1. 5-Choice Serial Reaction Time

The 5-choice serial reaction time (5-CSRT) task (Robbins, 2002) is commonly used to assess attention and impulsivity in non-human primates. As to the studies reviewed here, the task is conducted in an operant chamber that contains a monitor and a single response lever. On each trial, five circles connected by lines are presented on the monitor, and the trial begins when the animal presses and holds down the response lever. After a variable delay period of 0.75–2.5 seconds, a yellow circle is quickly presented on one of the five circles for 20, 100, or 1000 milliseconds. The animal must touch the circle that contained the yellow circle within 2 seconds for reinforcer delivery. The release latency (time to release the lever) and movement time (time to move from lever to target) are used to measure attentional performance, with longer release latencies/movement times representing poorer attention.

Taffe et al. (2001, 2002) investigated the effects of MDMA on the 5-CSRT task, and these studies are listed in Table 1. Taffe et al. (2001) trained adult male rhesus monkeys on the task for 4 weeks prior to drug treatment. Monkeys were then given two daily injections of 10 mg/kg i.m. MDMA at a 12-hour interval for 4 consecutive days. Testing continued during the treatment week (3–5 hours after the first injection of each day, so testing occurred after the peak drug effect) and also for the following 21 weeks. During all three testing periods (pretreatment weeks, treatment week, and posttreatment weeks), MDMA-treated monkeys and saline controls did not significantly differ in release latency or movement time. However, the release latency of MDMA-treated monkeys was significantly longer during the treatment week than during the pretreatment weeks. Taffe et al. (2002) tested the same group of rhesus monkeys 13 months later, and again MDMA-treated monkeys and saline controls did not significantly differ in release latency or movement time. Together, these findings indicate that treatment with repeated doses of 10 mg/kg MDMA may produce slight attentional deficits during the treatment period but have no residual effects on attention for more than 1 year later.

1. Delayed (Non)matching-to-Sample

The delayed matching-to-sample (DMS) and delayed nonmatching-to-sample (DNMS) tasks (see Dudchenko, 2004) are widely used to study working memory in many species, including rodents, birds, and non-human primates. These tasks assess recognition memory for a visual stimulus and can be conducted using stimuli such as retractable levers, color illuminated keys, or visual stimuli displayed on a press-plate or touchscreen. Each trial has three main phases: sample presentation, delay, and choice. During the sample presentation, a single visual stimulus is presented to the animal (i.e., right or left lever, red or green key, a geometric shape on the press-plate or touchscreen). After the animal makes an observing response (i.e., a press or nose-poke) to the sample stimulus, the stimulus is removed for a delay period of a specified duration. The delay period is followed by the choice phase, when two or three visual stimuli are presented to the animal, only one of which is identical to the sample stimulus. The animal must respond (i.e., a press or nose-poke) to the sample stimulus in the DMS task or the novel stimulus in the DNMS task for accuracy, food reinforcer delivery, and initiation of the next trial.

Sessions are typically conducted daily, and a range of delay periods are tested, with each animal performing multiple trials at each delay duration. Accuracy is determined at each delay by average percent correct (i.e., percent of trials that a correct response was made). Under normal working memory function, accuracy will decrease as the duration of the delay period increases. A working memory deficit is usually indicated by normal accuracy under no delay or ultra-short delays but a significant decrease in accuracy relative to normal at longer delays. A significant decrease in accuracy relative to normal across all delays [i.e., no/ultra-short delay(s) and long delays] does not represent impaired working memory, but rather a performance impairment.

Table 2 includes the seven studies that tested the effects of MDMA on the DMS (5 studies) and DNMS (2 studies) tasks. All of these studies trained animals on the task to a criterion level before beginning on-drug testing, and some of the studies continued testing after the on-drug trials.

Harper et al. (2005) and Harper (2011) trained and tested adult male Sprague-Dawley rats on a DMS task using retractable levers and delays of 0.1, 3, 9, and 18 seconds. Harper et al. (2005) gave rats 0, 0.3, 1, 2, or 3 mg/kg i.p. MDMA in a within-subjects design 10 minutes before on-drug test sessions. Relative to saline, doses of 0.3 and 1 mg/kg MDMA had no effect on accuracy, and doses of 2 and 3 mg/kg MDMA significantly decreased accuracy across all delays. Harper (2011) gave rats 0 or 3 mg/kg i.p. MDMA in a within-subjects design 5 min before on-drug test sessions. A dose of 3 mg/kg MDMA significantly decreased accuracy across all delays relative to saline. Together, these results indicate that while doses of 2 and 3 mg/kg MDMA impair performance on the task, doses of 0.3, 1, 2, and 3 mg/kg MDMA have no effect on working memory.

Frederick et al. (1995a) trained and tested adult male rhesus monkeys on a DMS task using a press-plate apparatus and delays of 2, 4, 8, 16, 32, and 48 seconds. Monkeys were given 0, 0.1, 0.3, or 1 mg/kg i.m. MDMA in a within-subjects design 30 minutes before on-drug test sessions. MDMA had no effect on overall accuracy relative to saline at any of the doses tested. In continuation of this study, Frederick et al. (1995b) tested the same rhesus monkeys on the same task under different MDMA treatments. Monkeys were given two daily injections of i.m. MDMA at an 8-hour interval for 14 consecutive days. The dose of MDMA was increased every 2 weeks, such that doses of 0, 0.1, 0.3, 1, 3, 5.6, 10, and 20 mg/kg MDMA were each given for 14 consecutive days in sequential order. On-drug testing took place 30 minutes after the first injection of each day, and MDMA had no effect on overall accuracy relative to saline at any dose. Five months later, monkeys were given 0, 0.3, 1, 1.75, 3, or 5.6 mg/kg i.m. MDMA in a within-subjects design 30 min before daily on-drug test sessions. Doses of 0.3, 1, 1.75, and 3 mg/kg MDMA had no effect on overall accuracy relative to saline, and the effect of 5.6 mg/kg MDMA could not be determined due to performance failure. In all, these studies indicate that doses of 0.1, 0.3, 1, 1.75, 3, 5.6, 10, and 20 mg/kg MDMA may have no effect on working memory.

LeSage et al. (1993) trained and tested adult White Carneau pigeons on a DMS task using color illuminated keys and delays of 0, 3, and 6 seconds. Pigeons were given 0, 0.32, 1, 1.7, 3.2, 4.2, or 5.6 mg/kg i.m. MDMA in a within-subjects design 10 minutes before on-drug test sessions. Relative to saline, doses of 0.32, 1, and 1.7 mg/kg MDMA had no effect on accuracy across all delays, doses of 3.2 and 4.2 mg/kg MDMA significantly decreased accuracy across all delays, and a dose of 5.6 mg/kg MDMA completely suppressed responding. Ten days later, pigeons were given i.m. MDMA at doses of 0 mg/kg for 2 days (baseline), followed by 3.2 mg/kg for 20 days, 4.2 mg/kg for 1 day (challenge dose), 3.2 mg/kg for 5 days, and 5.6 mg/kg for 1 day (challenge dose) (all consecutive days). The final dose of 3.2 mg/kg MDMA and the challenge doses of 4.2 and 5.6 mg/kg MDMA had no effect on accuracy relative to saline (baseline) across all delays. These findings suggest that doses of 3.2, 4.2, and 5.6 mg/kg MDMA initially impair performance on the DMS task but these impairments diminish after treatment with repeated doses of MDMA. Nevertheless, doses of 0.32, 1, 1.7, 3.2, 4.2, and 5.6 mg/kg MDMA appear to have no effect on working memory.

Taffe et al. (2001) trained adult male rhesus monkeys on a DNMS task using touchscreen stimuli. Monkeys were first tested under delays of 0, 16, 32, and 64 seconds for 4 weeks. The following week, monkeys were given two daily injections of 10 mg/kg i.m. MDMA at a 12-hour interval for 4 consecutive days. Testing continued during the MDMA treatment week (3–5 hours after the first injection of each day, so testing occurred after the peak drug effect) and for the 21 weeks following treatment. During all three testing periods (pretreatment weeks, treatment week, and posttreatment weeks), MDMA-treated monkeys and saline controls did not significantly differ in accuracy across all four delays. The accuracy of MDMA-treated monkeys was significantly reduced during the treatment week compared with the pretreatment weeks at delays of 0 and 64 seconds, but this effect can be attributed to performance deficits rather than working memory deficits as the reductions were seen at both no delay and a long delay. In all, these findings indicate that treatment with repeated doses of 10 mg/kg MDMA may have no effect on working memory during treatment and for at least 5 months later.

