individual rats were placed in standard operant chambers (30 × 50 × 25.5 cm) with three aluminum walls, one Perspex wall and a metal grid floor. The walls of the chambers were fitted with two passive infrared detectors that were triggered by movements of the head and body of the rats, as well as gross locomotion. Activity counts were recorded by a Macintosh computer running ‘WorkbenchMac’ data acquisition software. The chambers were enclosed in wooden sound attenuation boxes. Room temperature was maintained at an ambient temperature of 28°C by a reverse-cycle air conditioner. High temperatures may exacerbate the neurotoxic effects of MDMA in rats (Malberg and Seiden, 1998) and better simulate the conditions under which MDMA is often taken by humans.

The body temperature of all rats was recorded each hour with a Braun Thermoscan Instant Thermometer (IRT 1020) at the time of each injection. This procedure provides a rapid reading of body temperature and has been used previously with minimal stress compared to other procedures (Gurtman et al, 2002; McGregor et al, 2003b; Morley et al, 2001). Following the 4 h drug administration period, all rats were housed individually in the colony overnight and replaced back in their home cages the following morning. This procedure prevents the possible lethal effects of ‘aggregation toxicity’ sometimes seen with group housing following high-dose stimulant treatment (Green et al, 1995).

The MDMA administration phase was staggered over 3 weeks, controlling for age and weight at the time of MDMA treatment. The interval between MDMA treatment and subsequent fluoxetine treatment and behavioral testing of rats varied by up to 3 weeks across subjects.

Chronic fluoxetine treatment

At 9–12 weeks following MDMA treatment, the MDMA and vehicle groups were subdivided so that half received chronic fluoxetine (FLX) treatment, while the other half continued to receive standard drinking water. This resulted in four groups: VEH (n=12), VEH/FLX (n=13), MDMA (n=13), and MDMA/FLX (n=13). Rats were re-housed into cages of 6–7, in a way that ensured minimal weight differences between treatment groups. There were four fluoxetine-treated home cages and four receiving plain drinking water. Each cage contained approximately equal numbers of MDMA and vehicle pretreated rats.

Fluoxetine hydrochloride ((±)-N-methyl-γ-(4-[trifluoromethyl]-phenoxy)-benzenepropanamine) was obtained from Sigma (St Louis, USA). A target dose of 7 mg/kg/day fluoxetine was chosen on the basis of effective doses for modifying behavior with chronic administration in previous studies (Berton et al, 1999; Contreras et al, 2001; Durand et al, 1999; File et al, 1999; Griebel et al, 1999; Jones et al, 2002; Silva and Brandao, 2000; To et al, 1999). Estimating that a 500 g rat would drink approximately 20 ml of fluoxetine solution per day, the drug was dissolved in tap water at a concentration of 0.175 mg/ml, to approach the target dose of 7 mg/kg/day. The chronic fluoxetine regime was maintained throughout the testing period, a total of 37 days, until the rats were killed at the end of the experiment. Body weights and fluid intake were recorded regularly throughout the period of fluoxetine administration.

Social interaction test

At 3 weeks following the start of fluoxetine treatment and a total of 12–15 weeks following MDMA administration, pairs of rats were assessed in the social interaction test, as described previously (File and Hyde, 1978; McGregor et al, 2003a). Pairs of rats were tested together, with each pair of approximately equal body weight and from the same treatment condition, but from a different home cage. Owing to uneven group numbers, one rat from each of the MDMA and MDMA/FLX conditions was tested twice in the social interaction test, with a different partner each time.

Pairs of rats were placed in a square black Perspex box (52 × 52 × 40 cm3) dimly lit with red light (40 W). A video camera located above the apparatus allowed live scoring of the interactions in an adjacent room by an experimenter who was blind to group allocations. Each social interaction session lasted for 10 min during which the total duration of social interaction and number of interaction bouts were scored by the observer using ODLog software (www.macropodsoftware.com). The test arena was wiped down with 10% ethanol in between each test session. Behaviors that were recorded as social interaction included sniffing, adjacent lying, following, crawling over/under, and mutual grooming.

