Tripping the Light Fantastic: Modeling the Consequences of Recreational Use of MDMA or 5-MeO-DIPT in Humans Using Weekend "Rave" Exposures in Rat

Method Subjects The subjects consisted of 36 (n = 18 male & 18 female) Long-Evans rats (Charles River, Wilmington, MA). All rats were individually housed in standard stainless steel cages in a climate-controlled facility and were maintained on a 12-h light/dark cycle with food (Mazuri Rodent Chow) and water provided ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee of Palm Beach Atlantic University and the animals were maintained according to the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Drug exposure began when the rats are in the mid-adolescent period of development (i.e., 35 days old). All injections took place on five evenly spaced "weekends" spaced five days apart. All rats received a total of 10 injections, two per "weekend", of Foxy (5 mg/kg; Biosynth International, Naperville, IL), MDMA (5 mg/kg; Sigma-Aldrich, St. Louis, MO), or a corresponding injection volume of isotonic saline. The suppliers using HPLC verified purity of the Foxy and MDMA. During the period of all drug exposure sessions, the ambient temperature as maintained at approximately 24°c with the humidity between 45% and 50%. Prior to the commencement of the experiment male and female rats were randomly assigned to one of three drug treatment conditions: male-MDMA (n = 6), female-MDMA (n = 7), male-Foxy (n = 5), female-Foxy (n = 5), male-saline (n = 7), and female-saline (n = 6). The rats were exposed to MDMA (n = 13), Foxy (n = 10), or saline (n = 13) from 35 to 57 days of age. Behavioral testing occurred in adulthood when the rats were 131 days old and had been drug free for 66 days. Rave Sessions Each rave session took place in a sound deadened colony room. All rats remained in their home cages with food and water ad libitum. Each rave lasted for 6 hours. Following injections, music was from the commercial radio station Wild 95.5 of Juno Beach, FL was streaming through iHeartRadio (iHeartRadio, inc.) to a JBL Onbeat Venue speaker (30 watts, Harmon, Stanford, CT). The station offers contemporary hit radio with a playlist dominated by pop and dance music. The music was played at an average sound level 110dB as measured by Sound Meter Pro (Android, Mobile Essentials ver. 2.92). Two 5-inch strobe lights (MiniStrobe, Amscan, Elmsford, NY) flashed at 11 Hz. Last, during the rave, room temperature was raised from the normal colony temperature of 240C to 270C (+20C). Apparatus - Morris Water Maze (MWM) All spatial testing took place in a circular white acrylic plastic swimming pool with a diameter of 183 cm. Depending on the phase of the experiment, different extra-maze cues and escape parameters were used. With the exception of the cued water maze phase of the experiment (see following), the depth of the water was held constant at 30 cm and made opaque white in color using a nontoxic water-based paint (Sargant Art, Hazelton, P A). The swimming pool was located in a quiet testing room approximately 36.88 square meters in size. White curtain panels surrounded the pool, limiting the number of external stimuli available to aid navigation when viewed from the surface of the pool. With the exception of free swim “probe” trials, a 15 cm X 15 cm flat white escape platform was used throughout all phases of training and testing. For the cued water maze task described below, the platform protruded 15 mm above the surface of the water and was located a distance of 18 cm from the wall of the swimming pool. For all other phases of the experiment, the escape platform was submerged to a depth of 15 mm below the surface of the water. Assessment of General Activity and Exploration. General activity levels were evaluated for two 5 min periods (one per day) in a 60.96 cm X 60.96 cm chamber consisting of 10.16 cm squares (i.e., a checkerboard). Activity measures included the number of squares crossed during the measurement period and the number of times the rats rose onto their hind legs. In addition, motivational and sensorimotor deficits were assessed using a cued version of the MWM task described below. Step-Down Passive Avoidance Testing. The step-down passive avoidance testing occurred in a standard operant chamber (Lafayette Model 84022) with a stainless steel electrified grid floor. Located in the center of the 21-cm x 28-cm chamber was a 10.14-cm x 10.14 cm brick block. The rats experienced a 0.4mA current delivered to the feet whenever the animal left the block and touched the grid floor. Water Maze Tasks The series of water maze protocols we employed allowed us to evaluate different aspects of rat learning and memory without the provision of food deprivation associated with appetitive tests of memory. For all MWM elements, the platform was either 15 mm above the water's surface (cued memory task) or was submerged 15 mm below the surface of the water (the place & spatial learning set tasks). Across all spatial phases of the experiment, on a given trial the rat was gently released into the pool at one of four compass points, labeled north, south, east, or west, and allotted 60 sec per trial to reach the escape platform. The platform location varied at one of four compass positions - northeast, northwest, southeast, or southwest. If the rat was unable find the platform within 60 sec, it was gently placed on the platform. Escape times to the escape platform were recorded with a stopwatch and errors, operationally defined as crossing one of four quadrants associated with the four compass points, were tabulated by no fewer than teams of two experimenters. Simple (Cued) Place Learning. The cued place learning MWM navigation task was administered after the drug recovery period and assessment of general activity and exploration. This phase was used to determine whether nonassociative influences might develop over time and influence performance on subsequent place learning or learning set tasks. Thus, using a visible platform, the cued place learning phase was used to examine general swimming ability, motivational deficits, and nondeclarative memory ability that could influence performance during the spatial place and learning set tasks. The rats received 10 trials per day for two days of testing. On each trial, the escape platform was located in one of four possible locations. After successfully locating the platform, the rats were allowed to rest on the platform for about 15 sec at the completion of each trial. Spatial Water Maze Tasks. The next two phases involved an assessment of