Marston et al. (1999) trained and tested adult male Lister Hooded rats on a DNMS task using retractable levers and delays of 0.3, 1, 3, 5.6, 10, 17.6, and 30 seconds. Rats were given two daily injections of i.p. MDMA at a 10-hour interval for 3 consecutive days at doses of 10 mg/kg MDMA on day 1, 15 mg/kg MDMA on day 2, and 20 mg/kg MDMA on day 3. Testing continued during MDMA treatment (45–130 min after the first injection of each day) and for the 3–16 days following treatment. MDMA suppressed responding during the treatment days, so the results on accuracy were not reported. During the posttreatment days, the accuracy of MDMA-treated rats was significantly reduced relative to saline controls at the longer delays of 17.6 and 30 seconds (but not the shorter delays) during the last few days of testing. These results suggest that treatment with increasing doses of 10–20 mg/kg MDMA impairs working memory for up to about 2 weeks later.

2. Spontaneous and Delayed Alternation

The spontaneous alternation (SA) and delayed alternation (DA) tasks (see Dudchenko, 2004; Hughes, 2004) are used to assess spatial working memory in rodents, typically on a T- or Y-maze. The main difference between these two tasks is that SA responses are driven by the natural tendency for rodents to explore novel environments, and DA responses are driven by food reinforcement. In both tasks, the goal of the animal is to investigate a new arm of the T- or Y-maze rather than one that they recently visited.

There are two main versions of the SA task, continuous SA and two-trial SA. The continuous SA task is completed in one trial, during which the animal is allowed to freely explore all three arms of the maze for the entire duration (usually several minutes). Number of alternations, defined as consecutive entries into all three arms without repeated entries, is scored for each animal and converted to percent alternation (ratio of actual to possible alternations given number of arm entries). The two-trial SA task consists of a forced trial and a test trial. On the forced trial, the animal is placed at the end of the “start” arm and is only allowed to enter one other arm (the “familiar” arm), as the third arm (the “novel” arm) is blocked by a door. Normally, a delay period is placed after the forced trial and before the test trial. On the test trial, the animal is returned to the end of the “start” arm and allowed to enter either the “familiar” arm or the “novel” arm (all three arms are open). A correct response or alternation is defined as an entry into the “novel” arm on the test trial.

The DA task is quite similar to the two-trial SA task. Each session usually consists of one forced trial followed by several choice trials. The forced trial is conducted in the same manner as the SA task, except a food reinforcer is placed at the end of the “familiar” arm. On the first choice trial, the food reinforcer is placed at the end of the “novel” arm, and for all subsequent choice trials, the food reinforcer is placed at the end of the arm that was not entered on the previous trial. Only one entry is permitted per trial, and a correct response or alternation is defined as a reinforced response, an entry into the arm that was not entered on the previous trial.

Table 2 includes the seven studies that explored the effects of MDMA on the SA (four studies) and DA (three studies) tasks. Of these studies, only Costa et al. (2014) conducted the continuous SA task. In this study, male C57BL/6 mice were given two daily injections of 10 mg/kg i.p. MDMA at a 4- to 6-hour interval on the 2nd and 5th days of each week for 9 weeks, which began in adolescence and extended into adulthood. Mice were tested on a Y-maze, off-drug, on the 7th day of drug treatment weeks 1, 4, and 9 and postdrug treatment weeks 2 and 3. The percent alternations of MDMA-treated mice and saline controls did not significantly differ at any time point, suggesting that treatment with repeated doses of 10 mg/kg MDMA has no effect on spatial working memory for up to 3 weeks after treatment.

Edut et al. (2011) and Cassel et al. (2005) tested rodents on the two-trial SA task at least 1 week after MDMA treatment. Edut et al. gave adult male ICR mice a single injection of 10 mg/kg i.p. MDMA and tested them 7 and 30 days later on a Y-maze. Mice were permitted to enter multiple arms during a 5-minute forced trial and a 2-minute choice trial, which were separated by a 2-minute delay period. The preference index [(time at “novel” arm – time at “familiar” arm)/(time at “novel” arm + time at “familiar” arm)] of MDMA-treated mice and saline controls did not significantly differ at both 7 and 30 days later. Cassel et al. gave adult male Long Evans rats a daily injection of 10 mg/kg i.p. MDMA for 4 consecutive days. Four days later, rats began testing on a T-maze, and were tested once daily for 4 days and then twice on a 5th day. Rats were permitted to enter only one arm during each trial, which were separated by a 30-second delay period. The overall percent alternation of MDMA-treated rats and saline controls did not significantly differ. These two studies suggest that pretreatment with a dose of 10 mg/kg MDMA, whether administered once or repeatedly, has no subsequent effect on spatial working memory.

Kolyaduke and Hughes (2013) performed a variation of the two-trial SA task in which all three arms of a Y-maze were open during both trials (now referred to as the acquisition and retention trials). During the acquisition trial, one arm contained a black insert and one arm contained a white insert, and during the retention trial, both arms contained a black insert (the changed arm = the novel arm). Multiple choices were allowed during the 6-minute acquisition trial and the 3-minute retention trial, and there was no delay period between the two trials. Male and female PVG/c hooded rats were given a daily injection of 10 mg/kg i.p. MDMA for 10 consecutive days during early adolescence (postnatal days 35–45) or late adolescence (postnatal days 45–55). Rats were tested as adults on two separate days at least 35 days after MDMA treatment (after postnatal day 90). Both early and late adolescence MDMA-treated rats did not significantly differ from saline controls in percent novel entries and percent time spent in the novel arm, indicating that pretreatment with repeated doses of 10 mg/kg MDMA may have no subsequent effect on spatial working memory.

Ricaurte et al. (1993) gave adult male Long Evans rats two daily injections of 20 mg/kg s.c. MDMA at an 8-hour interval for 4 consecutive days, and this treatment was repeated again about 1 week later. About 1 month later, rats began training for a DA task on a T-maze. Seven weeks after MDMA treatment, rats began daily test sessions consisting of one forced trial followed by 10 choice trials under a constant delay of 5 seconds. The percent correct of MDMA-treated rats and saline controls increased at a similar rate over the 20 test sessions, and there were no significant differences between groups. After 5 weeks of testing under a constant delay, variable delays of 5, 30, 60, 120, and 180 seconds were introduced, and testing continued for an additional 3 weeks. The percent correct of MDMA-treated rats and saline controls decreased at a similar rate as the duration of the delay period increased, and again there were no significant differences between groups. Findings from this study indicate that pretreatment with repeated doses of 20 mg/kg MDMA may have no subsequent effect on spatial working memory.

Young et al. (2005) performed a two-part task on a double Y-maze: the first part was a spatial discrimination (SD) task (described in section V.C.3.a) and the second part was a DA task (summarized here). Young adult male Wistar rats were trained to criterion on the task prior to being introduced to delays of 0, 15, or 60 second and then on-drug testing. Rats were injected with 0, 1.25, 2.25, or 5 mg/kg i.p. MDMA in a within-subjects design 20 minutes before on-drug test sessions. Each test session consisted of one forced trial followed by 24 choice trials with randomly allocated delays. Overall, the percent correct of all rats significantly decreased as the duration of the delay increased. Relative to saline, doses of 1.25 and 2.25 mg/kg MDMA had no effect on percent correct at any delay, a dose of 2.25 mg/kg MDMA produced a small increase in percent correct under a 60-second delay, and a dose of 5 mg/kg MDMA significantly decreased percent correct at all delays. Typically, this deficit would be attributed to a performance impairment, but since 5 mg/kg MDMA had no effect on accuracy in the SD component, which required the same performance abilities (see section V.C.3.a), this may be due to a working memory impairment. In all, these findings suggest that a dose of 1.25 or 2.25 mg/kg MDMA has no effect on spatial working memory, but a dose of 5 mg/kg MDMA may produce spatial working memory deficits.

Viñals et al. (2012) performed an operant/nonspatial version of the DA task in which adult male C57BL/6 mice were trained to alternate nose-poking between two nose-poking holes. Mice were trained to a criterion level on the task, and then given two daily injections of 3 or 30 mg/kg i.p. MDMA at a 4-hour interval for 4 consecutive days. Mice were tested off-drug for 7 days after MDMA treatment and introduced to delays of 2, 4, 6, or 8 seconds in a random order. Mice given 3 mg/kg MDMA injections and saline controls did not significantly differ in percent correct over all 7 days of testing. The percent correct of mice given 30 mg/kg MDMA injections was significantly higher than saline controls on the 1st day of testing but did not significantly differ from saline controls for the remaining 6 days. The increased accuracy on the 1st day of testing may be because of a slowed reaction time rather than working memory enhancements, as mice given 30 mg/kg MDMA injections also demonstrated a significantly longer response latency compared with saline controls. In all, these results suggest that pretreatment with repeated doses of 3 or 30 mg/kg MDMA has no subsequent effect on nonspatial working memory.

3. Radial Arm Maze

The radial arm maze (RAM) (see Dudchenko, 2004; Quillfeldt, 2016) is a useful tool to study spatial working and reference memory in rodents. The goal of this task is to learn and remember the location of food pellets using spatial cues. Performance can be separated by type of memory (working vs. reference). The effects of MDMA on working memory in the RAM task will be discussed here, while the effects on reference memory will be reviewed in section V.C.2.