Emergence test

At 1 day following the social interaction test, the rats were tested in the emergence test as described previously (McGregor et al, 2003a; Minor et al, 1994). The apparatus consisted of a black wooden rectangular arena (96 × 100 × 40 cm3) with a black wooden hide box (24 × 40 × 15 cm3) placed in the top right corner of the arena. The open part of the arena was illuminated with a fluorescent light. A video camera was mounted above the arena and connected to a video recorder, allowing live scoring by an observer in an adjacent room. Analysis was accomplished using ODLog data-logging software with an observer blind to group assignment. Rats were initially placed inside the wooden hide box (which had a hinged lid through which the rat could be placed inside the box). Testing continued for 5 min, with the following behaviors scored: (a) Emergence latency: the time taken for the rat to fully emerge from the hide box, (b) Open field time: the time spent exploring the open field, and (c) Risk assessment: the time spent with part but not all of the head/body protruding from the hide box. After each test session, the apparatus was thoroughly wiped down with a damp cloth containing 10% ethanol.

Forced swim test

At 8 days following the emergence test, the rats were exposed to the forced swim test as described previously (Blokland et al, 2002; McGregor et al, 2003b; Porsolt et al, 1978). Rats were placed in cylindrical clear Perspex tubes (40 cm high × 17 cm diameter) filled to a height of 27.5 cm with water at a temperature of 23°C. This water height was chosen to prevent the animal from touching the bottom of the container, while at the same time preventing escape from the apparatus (Detke and Lucki, 1996). The tubes were located in a room illuminated with a 40 W dim red light, and were cleaned and refilled with fresh water in between each trial. A miniature video camera was located near the apparatus with pictures relayed to a ‘blind’ observer in an adjacent room, who scored using ODlog software. Behaviors scored included swimming, climbing, and immobility. Rats were tested for 5 min on each of 2 consecutive days.

Neurochemical analysis

At 1 week after the forced swim test, all rats were decapitated using a guillotine, their brains rapidly removed, and five brain regions of interest manually dissected out over dry ice, using methods previously reported (McGregor et al, 2003b). Samples from the prefrontal cortex, striatum, hippocampus, amygdala, and hypothalamus were stored in a freezer at −80°C, until assayed.

Tissue samples were weighed and then homogenized with a 500 μl ice-cold solution of 0.2 M perchloric acid containing 0.1% cysteine and 200 nmol/l of internal standard 5-hydroxy-N-methyltryptamine (5-HMeT). The homogenate was centrifuged at 15 000g for 10 min at 4°C and a 20 μl aliquot of the resulting supernatant fluid was then analysed by high-performance liquid chromatography (HPLC).

The HPLC system consisted of a Shimadzu ADVP module (Kyoto, Japan) equipped with SIL-10 autoinjector with sample cooler and LC-10 on-line vacuum degassing solvent delivery unit. Chromatographic control, data collection, and processing were carried out using Shimadzu Class VP data software. The mobile phase consisted of 0.1 mol/l phosphate buffer (pH 3.0), PIC B-8 octane sulfonic acid (Waters, Australia) 0.74 mmol/l, sodium EDTA (0.3 mmol/l), and methanol (12% v/v). The flow rate was maintained at 1 ml/min. Dopamine, 5-hydroxyindole acetic acid (5-HIAA), 5-HT, and 5-HMeT were separated by a Merck LiChrospher 100 RP-18 reversed-phase column. Quantification was achieved via a GBC LC-1210 electrochemical detector (Melbourne, Australia) equipped with a glassy carbon working electrode set at +0.75 V. The calibration curve of each standard was obtained by the concentration vs the area ratio of the standard and internal standard.