spatial reference memory. Both were considered tests of spatial reference memory but differed in terms of difficulty. The place learning tasks involved learning the location of a submerged platform that remained constant across all trials within a given phase of the experiment. Two variations of the task were used because often only minor deficits at most are reported using the standard version of this test (Hartman, Lee, Zipfel, & Wozniak, 2005). Therefore, a more difficult version of the place-learning task was included, as the latter of the two was considered more sensitive for detecting spatial learning/memory impairments following adolescent drug exposure to MDMA or Foxy. The simple (high cue) version of the place-learning task consisted of training the rats for 10 trials per day for two days. At the completion of each trial, the rats were allowed to remain on the platform for 15 sec. In addition, a test of retention was evaluated with a probe trial on the second day. This consisted of removing the escape platform and testing the subject for a 60 sec “free swim”. This assessment took place approximately two hours after the last place learning trial. Both the time spent swimming in the target quadrant and the number of crossings over the former platform location were recorded. The next phase, a task considered more difficult, was a low cue version of the place-learning task. Here, the task involved reducing the availability of extra-maze cues to aid navigation. For this phase of place learning, the rats were trained four consecutive trials per day for five consecutive days. As in previous MWM phases, task difficulty was increased by placing a curtain around the water maze. Additional task difficulty was achieved by indirectly lighting the room with a single 60-watt red light bulb located beyond the curtain, below the horizon of the pool, and approximately three meters from the water maze. As a result, few visual cues remained to aid navigation. Rats were allowed to remain on the platform for 15 sec after each trial. Daily probe trials were administered not less than two hours after the last trial of the daily four-trial series. As a phase of testing, learning set acquisition required the animals to learn a new location for the escape platform each day for five consecutive days. All animals received four consecutive trials per day. The averaged performance on Trial 2 of each day was used as an index of working (short-term) memory because in the task the animal is required to recall its response on the immediately preceding trial. The rats were allowed to sit on the platform for 15 sec at the completion of each trial. Plus Maze Response Learning. The final phase of the experiment involved the use of a plus maze response-learning task similar to that discussed elsewhere (Compton, Selinger et al., 2011). In this task, the animal was faced with three response alternatives - to tum left, to tum right, or to swim straight ahead. Using a Fellows (1967) series, the ordering of placement included one of two starting points. Consistent with all earlier tests, the trials in this phase began by gently lowering the animal to the surface of the water facing the wall of the tank. As a result, the animal was required to turn 180° and swim towards the choice point located at the center of the plus maze. This phase of assessment protocol was designed to assess nonspatial response learning as well as perseverative behaviour. Thus, within a given set of trials, the configuration of the available allocentric information differed as a function of each trial. Therefore, in order to master the task, (i.e., "turn right vs. left"), the animal was required to learn a rule to turn in a specific direction regardless of the starting location (McDaniel et al., 1995). While the goal remained fixed for each animal and a series of reversals were not considered here, we have found that the ability to adjust flexibly their behaviour as a function of available allocentric cues has proven to be an effective measure of perseverative behaviour. Assessment of brain serotonin (5-HT) levels In order to assess brain serotonin (5-HT) levels, one month following the completion of the behavioral testing, the rats were euthanized. 5-HT levels were determined in the saline, MDMA, and Foxy using high performance liquid chromatography (HPLC; Waters Model 600 with electrochemical detection). Procedures in the present experiment were based on a modified version of that described elsewhere (Chapin, Lookingland, & Moore, 1986). Briefly, the raw data were integrated and analyzed to determine 5-HT levels in three regions of the brain - the hippocampus, the prefrontal cortex, and the striatum. Concentrations in the amounts of 0.04% sodium octyl sulfate, 0.1 mM disodiumethylenediamine-tetraacetate, 0.05 M sodium phosphate were dissolved in HPLC grade H2O. As part of the protocol 0.03 M citric acid acted as a buffer. The aqueous portion of the mobile phase was maintained at pH levels between 2.7 and 2.9 and the mobile phase consisted of 20% methanol and 80% aqueous phase. The HPLC column was a Waters C18 reverse phase analytical column (3.9 X 300 mm; 4 micrometer). Serotonin levels were calculated and reported as ng/g tissue. Data Analysis For the step-down passive avoidance task, step down latencies were assessed for each day. For all MWM tasks and the plus maze response learning task escape latencies and navigation errors were the two primary measures of performance. For the plus maze phase of the experiment, total errors was divided into working and reference memory errors (see below, nonspatial response learning section). When considering the MWM tasks, the optimal swim path distances differed depending on the start and escape loci. Therefore, the recorded escape latencies for the four start locations were normalized. Normalization involved computation of the ratio of the minimum swim distance in cm for each of the two longer swim paths to the escape platform (e.g., north start location and a southwest goal location) to the minimum swim of the two shorter swim paths (e.g., a north start location and a northeast goal location) trials in cm. Statistical analyses involved mixed analysis of variance (ANOVAs), with drug group as the between-subjects factor and days, or blocks of trials and days as within-subjects factors. Post-hoc analyses were performed using TukeyHSD or paired t-tests with a Bonferroni correction to control for multiple comparisons. The alpha level for acceptance was set at p < .05 and analyses were performed using SPSS (IBM, 2013).