The RAM consists of a central hub that provides access to eight radiating arms. All eight arms are equal in length, and a food well is attached to the end of each arm. The entire maze is typically elevated above the floor in a room with many distal spatial cues at fixed locations. Prior to training, each animal is randomly assigned a set of four baited arms and four nonbaited arms, which remains fixed for the remainder of the experiment. Training is usually conducted daily (or sometimes spaced by 1 to 2 days), with all animals completing several trials per day (2–6 trials/day for the studies reviewed). Before each trial, food pellets are placed in the food wells of the four baited arms assigned to that animal. The trial then begins by placing the animal in the central hub facing arm number one. The animal is typically allowed to enter four arms per trial before being removed from the maze. The number of working memory errors, defined as entries into a baited arm that has already been visited in that same trial, is scored for each trial. A single entry into each baited arm reflects accurate spatial working memory of the food pellet locations.

Table 2 includes the six studies that used the RAM to evaluate the effects of MDMA on spatial working memory. Five of the studies used the general methods outlined above, while Braida et al. (2002) used an alternative procedure that is described below.

Hernandez-Rabaza et al. (2010) and Ros-Simó et al. (2013) treated adolescent male rodents with two injections of MDMA on a single day. Hernandez-Rabaza et al. gave Long Evans rats two injections of 10 mg/kg i.p. MDMA at a 6-hour interval, 12 days prior to training. MDMA-treated rats and saline controls exhibited a decrease in working memory errors over the 5 days of training, and the number of working memory errors did not significantly differ between groups. Ros-Simó et al. began training CD1 mice prior to any MDMA administration. Each animal was assigned only three baited arms, and animals were not limited to a certain number of arm entries within each trial. Mice were trained for a total of 12 consecutive days and were given two injections of 20 mg/kg i.p. MDMA on the 12th training day, one immediately after training and another 2 hours later. Three days later, mice were subject to an additional training session, during which the number of working memory errors produced by MDMA-treated mice and saline controls did not significantly differ. The findings of these two studies suggest that pretreatment with two doses of 10 or 20 mg/kg MDMA has no later effect on spatial working memory.

Figure 5 presents the findings of Kay et al. (2010), which exemplify dose-dependent effects of MDMA on working memory. Kay et al. trained adult male Sprague-Dawley rats off-drug until all rats reached a criterion of at least 75% correct arm entries for 7 days. After reaching criterion, rats began on-drug training. Rats were given 0, 0.75, 3, or 4 mg/kg i.p. MDMA in a within-subjects design 15 minutes before each day of training. Relative to saline, a dose of 0.75 mg/kg MDMA had no effect on the mean overall percent correct, but doses of 3 and 4 mg/kg MDMA significantly decreased the mean overall percent correct (Fig. 5A). The deficits produced by 3 mg/kg MDMA were not due to working memory impairments, as this dose did not significantly impact the percent of working memory errors (number of errors/number of errors possible per day). A dose of 4 mg/kg MDMA did significantly increase the percent of working memory errors relative to saline; however the percent of working memory errors was still significantly smaller than the percent of reference memory errors (Fig. 5B). These findings indicate that doses of 0.75 and 3 mg/kg MDMA have no effect on spatial working memory, but a larger dose of 4 mg/kg MDMA slightly impairs spatial working memory.

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Fig. 5.

Dose-dependent effects of MDMA on the radial arm maze task. 0.75 mg/kg MDMA had no effect on overall accuracy (A) or errors (B), while 3 and 4 mg/kg MDMA impaired overall accuracy (A) and increased working (4 mg/kg only) and reference memory errors (B). Data redrawn with permission from Figs. 1 and 3 in Kay et al. (2010).

Kay et al. (2011) and Harper et al. (2013) conducted similar experiments in which adult male Sprague-Dawley rats were treated with MDMA before and/or during training. Kay et al. gave rats four injections of 10 mg/kg i.p. MDMA at 2-hour intervals, 2 days prior to training. Rats were trained off-drug until all animals reached training criterion (28 days later). Over the 24 off-drug training sessions, the average percent correct of MDMA-treated rats increased at a slower rate than saline controls. Two days after off-drug training, on-drug training began and continued for a total of 12 days. Rats were given 0 or 4 mg/kg i.p. MDMA in a within-subjects design 20 minutes before each on-drug training session. MDMA significantly decreased the average percent correct relative to saline, but this impairment was significantly smaller in rats treated with MDMA prior to off-drug training relative to saline controls. Nonetheless, the impairments observed during off- and on-drug training were not due to working memory deficits, as working memory error percentage was not significantly affected by any MDMA treatment.

Harper et al. also gave rats four injections of 10 mg/kg i.p. MDMA at 2-hour intervals. Training began after MDMA treatment and lasted for 47 days. Most training sessions took place off-drug, except some rats were given 4 mg/kg i.p. MDMA before training sessions on days 8, 15, 22, 28, 34, and 41. The average percent correct of rats pretreated with MDMA prior to all training sessions was significantly lower than saline controls on both off-drug and on-drug training days. MDMA also significantly reduced average percent correct during on-drug training days relative to saline. Again, none of these impairments were due to working memory deficits, as working memory error percentage was not significantly affected by any MDMA treatment. The findings of Kay et al. and Harper et al. suggest that pretreatment with four doses of 10 mg/kg MDMA has no subsequent effect on spatial working memory, and a dose of 4 mg/kg MDMA also has no on-drug effect on spatial working memory.

Braida et al. (2002) conducted an alternative working memory task on the RAM. In this version, all eight arms of the maze are baited, and the animal’s goal is to enter all eight arms only one time during each trial. The number of errors are scored for each trial, which is synonymous with working memory errors on the typical RAM task (i.e., re-entries into a baited arm). Here, the task was conducted both without a delay and with a 2-hour delay between the fourth and fifth arm entry. Adult male Wistar rats were trained on the task to a criterion level, and then began on-drug training for 3 consecutive days. Rats were given a single injection of 1, 2, or 3 mg/kg i.p. MDMA 20 minutes before each on-drug training session. Without a delay, MDMA had no effect on the total number of errors relative to saline. With a 2-hour delay, doses of 1 and 2 mg/kg MDMA had no effect on the total number of errors during the pre-delay period (first 4 choices) and post-delay period (last 4 choices) relative to saline. A dose of 3 mg/kg MDMA also had no effect on the total number of errors during the pre-delay period, but significantly increased the total number of errors during the post-delay period relative to saline. These results suggest that doses of 1 and 2 mg/kg MDMA have no effect on spatial working memory, but a dose of 3 mg/kg MDMA impairs spatial working memory.

4. Other Working Memory Tasks

1. Morris Water Maze

The Morris water maze (MWM) (Morris, 1984) is one of the most widely used tasks for studying spatial learning and memory in rodents. The objective of this task is to learn to navigate to a hidden platform in a large circular pool of water using spatial cues. The pool is arbitrarily divided into four quadrants and is in a room with many distal visual cues at fixed locations (e.g., furniture, wall art, etc.). There are two main stages of the task: acquisition training and the probe test, which assess spatial learning and spatial reference memory, respectively.

Acquisition training takes place over a few consecutive days (3–5 days for the studies reviewed), with all animals completing several trials per day (3–8 trials/day for the studies reviewed). On each trial, an animal is placed into the water facing the wall of the pool and is expected to swim and escape onto the hidden platform. The hidden platform remains in the same location throughout acquisition training, but the starting location of the animal is varied between trials. As a result, spatial memory of the distal visual cues is required to identify the location of the hidden platform. The escape latency (i.e., time taken to reach the platform), and often the path length (i.e., distance swam to reach the platform), is recorded for all trials. A significant decrease in escape latency/path length over days of acquisition training suggests spatial learning of the platform location.

The probe test takes place after the last acquisition training session, either the same day or the following day. The procedure is similar to acquisition training, except the hidden platform is removed from the pool and each animal performs only one trial. The total time spent swimming in each quadrant of the pool, or sometimes the average distance from the platform location, is recorded. A significantly greater amount of time spent swimming in the target quadrant (i.e., quadrant where the hidden platform used to be located) relative to the other three quadrants indicates spatial reference memory of the platform location.

Table 3 presents the 14 studies that report the effects of MDMA on the standard MWM task. Most of these studies completed both acquisition training and the probe test. Some studies do not report the change in escape latency/path length over days of acquisition training, and a few other studies do not report findings on the probe test. In these particular studies, effects on spatial learning or spatial reference memory (respectively) cannot be properly assessed. All 14 studies used high doses of 5–20 mg/kg MDMA, and none used lower, typical doses of less than 3 mg/kg MDMA.

Taghizadeh et al. (2016) were the only group to conduct on-drug acquisition training. Adult male Wistar rats were given 5, 10, or 15 mg/kg i.p. MDMA 30 minutes before the first trial of acquisition training on all 4 days. During the probe test on the following day (off-drug), MDMA-treated rats spent significantly less time in the target quadrant than saline controls. These results suggest that doses of 5, 10, and 15 mg/kg MDMA impair spatial reference memory when acquisition occurs on-drug.