SERT binding

Samples of prefrontal cortex from the contralateral side of the brain to that used for HPLC analysis were used to assay SERT density. This analysis was performed for four randomly selected rats from each of the four groups. The samples were individually homogenized in 40-vol ice-cold Tris/HCl buffer (120 mM NaCl, 5 mM KCl, pH 7.4) and centrifuged (20 000g, 20 min, 4°C). Supernatants were discarded and the pellet re-suspended in 40-vol Tris/HCl and centrifuged again at 15 000g (10 min, 4°C). Prior to the third centrifugation (20 000g, 10 min, 4°C), re-suspended pellets were incubated for 15 min at 35°C to remove endogenous 5-HT. Final pellets were re-suspended in 15-vol Tris/HCl buffer and added to the reaction mix, which consisted of one of six concentrations of 3H-citalopram (84.2 Ci/mmol, Perkin-Elmer, Australia) ranging from 0.3 to 11 nM. Nonspecific binding was detected in the presence of 1 μM Fluoxetine HCl. The reaction was carried out for 1 h at room temperature, and terminated by the addition of 4 ml ice-cold Tris/HCl, followed by rapid filtration through a Whatman GF/B filter paper (presoaked in 0.01% polyethyleneimine, 1 h, 4°C). Filters were washed twice, transferred to scintillation vials, liquid scintillant added, and samples counted the next day.

Analysis of serum fluoxetine

At the time of decapitation, blood was collected in prechilled tubes, allowed to clot, and serum separated from cells by centrifuging (3300g, 4°C, 15 min). Serum samples were stored at −20°C and thawed just prior to analysis. For analysis, 0.5 ml of serum was spiked with 20 μl of internal standard in an Eppendorf tube, to give a final serum concentration of 2 μmol/l. The sample was then diluted with 0.5 ml of 0.1 M KH 2 PO 4 buffer (pH 6.0) and mixed gently. The SPEC-DAU micro-disc SPE cartridges (Varian, Melbourne, Australia) were connected to a Vac Elut and conditioned with 0.5 ml methanol, followed by 0.5 ml of 0.1 M KH 2 PO 4 buffer (pH 6.0). Serum samples were then applied to each cartridge. The sample was allowed to run through the disc at a low flow rate of no more than 1 ml/min. The cartridge was then rinsed with 0.5 ml 1 M acetic acid, followed by 0.5 ml methanol. The disc was dried under vacuum for about 2 min. The tips of the Vac Elut delivery needles were wiped and a rack with labeled collection microtubes was placed in the Vac Elut. The analytes were eluted with 0.5 ml of dichloromethane–isopropanol–ammonia (80 : 20 : 2 v/v), at a flow rate of no more than 1 ml/min. The elutant was then dried under vacuum in a SpeedVac vacuum evaporator (Savant Instruments, Farmingdale, NT, USA) and the dried residue was re-dissolved in 50 μl of mobile phase. The mixture was then vortexed and centrifuged to remove particulates, the supernatant transferred to micro insert vials and 20 μl of reconstituted solution was automatically injected into the HPLC system. Serum calibration curves of 100–4000 nmol/l of each analyte were also prepared and extracted similarly. The concentrations of fluoxetine and norfluoxetine in the unknown samples were calculated from the least-squares linear regression equation of the calibration curve.

Chromatographic separation of fluoxetine, norfluoxetine, and the internal standard clomipramine was accomplished using the previously described HPLC system on a Waters Symmetry C 8 5 μm (2.1 × 150 mm) micro-bore reverse-phase column (Waters, Australia) coupled with a 3 mm Opti-Guard C 8 pre-column (Optimize Technologies, Alpha Resources, Thornleigh, Australia). The mobile phase consisted of a mixture of 67 mmol/l potassium phosphate buffer (pH 3.0) and acetonitrile (67 : 33 v/v). The flow rate was maintained isocratically at 0.3 ml/min. The eluate from the HPLC column was directed via a GBC LC1200 UV-VIS detector (Melbourne, Australia) monitored at 226 nm. The total run time was 15 min.

Statistical analysis

For the MDMA treatment phase, repeated-measures ANOVA was used to compare locomotor activity and body temperature in MDMA, and vehicle-treated rats, across the 4 h of testing on each day of treatment. Differences between groups for each hour of testing were subsequently analysed using post hoc contrasts.

For subsequent behavioral and neurochemical variables, data from the four experimental groups (VEH, VEH/FLX, MDMA, MDMA/FLX) were compared using one-way analysis of variance (ANOVA), followed by Fisher's PLSD post hoc comparisons. Fluid consumption and body weight during fluoxetine administration were analysed via repeated-measures ANOVA with group and time as the independent variables. Log transformation of data was occasionally performed when significant skew was evident in the raw data.

Data were analysed using Statview 5.0 software for Macintosh, with significance levels set at 0.05 for all tests.