The remaining studies explored the effects of administering MDMA one or more days prior to acquisition training. Mirzaei et al. (2013) gave adult male Wistar rats a single injection of 10 mg/kg i.p. MDMA, 3 days prior to acquisition training. The escape latency and path length of MDMA-treated rats were significantly higher than that of saline controls on the 1st day of training, but these values decreased significantly over the 2nd and 3rd day to a level comparable to that of the saline controls. This suggests that pretreatment with a dose of 10 mg/kg MDMA has no subsequent effect on spatial learning.

Sprague et al. (2003), Cohen et al. (2005), Able et al. (2006), Skelton et al. (2008), and Cunningham et al. (2009) all gave adult male Sprague-Dawley rats multiple injections of MDMA on a single day prior to acquisition training. Sprague et al. gave rats two injections of 20 mg/kg s.c. MDMA at a 12-hour interval, 1 week prior to acquisition training. The escape latency and path length of MDMA-treated rats decreased significantly over 3 days of acquisition training, and there were no significant differences between MDMA-treated rats and saline controls during acquisition. During the probe test directly after the last acquisition session, MDMA-treated rats spent significantly less time in the target quadrant than saline controls, yet significantly more time in the target quadrant than two of the other three quadrants. Similarly, Cunningham et al. gave rats four injections of 7.5 mg/kg i.p. MDMA at 2-hour intervals, 24 days prior to acquisition training. There were no significant differences between MDMA-treated rats and saline controls in the decrease in escape latency and path length over 5 days of acquisition training. During the probe test directly after the last acquisition session, MDMA-treated rats spent the same amount of time in all four quadrants, unlike the saline controls that spent significantly more time in the target quadrant than the other three quadrants. Able et al. gave rats four injections of 15 mg/kg s.c. MDMA at 2-hour intervals, 12 days prior to acquisition training. Again, there were no significant differences between MDMA-treated rats and saline controls during acquisition training, as both groups exhibited similar decreases in escape latency over all 5 days. During the probe test on the following day, the average distance from the platform location of MDMA-treated rats was significantly greater than that of saline controls. Cohen et al. also gave rats four injections of 15 mg/kg s.c. MDMA at 2-hour intervals, but at least 2 weeks prior to 5 days of acquisition training. On the probe test the day after the last acquisition session, there were no significant differences between MDMA-treated rats and saline controls in percent time spent in the target quadrant. Skelton et al. also gave rats four injections of 15 mg/kg s.c. MDMA at 2-hour intervals, 14 days prior to acquisition training. The latency of MDMA-treated rats and saline controls decreased in a similar manner over 5 days of acquisition training. During the probe test on the following day, the average distance from the platform location of MDMA-treated rats and saline controls did not significantly differ.

The findings from the five studies above suggest that pretreatment with two doses of 20 mg/kg MDMA or four doses of 7.5 or 15 mg/kg MDMA has no later effect on spatial learning. The results from the probe test of these studies suggest that pretreatment with two doses of 20 mg/kg MDMA or four doses of 7.5 mg/kg MDMA subsequently impairs spatial reference memory, while pretreatment with four doses of 15 mg/kg MDMA has no later effect on spatial reference memory (apart from Able et al.’s findings that this dose produces spatial reference memory impairments).

The remainder of the studies investigated the consequences of administering multiple daily injections of MDMA on multiple days prior to acquisition training. Camarasa et al. (2008) and Abad et al. (2014) treated rats with two daily injections of MDMA for 4 consecutive days. Camarasa et al. gave adult male Long Evans rats two daily injections of 15 mg/kg s.c. MDMA at a 7-hour interval for 4 consecutive days, 9 days prior to acquisition training. Unlike saline controls, the escape latency of MDMA-treated rats did not significantly decrease over 4 days of acquisition training. On the probe test the following day, MDMA-treated rats also did not spend significantly more time in the target quadrant than that predicted by random (1/4th of the total time). Abad et al. gave adolescent male Sprague-Dawley rats two daily injections of 20 mg/kg s.c. MDMA for 4 consecutive days, 1 week prior to acquisition training. The escape latency of MDMA-treated rats decreased at a faster rate than saline controls over 4 days of acquisition training. MDMA-treated rats and saline controls spent significantly more time in the target quadrant than the opposite quadrant on the probe test the next day. These two studies have opposing findings. The results of Camarasa et al. suggest that pretreatment with repeated doses of 15 mg/kg MDMA later impairs both spatial learning and spatial reference memory, while the results of Abad et al. suggest that pretreatment with repeated doses of 20 mg/kg MDMA later enhances spatial learning and has no effect on spatial reference memory. The use of different rat strains or ages (Camarasa et al. tested adult Long Evan rats and Abad et al. tested adolescent Sprague-Dawley rats) may account for this discrepancy in findings.

Busceti et al. (2008) used a similar MDMA regimen as Camarasa et al. and Abad et al., but instead gave adult male C57BL/6 mice two daily injections of 5 or 15 mg/kg i.p. MDMA at a 2-hour interval for 6 consecutive days. Acquisition training began 7 or 40 days after MDMA treatment, and at both time points, the escape latency of MDMA-treated mice did not significantly decrease over the 4 days. On the probe test, mice given 5 mg/kg MDMA injections spent significantly less percent time than saline controls in the target quadrant when tested 7 days later but not when tested 40 days later. Mice given 15 mg/kg MDMA injections spent significantly less percent time than saline controls in the target quadrant when tested 7 or 40 days later. These results suggest that pretreatment with repeated doses of 5 or 15 mg/kg MDMA subsequently results in spatial learning and spatial reference memory deficits, but spatial reference memory may return to normal by 40 days after treatment with repeated doses of 5 mg/kg MDMA only.

The next group of studies treated adult rats with MDMA for 7 consecutive days. Kermanian et al. (2012) gave adult male Sprague-Dawley rats a daily injection of 10 or 20 mg/kg i.p. MDMA for 1 week. Acquisition training began 1 week later, and unlike saline controls, the escape latency of MDMA-treated rats did not significantly decrease over the 4 days. Soleimani Asl et al. (2015) gave male and female adult Sprague-Dawley rats two daily injections of 5 mg/kg i.p. MDMA for 1 week. On the following probe test, MDMA-treated rats spent significantly less percent time in the target quadrant than saline controls. Soleimani Asl et al. (2011) and Soleimani Asl et al. (2013) gave adult male Sprague-Dawley rats two daily injections of 5, 10, or 20 mg/kg i.p. MDMA at an 8-hour interval for 1 week. Soleimani Asl et al. (2011) began 3 days of acquisition training 1 week after MDMA treatment. The probe test took place the day after acquisition training, during which MDMA-treated rats and saline controls spent the same percent time in the target quadrant. Soleimani Asl et al. (2013) began 3 days of acquisition training the day after MDMA treatment. The probe test also took place the day after acquisition training, but MDMA-treated rats spent significantly less percent time in the target quadrant than saline controls. In addition to testing the effects of a single dose of MDMA (described above), Mirzaei et al. (2013) gave another group of adult male Wistar rats two daily injections of 10 mg/kg i.p. MDMA for 1 week and began acquisition training the following day. The escape latency and path length of MDMA-treated rats were significantly higher than that of saline controls on the 1st day of training, but these values decreased significantly over the 2nd and 3rd day to a level comparable to that of saline controls.

The findings from the above studies are mixed. The results of Kermanian et al. suggest that pretreatment with repeated doses of 10 or 20 mg/kg MDMA leads to spatial learning deficits, while the results of Mirzaei et al. suggest that pretreatment with repeated doses of 10 mg/kg MDMA has no effect on spatial learning. Likewise, the results of Soleimani Asl et al. (2013, 2015) suggest that pretreatment with repeated doses of 5, 10, or 20 mg/kg MDMA leads to spatial reference memory deficits. The results of Soleimani Asl et al. (2011), however, suggest that pretreatment with repeated doses of 5, 10, or 20 mg/kg MDMA has no effect on spatial reference memory. These differences in findings could be due to the timing of training/testing relative to MDMA treatment. Soleimani Asl et al. (2013) and Mirzaei et al. began acquisition training 1 day after MDMA treatment and found learning and memory deficits, while Soleimani Asl et al. (2011) and Kermanian et al. (2012) began acquisition training 1 week after MDMA treatment and found no effects. The differences between the findings of Kermanian et al. and Mirzaei et al. could also be due to the use of different rat strains (Sprague-Dawley vs. Wistar). In all, it appears that pretreatment with MDMA for 7 consecutive days may produce spatial learning and memory deficits within the week after treatment, but not after 1 week.

Skelton et al. (2008), in addition to studying the effects of multiple MDMA injections on a single day (above), gave another group of animals the same treatment weekly. Adult male Sprague-Dawley rats were given four daily injections of 15 mg/kg s.c. MDMA at 2-hour intervals once weekly for 5 weeks. Acquisition training began 14 days later, during which the latency of MDMA-treated rats and saline controls decreased in a similar manner over all 5 days. During the probe test on the following day, the average distance from the platform location of MDMA-treated rats and saline controls did not significantly differ. These results suggest that pretreatment with repeated doses of 15 mg/kg MDMA has no subsequent effect on spatial learning or spatial reference memory.

a. Morris water maze variations

Four studies included in Table 3 used variations of the water maze to assess the effects of MDMA on spatial learning and memory. Robinson et al. (1993) conducted a spatial navigation task that is similar to the standard MWM task. Adult male Sprague-Dawley rats were given two daily injections of 10 mg/kg i.p. MDMA at a 12-hour interval for 4 consecutive days, 2 days prior to training. The location of the hidden platform changed each day of training, and therefore this phase measured working memory rather than spatial learning (see section V.B.4.a). Training lasted for 3 days, and on the 4th day the hidden platform was left in the same location as the previous day for a retention test of spatial reference memory of the platform location (similar to the probe test in the standard MWM task). The escape latencies of MDMA-treated rats and saline controls showed no significant differences over all four trials of the retention test, which suggests that pretreatment with repeated doses of 10 mg/kg MDMA has no subsequent effect on spatial reference memory.

Figure 6 portrays the findings of Galizio et al. (2014), which exemplify dose-dependent effects of MDMA on spatial learning and memory. Galizio et al. conducted a repeated acquisition/performance procedure on the MWM. Adult male Sprague-Dawley rats were trained on the acquisition and performance components of the task prior to any MDMA administration. Each training day consisted of 12 trials that alternated between acquisition and performance trials. The acquisition component (see section V.B.4.a) assessed working memory, as the platform location changed each training day but remained fixed for all trials on a particular day. The performance component corresponded to acquisition training on the standard MWM, as the platform location remained fixed over all days of training. Once rats reached criterion on training, the same procedure was repeated on-drug. Unlike the other MWM studies, MDMA was tested across a wide range of doses. Rats were given 0, 0.3, 1.0, 1.7, 3.0, or 5.6 mg/kg i.p. MDMA in a within-subjects design 15 minutes before each on-drug session. On the performance component, doses of 0.3, 1.0, and 1.7 mg/kg MDMA had no effect on escape latency while doses of 3.0 and 5.6 mg/kg MDMA significantly increased escape latency relative to saline. These results suggest that doses of 0.3, 1.0, and 1.7 mg/kg MDMA have no effect on spatial learning but doses of 3.0 and 5.6 mg/kg MDMA impair spatial learning.

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Fig. 6.

Dose-dependent effects of MDMA on a variation of the Morris water maze task. 0.3, 1.0, 1.7 mg/kg MDMA had no effect on working memory (acquisition) or spatial learning (performance), while 3 mg/kg MDMA impaired spatial learning (performance). Data redrawn with permission from Fig. 4 (middle) in Galizio et al. (2014).

Compton et al. (2011) used a constant-start training and novel-start testing procedure on the MWM. Adolescent male Long Evans rats were given a daily injection of 10 mg/kg i.p. MDMA for 6 alternating days. Rats were trained and tested as adults about 3 months later. The experiment started with constant-start training, during which the starting location of each rat and the platform location were invariable. Novel-start testing began after rats reached training criterion, which is executed in the same manner as acquisition training on the standard MWM (variable starting locations and a fixed platform location). Rats were tested for 3 days, with each day consisting of six trials, trials one, two, four, and five were constant-start trials and trials three and six were novel-start trials. The escape latency of MDMA-treated rats on novel-start trials were significantly greater than that of saline controls, suggesting that pretreatment with repeated doses of 10 mg/kg MDMA during adolescence will impair spatial learning as adults.

Edut et al. (2011) tested the effects of MDMA treatment on the dry maze test, a variation of the MWM that does not require swimming. The dry maze consists of a circular arena with 20 tiny wells arranged in a circular manner. The goal of the task is to learn the location of the single well that is filled with water. Adult male ICR mice were first trained to drink from all 20 wells, and then introduced to a procedure identical to acquisition training on the standard MWM to learn the water well location. Mice were given a single injection of 10 mg/kg i.p. MDMA and tested 7 and 30 days later. Seven days after MDMA treatment, the latency to reach the water well of MDMA-treated mice and saline controls decreased in a similar manner over all 7 days of testing. Thirty days after MDMA treatment, the latency of MDMA-treated mice was significantly higher than saline controls on days 4 and 6 of acquisition, but both groups showed significant decreases in latency over all 7 days and by the last day of acquisition there were no significant differences. These results suggest that pretreatment with a dose of 10 mg/kg MDMA has no subsequent effect on spatial learning.

2. Radial Arm Maze

As described in section V.B.3, the radial arm maze (RAM) (see Dudchenko, 2004; Quillfeldt, 2016) is a useful tool to study spatial working and reference memory in rodents. Here, we review the five studies that examined the effects of MDMA on spatial reference memory using the RAM task, which are outlined in Table 3. The methods for these studies are as previously explained; however, now the outcome variable of interest is the number of reference memory errors per trial. Reference memory errors are defined as entries into a nonbaited arm. Entries into only baited arms reflect accurate spatial reference memory of the food pellet locations.

Hernandez-Rabaza et al. (2010) gave adolescent male Long Evans rats two injections of 10 mg/kg i.p. MDMA at a 6-hour interval, 12 days prior to training. MDMA-treated rats and saline controls exhibited a similar decrease in reference memory errors over 5 days of training. The total reference memory errors during all 5 days also did not significantly differ between groups. Similarly, Ros-Simó et al. (2013) gave adolescent male CD1 mice two injections of 20 mg/kg i.p. MDMA, but on the 12th and last day of training (using the alternative methods described in section V.B.3). One injection was given immediately after training and the second was given 2 hours later. Three days later, mice were subject to an additional training session, during which MDMA-treated mice produced significantly more reference memory errors than saline controls. Although these two studies administered similar MDMA treatments, the findings of Hernandez-Rabaza et al. suggest that pretreatment with a two doses of 10 mg/kg MDMA has no later effect on spatial reference memory, while the findings of Ros-Simó et al. suggest that treatment with two doses of 20 mg/kg MDMA impairs consolidation of spatial reference memory.

Kay et al. (2010) gave adult male Sprague-Dawley rats (that were pretrained on the task) 0, 0.75, 3, or 4 mg/kg i.p. MDMA in a within-subjects design 15 minutes before each day of training. As summarized previously, a dose of 0.75 mg/kg MDMA had no effect on the mean overall percent correct, but doses of 3 and 4 mg/kg MDMA significantly decreased the mean overall percent correct relative to saline. The deficits produced by doses of 3 and 4 mg/kg MDMA are primarily attributed to reference memory impairments, as both doses significantly increased the percent of reference memory errors relative to saline, and the percent of reference memory errors were significantly higher than the percent of working memory errors. As illustrated in Fig. 5, these findings indicate that a dose of 0.75 mg/kg MDMA has no effect on spatial reference memory but doses of 3 and 4 mg/kg MDMA impair spatial reference memory.

Kay et al. (2011) and Harper et al. (2013) gave adult male Sprague-Dawley rats MDMA before and/or during training. Kay et al. gave rats four injections of 10 mg/kg i.p. MDMA at 2-hour intervals, 2 days prior to off-drug training. After 28 days of off-drug training, on-drug training began and continued for a total of 12 days. Rats were given 0 or 4 mg/kg i.p. MDMA in a within-subjects design 20 minutes before each on-drug training session. The effects of MDMA on average percent correct that were previously reported in section V.B.3 can be attributed to reference memory deficits, as the effects on average percent correct and reference memory error percentage follow the same pattern. During off-drug training, the reference memory error percentage of MDMA-treated rats decreased at a slower rate than saline controls. During on-drug training, MDMA significantly decreased the reference memory error percentage relative to saline (this impairment was also significantly smaller in rats treated with MDMA prior to off-drug training vs. saline controls).

Harper et al. (2013) gave rats four injections of 10 mg/kg i.p. MDMA at 2-hour intervals and began training after MDMA treatment. Training lasted for 47 days, with a mix of on-drug sessions (days 8, 15, 22, 28, 34, and 41) and off-drug sessions (all other days). Before the on-drug training sessions, some rats were given 4 mg/kg i.p. MDMA. Again, the effects of MDMA on average percent correct that were previously reported in section V.B.3 can be attributed to reference memory deficits, as the effects on average percent correct and reference memory error percentage follow the same pattern. The reference memory error percentage of rats pretreated with MDMA prior to all training sessions was significantly higher than saline controls on both off-drug and on-drug training days. MDMA also significantly increased reference memory error percentage during on-drug training days relative to saline. The findings of Kay et al. and Harper et al. suggest that pretreatment with four doses of 10 mg/kg MDMA subsequently impairs spatial reference memory, and a dose of 4 mg/kg MDMA also impairs spatial reference memory when on-drug.

3. Other Spatial Tasks

1. Novel Object Recognition

The novel object recognition (NOR) task (Ennaceur and Delacour, 1988) is a relatively simple test of nonspatial memory. This method is based on the natural tendency for rodents to explore a novel object more than a familiar object. Animals are first habituated to the testing environment, a box or circular arena that is typically under dim lighting, on 1 or more days prior to testing. Testing consists of two trials, a training trial and a test trial, separated by a delay ranging from 1 minute to 24 hours. This task can measure short-term memory or long-term memory, depending on the duration of the delay. Short-term memory does not require protein synthesis but long-term memory does require protein synthesis, and the transition from protein synthesis-independent to protein synthesis-dependent long-term potentiation begins about 2 hours after memory acquisition (Frey and Morris, 1997; Lu et al., 2008). Therefore, we can consider that delays of less than 2 hours measure short-term recognition memory, and delays of 2 hours or more measure long-term recognition memory.

During the training trial, the animal is presented with two identical objects (“A”), and the total time spent exploring the two objects is measured. During the test trial (following the delay), the animal is presented with one familiar object (“A”) and one novel object (“B”), and the time spent exploring each object is measured. Object exploration is defined as touching, sniffing, or directing the nose and vibrissae toward the object at a distance of less than 1 to 2 cm. Significantly more exploration of the novel object B than of the familiar object A in the test trial is an indicator of object recognition memory. A “discrimination index” or “discrimination ratio” is usually calculated to capture this data. The “discrimination index” is the difference in exploration times of the novel object B and the familiar object A, divided by the total exploration time of the two objects in the test trial. The discrimination ratio is the exploration time of the novel object B divided by the total exploration time of the two objects in the test trial. A higher discrimination index or discrimination ratio reflects greater memory retention of the familiar object.

Table 4 outlines the 22 studies that investigated the effects of MDMA on the NOR task. Similar to the MWM studies, most of the NOR studies used high doses of 3–20 mg/kg MDMA, and none used lower, typical doses of less than 3 mg/kg MDMA. For most of these studies, the rodents were pretreated with MDMA and then trained and tested on the task at a later time point. Two of the 22 studies (Ros-Simó et al., 2013; Shortall et al., 2013), however, administered MDMA on the same day as the training and/or test trials, and these studies will be discussed first.

Instead of conducting only one training trial, Ros-Simó et al. (2013) trained adolescent male CD1 mice daily for 3 days. Mice were given two injections of 20 mg/kg i.p. MDMA, one directly after the third training trial and another 2 hours later. The test trial took place 72 hours later. The discrimination index of MDMA-treated mice was significantly less than that of saline controls. These results suggest that two doses of 20 mg/kg MDMA administered after memory acquisition leads to impairments in long-term recognition memory. Shortall et al. (2013) gave young adult male Lister Hooded rats a daily injection of 10 mg/kg i.p. MDMA for 2 consecutive days. The training trial began 30 minutes after the drug injection on the 2nd day, which was followed by a 2-hour delay and then the test trial. Rats treated with MDMA did not explore the novel object more than the familiar object, and the discrimination ratio of MDMA-treated rats was significantly less than that of saline controls. This suggests that a dose of 10 mg/kg MDMA impairs long-term recognition memory when administered on the day before and the day of memory acquisition/retrieval. The findings of Ros-Simó et al. and Shortall et al. together suggest that MDMA impairs long-term recognition memory when on-drug during the memory consolidation phase.

Nawata et al. (2010) and Edut et al. (2011) gave adult male CD1 and ICR (respectively) mice a single injection of 10 mg/kg i.p. MDMA. Nawata et al. ran the training and test trials 1 or 7 days after MDMA treatment, with a 3-hour delay between trials. The discrimination indexes of MDMA-treated mice and saline controls were comparable at both 1 and 7 days posttreatment. Edut et al. ran the training and test trials 7 or 30 days after MDMA treatment, with a 24-hour delay between trials. The discrimination indexes of MDMA-treated mice and saline controls were comparable at 7 days posttreatment, but MDMA-treated mice showed significant reductions relative to saline controls at 30 days posttreatment. Together, these findings suggest that pretreatment with a dose of 10 mg/kg MDMA has no effect on long-term recognition memory up to 1 week after treatment, but deficits may arise 30 days after treatment.

The following group of studies treated rats with multiple injections of MDMA on a single day prior to testing. Figure 7 depicts the findings of Rodsiri et al. (2011), which exemplify dose-dependent effects of MDMA on nonspatial learning and memory. Rodsiri et al. gave adult male Lister Hooded rats three injections of 3 or 6 mg/kg i.p. MDMA at 2-hour intervals. Rats were tested 2 weeks later, with a 2-hour delay between trials. Rats given 3 mg/kg MDMA injections and saline controls did not exhibit differences in discrimination ratios, but the discrimination ratio of rats given 6 mg/kg MDMA injections was significantly less than that of saline controls (Fig. 7B). These findings indicate that there may be dose-dependent effects of MDMA on long-term recognition memory, as pretreatment with three doses of MDMA had no later effect at 3 mg/kg MDMA but led to memory impairments at 6 mg/kg.

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Fig. 7.

Dose-dependent effects of MDMA on novel object recognition. Pretreatment with three doses of 3 mg/kg MDMA had no effect on exploration time of the novel and familiar objects during the test trial (A) or the discrimination ratio [novel/(novel+familiar)] (B), while pretreatment with three doses of 6 mg/kg MDMA significantly decreased exploration time of the novel object (A) and the discrimination ratio (B). Data redrawn with permission from Fig. 3, A and C in Rodsiri et al. (2011).

In a similar study, Piper et al. (2008) gave young adult male Sprague-Dawley rats four injections of 10 mg/kg s.c. MDMA at 1-hour intervals. Rats were tested at 15–17 and 17–19 days after MDMA treatment with shorter 15- and 60-minute delays, respectively. The discrimination ratios of MDMA-treated rats and saline controls did not significantly differ during either test. Cohen et al. (2005), Able et al. (2006), and Skelton et al. (2008) all gave adult male Sprague-Dawley rats four injections of 15 mg/kg s.c. MDMA at 2-hour intervals. Able et al. tested their rats 30 days after MDMA treatment and Cohen et al. and Skelton et al. tested their rats at least 5 weeks after MDMA treatment. All three studies used a 1-hour delay period and found that during the test trial MDMA-treated rats and saline controls explored the novel object more than the familiar object, and both groups explored the novel object for a similar amount of time. The findings from the above studies suggest that pretreatment with four doses of 10 or 15 mg/kg MDMA has no subsequent effect on short-term recognition memory.

The remaining studies treated animals with MDMA over several days prior to testing. Morley et al. (2001) and McGregor et al. (2003) treated adult male Wistar rats with MDMA for 2 consecutive days. Morley et al. gave rats one or four (at 1-hour intervals) daily injections of 5 mg/kg i.p. MDMA for 2 consecutive days. Rats were trained and tested 14 weeks later with a 15-minute delay and again 1 more week later with a 60-minute delay. The discrimination ratio of rats given one daily injection did not significantly differ from that of saline controls at either delay. The discrimination ratio of rats given four daily injections did not significantly differ from that of saline controls at the 60-minute delay, but was significantly less than saline controls at the 15-minute delay. McGregor et al. also gave rats four daily injections of 5 mg/kg i.p. MDMA at 1-hour intervals for 2 consecutive days. Approximately 10–12 weeks later, rats were tested with a 1-hour delay between trials. Two “preliminary” days of testing were conducted followed by a third identical day of testing that provided the reported data. The discrimination ratio of MDMA-treated rats was significantly less than that of saline controls. In all, the findings from these two studies suggest that treatment with one dose of 5 mg/kg MDMA for 2 consecutive days has no subsequent effect on short-term recognition memory, while treatment with four doses of 5 mg/kg MDMA for 2 consecutive days may impair short-term recognition memory (with the exception of Morley et al.’s findings at the 60-minute delay).

Abad et al. (2014) gave adolescent male Sprague-Dawley rats two daily injections of 20 mg/kg s.c. MDMA for 4 consecutive days. Rats were tested 1 week later with a 1-hour delay between trials. MDMA-treated rats and saline controls explored the novel object for significantly more percent of the total exploration time than the familiar object. These findings indicate that pretreatment with repeated doses of 20 mg/kg MDMA may have no subsequent effect on short-term recognition memory.

In addition to testing a single dose of MDMA, Nawata et al. (2010) gave another group of adult male CD1 mice a daily injection of 10 mg/kg i.p. MDMA for 1 week. These mice were also tested 1 or 7 days later with a 3-hour delay between trials. The discrimination index of MDMA-treated mice was significantly less than that of saline controls at 1 and 7 days posttreatment; however this value was significantly above chance at 1 day posttreatment. Although this study found that pretreatment with a single dose of 10 mg/kg MDMA had no effect on long-term recognition memory (see above), these additional findings suggest that pretreatment with repeated doses of 10 mg/kg MDMA may lead to long-term recognition memory deficits, with more pronounced deficits 1 week after treatment versus 1 day.

van Nieuwenhuijzen et al. (2010) and Kolyaduke and Hughes (2013) treated rats with a daily injection of MDMA for 10 consecutive days. van Nieuwenhuijzen et al. treated adult male Wistar rats with a daily dose of 5 mg/kg i.p. MDMA. Rats were tested 6 weeks after MDMA treatment with a 1-hour delay between trials. The discrimination ratio of MDMA-treated rats was significantly less than that of saline controls. Kolyaduke and Hughes treated male and female adolescent PVG/c hooded rats with a higher daily dose of 10 mg/kg i.p. MDMA during early adolescence (postnatal days 35–45) or late adolescence (postnatal days 45–55). Both groups were tested as adults at no less than 90 days old (around 5–8 weeks postdrug) with a short 15-minute delay between trials. The exploratory behavior of MDMA-treated rats and saline controls during the test trial led to similar discrimination indexes. The findings from these two studies demonstrate that pretreatment with repeated doses of MDMA may lead to short-term recognition memory impairments at doses of 5 mg/kg but surprisingly may have no effect at doses of 10 mg/kg. This unexpected outcome may be because van Nieuwenhuijzen et al. and Kolyaduke and Hughes tested rats from different strains, ages, and sexes.

García-Pardo et al. (2017) gave adolescent male OF1 mice four injections of 10 mg/kg i.p. MDMA over 2 weeks, one on each of postnatal days 55, 57, 60, and 62. Testing took place on postnatal day 64, 2 days after MDMA treatment, with an ultra-short 1-minute delay between trials. The discrimination indexes of MDMA-treated mice and saline controls did not significantly differ, suggesting that pretreatment with four doses of 10 mg/kg MDMA may have no later effect on short-term recognition memory.

The following group of studies treated adolescent rats with MDMA every 5 days (for a specific number of total days), with multiple injections given on each treatment day. Llorente-Berzal et al. (2013) gave male and female Wistar rats two injections of 10 mg/kg s.c. MDMA at a 4-hour interval every 5 days from postnatal day 30–45. Rats were tested 1 month later as adults on postnatal day 75 with a 4-hour delay between trials. There were no significant differences between the discrimination indexes of MDMA-treated rats and saline controls. These findings demonstrate that pretreatment with repeated doses of 10 mg/kg has no subsequent effect on long-term recognition memory. Piper and Meyer (2004) gave male Sprague-Dawley rats two injections of 10 mg/kg s.c. MDMA at a 4-hour interval every 5 days from postnatal day 35 to 60. Rats were tested 1 week later with a 15-minute delay between trials. The discrimination ratio of MDMA-treated rats was significantly less than that of saline controls. Piper et al. (2005) gave male Sprague-Dawley rats four injections of 5 mg/kg s.c. MDMA at 1-hour intervals every 5 days from postnatal day 35 to 60. Rats were tested 1 week later (postnatal day 67) with a 15-minute delay and again 1 to 2 days later (postnatal day 68 or 69) with a 30-minute delay. There were no significant differences between the discrimination ratios of MDMA-treated rats and saline controls under either delay condition. The findings from these two studies are mixed; the results of Piper et al. suggest that pretreatment with repeated doses of 5 mg/kg MDMA has no subsequent effect on short-term recognition memory, while the findings of Piper and Meyer suggest that pretreatment with repeated doses of 10 mg/kg produces short-term recognition memory deficits. Although the same cumulative daily doses were given, the difference in number and dose of injections (two daily injections of 10 mg/kg vs. four daily injections of 5 mg/kg) could account for this discrepancy in findings.

The next group of studies treated animals with MDMA over several weeks. Clemens et al. (2007) gave adult female Wistar rats a single injection of 8 mg/kg i.p. MDMA once weekly for 16 weeks. Two days of testing were performed 8 weeks after MDMA treatment (with 1 day between the 2 days), and a 20-minute delay was used for both tests. The discrimination ratio of MDMA-treated rats and saline controls did not significantly differ during either test. In addition to testing the effects of multiple MDMA injections on a single day (see above), Skelton et al. (2008) gave another group of adult male Sprague-Dawley rats the same treatment of four injections of 15 mg/kg s.c. MDMA at 2-hour intervals once weekly for 5 weeks. Again, rats were tested 5 weeks after the MDMA treatment with a 1-hour delay period. MDMA-treated rats and saline controls explored the novel object more than the familiar object, and there were no significant differences between groups. Costa et al. (2014) gave male C57BL/6 mice two injections of 10 mg/kg i.p. MDMA at a 4- to 6-hour interval on the 2nd and 5th days of each week for 9 weeks (which started in adolescence and continued into adulthood). Mice completed a total of 5 days of testing—on the 6th day of drug treatment weeks 1, 4, and 9 and posttreatment weeks 2 and 3. A 1-hour delay was used for all five tests. The discrimination ratio of MDMA-treated mice and saline controls did not significantly differ during drug treatment weeks 1, 4, or 9 but was significantly reduced in MDMA-treated mice compared with saline controls during posttreatment weeks 2 and 3. The findings of Clemens et al. and Skelton et al. suggest that pretreatment with repeated doses of 8 or 15 mg/kg MDMA has no subsequent effect on short-term recognition memory. On the other hand, the findings of Costa et al., suggest that pretreatment with repeated doses of 10 mg/kg MDMA has no effect on short-term recognition memory 1 day posttreatment but produces impairments by 2 weeks posttreatment. This discrepancy may be because Costa et al. used mice as subjects rather than rats, or possibly because the mice were tested repeatedly throughout drug treatment.

Schulz et al. (2013) treated adolescent and adult male Wistar rats with a varying number of s.c. MDMA injections over 25 days. A single injection of 7.5 mg/kg MDMA was given on 10 of the 25 days, two injections of 7.5 mg/kg MDMA were given at a 4-hour interval on 5 of the 25 days, and no drug was given on 10 of the 25 days (treatment schedule was randomized). All rats were tested 10 days after the 25-day treatment period, and the adolescent rats were tested again as adults 6 weeks after the first test. Unlike the other NOR studies reviewed here, only one object was presented during the training trial, but the remainder of the methods were as described above. A 25-minute delay was placed between the training and test trials. The adult saline controls explored the familiar object significantly less in the test trial than the same object in the training trial, and significantly less than the novel object in the test trial. The adolescent saline controls, however, explored all three objects for a comparable amount of time during the first test. Because the adolescent saline controls did not exhibit normal recognition memory, the effects of MDMA cannot be accurately determined. During the second test, the adolescent saline controls explored the familiar object significantly less in the test trial than the same object in the training trial (but not significantly less than the novel object in the test trial). The adult and adolescent MDMA-treated rats explored all three objects for comparable amounts of time during all tests. These results suggest that pretreatment with repeated doses of 7.5 mg/kg MDMA during adulthood impairs short-term recognition memory, and the same treatment during adolescence may produce some deficits as in adults (but the effects during adolescence cannot be determined).

Abad et al. (2016) gave adolescent male C57BL/6 mice three injections of s.c. MDMA at 1-hour intervals once weekly for 8 weeks—at doses of 5 mg/kg MDMA for the first 2 weeks, 7.5 mg/kg MDMA for the next 3 weeks, and 10 mg/kg MDMA for the last 3 weeks. Mice were tested as adults, 1 week and 3 months after MDMA treatment with 1- and 24-hour delays. The discrimination indexes of MDMA-treated mice and saline controls did not significantly differ with a 1-hour delay but was significantly reduced in MDMA-treated mice compared with saline controls with a 24-hour delay at both 1 week and 3 months posttreatment. These findings suggest that pretreatment with repeated doses of MDMA (increasing from 5 to 10 mg/kg) has no subsequent effect on short-term recognition memory but may lead to long-term recognition memory deficits.

2. Other Nonspatial Tasks

1. Passive Avoidance

The passive avoidance (PA) task is a fear-motivated task that is used to evaluate learning and memory in rodents. A common version of this task is the step-through PA task (Jarvik and Kopp, 1967), which takes place in a two-compartment chamber consisting of one bright (e.g., illuminated, white walls) compartment and one dark (e.g., nonilluminated, black walls) compartment connected by a guillotine door. The task requires animals to inhibit their natural tendency to prefer dark areas/avoid bright areas to avoid an aversive stimulus. Each animal is first habituated to both compartments of the chamber as well as crossing through the guillotine door prior to training. Training is usually completed in a single trial, which begins by placing the animal in the bright compartment with the guillotine door closed. After a brief period, the guillotine door is opened, and once the animal enters the dark component, the guillotine door is closed, and the animal receives an inescapable foot shock. Testing typically takes place 24 hours after training, during which the animal is returned to the bright compartment, and again the guillotine door is opened after a brief period. If the animal remembers that entering the dark compartment lead to a foot shock during training, then the animal will inhibit its natural tendency to enter the dark compartment.

The step-through latency (i.e., time taken to enter the dark compartment once the guillotine door is opened) is measured during both the training and test trials, and the cutoff time/maximum latency recorded is usually 300 seconds. A significant increase in step-through latency from training to testing reflects normal memory retention, whereas the lack of this increase reflects memory deficits. A significantly lower step-through latency relative to normal during testing is also an indicator of memory deficits. The type of memory measured here involves both explicit memory (i.e., association with the context) and implicit memory (i.e., operant conditioning to the shock).

Table 5 lists the 12 studies that explored the effects of MDMA on the PA task. Eleven of these studies conducted the step-through PA task, whereas only one study (McNamara et al., 1995) performed another version, the step-down PA task (methods described below). The animals from most of these studies were treated with MDMA 1 or more days prior training, 30 minutes before training, and/or immediately after training. All of the studies evaluated long-term memory as delays of 24 hours or more were placed between the training and test trials.

Moyano et al. (2004, 2005) and Barrionuevo et al. (2000) all tested the effects of on-drug training. Adult male Wistar rats were given a single injection of MDMA 30 minutes before training and then tested 24 hours later. Moyano et al. (2004, 2005) found that rats injected with 10 mg/kg i.p. MDMA before training exhibited a significantly lower step-through latency than saline controls during testing, and Barrionuevo et al. (2000) found the same results with a dose of 20 mg/kg i.p. MDMA. These findings suggest that doses of 10 and 20 mg/kg MDMA produce long-term memory deficits when memory acquisition occurs on-drug.

Shariati et al. (2014) and Budzynska et al. (2017) explored the effects of administering the drug immediately after training. Figure 8 exhibits the findings of Budzynska et al., which exemplify the dose-dependent effects of MDMA on fear-motivated learning and memory. Budzynska et al. gave adult male Swiss Webster mice a single injection of 1, 2.5, 5, or 10 mg/kg i.p. MDMA immediately after training and tested the mice 24 hours later. The step-through latency of mice treated with 1 or 10 mg/kg MDMA did not significantly differ from that of saline controls, while mice treated with 2.5 or 5 mg/kg MDMA showed a significantly higher step-through latency than saline controls. Shariati et al. tested two groups of adult male Wistar rats—one group received a single injection of 10 mg/kg i.p. MDMA following two training trials, and another group received a daily injection of 10 mg/kg i.p. MDMA on 2 consecutive days per week for 3 weeks, with the last injection administered immediately following training. All rats were tested 24 hours after MDMA treatment, and both groups of MDMA-treated rats demonstrated a significantly shorter step-through latency than saline controls. Together, the above results indicate that administering MDMA immediately after memory acquisition has no effect on long-term memory retention at a dose of 1 mg/kg but enhances long-term memory retention at doses of 2.5 or 5 mg/kg. The findings regarding a dose of 10 mg/kg MDMA are mixed, as Budzynska et al. found that this dose has no effect on long-term memory retention, while Shariati et al. found that single or repeated administration of this dose impairs long-term memory retention. This discrepancy could be due to the use of different species (mice vs. rats) or the number of training trials (one vs. two).

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Fig. 8.

Dose-dependent effects of MDMA on passive avoidance. Posttraining doses of 2.5 and 5 mg/kg MDMA enhanced long-term memory, while posttraining doses of 1 and 10 mg/kg MDMA had no effect on long-term memory. Data redrawn with permission from Fig. 5 in Budzynska et al. (2017).

Jahanshahi et al. (2013) also treated young adult male Wistar rats with MDMA between training and testing, but the MDMA treatment began 24 hours after two training trials and lasted for 4 weeks. Rats were given three injections of 2.5, 5, or 10 mg/kg i.p) MDMA at 3-hour intervals once weekly for 4 weeks. Testing took place following drug treatment, and all three groups of MDMA-treated rats exhibited a significantly longer step-through latency than saline controls. These results reveal that treatment with doses of 2.5, 5, and 10 mg/kg MDMA after acquisition may enhance long-term memory retention.

The next group of studies treated animals with a specific MDMA regimen prior to training and testing. Timár et al. (2003) and Murnane et al. (2012) gave animals four injections of MDMA at 2-hour intervals. Timár et al. gave adolescent male Wistar rats doses of 10 mg/kg s.c. MDMA and tested them 3 days and 4 weeks after MDMA treatment, with 48 hours separating training and testing. During both tests, MDMA-treated rats and saline controls did not significantly differ in step-through latency. Murnane et al. gave adolescent male Swiss Webster mice doses of 10 or 20 mg/kg i.p. MDMA. Rats were trained 2 days after MDMA treatment and tested 2 days after training. Again, MDMA-treated rats and saline controls did not significantly differ in step-through latency. In all, these studies reveal that pretreatment with four doses of 10 or 20 mg/kg MDMA has no subsequent effect on long-term memory retention.

In addition to testing the effects of on-drug training (see above), Moyano et al. (2005) gave another group of adult male Wistar rats two daily injections of 10 mg/kg i.p. MDMA for 4 consecutive days. Rats were trained 1 week after MDMA treatment and tested 24 hours after training. The step-through latency of MDMA-treated rats and saline controls did not significantly differ. Moyano et al. tested an additional group, which received both of the previously described treatments, two daily injections of 10 mg/kg i.p. MDMA for 4 consecutive days, 1 week prior to training, and a single injection of 10 mg/kg i.p. MDMA, 30 minutes before training. This group of MDMA-treated rats exhibited a significantly slower step-through latency than saline controls. Together these findings suggest that pretreatment with repeated doses of 10 mg/kg MDMA has no subsequent effect on long-term memory retention, while a single dose of 10 mg/kg MDMA impairs long-term memory retention when memory acquisition occurs on-drug.

García-Pardo et al. (2015, 2017) gave adolescent male OF1 mice a single injection of 10 mg/kg i.p. MDMA on four alternating days (2 to 3 days between each injection). García-Pardo et al. (2015) trained their mice 4 days after MDMA treatment and García-Pardo et al. (2017) trained their mice 5 days after MDMA treatment, and all mice were tested at 24 hours and 1 week after training. Both studies had identical findings—the step-through latency of MDMA-treated mice did not change significantly from training to testing (24 hours and 1 week later), and the step-through latency of MDMA-treated mice was significantly shorter than that of saline controls at 1 week after training (but not at 24 hours). These findings suggest that pretreatment with four doses of 10 mg/kg MDMA leads to long-term memory impairments that are more significant at 1 week versus 24 hours after acquisition.

Rodríguez-Arias et al. (2011) gave adolescent male OF1 mice two daily injections of 10 or 20 mg/kg i.p. MDMA at a 4-hour interval on 2 consecutive days per week for 2 weeks. Mice were trained 22 days after MDMA treatment and tested 24 hours later. The step-through latency of MDMA-treated mice and saline controls did not significantly differ, revealing that pretreatment with repeated doses of 10 or 20 mg/kg MDMA may have no subsequent effect on long-term memory.

Unique to the other studies reviewed here, McNamara et al. (1995) tested adult male Sprague-Dawley rats on the step-down version of the PA task. Rats were given two daily injections of 5, 10, or 20 mg/kg i.p. MDMA at a 12-hour interval for 4 consecutive days and tested 6 days later. On each trial, a rat was placed on a triangular platform that was mounted above a grid floor. When the rat stepped off the platform, it received a foot shock. Repeated trials were conducted until the rat remained on the platform for at least 2 minutes. MDMA-treated rats and saline controls did not significantly differ in the number of trials it took for them to reach this threshold, suggesting that pretreatment with repeated doses of 5, 10, or 20 mg/kg MDMA has no effect on memory function.

2. Contextual Fear Conditioning

The contextual fear conditioning (FC) paradigm (Fanselow, 1986; Anagnostaras et al., 1999, 2010, 2015) is an efficient model to measure hippocampal-dependent learning and memory in rodents. In contextual FC, an animal learns to associate an aversive stimulus (typically a foot shock) with a specific context. As a result, the initially neutral context elicits a fear response in the animal. In rodents, this fear response arises as freezing behavior, which is a measure of contextual fear memory. Thus a significant decrease in freezing is indicative of memory deficits. Two studies explored the effects of MDMA on contextual FC and are outlined in Table 5. These studies used two different variations of the typical FC procedure, which are described below.

Shortall et al. (2013) conducted a “conditioned emotional response” task, which is a variation of the contextual FC task. The task took place in a two-compartment box that consisted of a dark side and a light side separated by a computer-operated door. For training, each animal was placed on the light side of the box, and after 30 seconds the door was opened. When the animal entered the dark side of the box, the door was closed, and the animal was subject to two light/tone and foot-shock pairings (a 5-second light and tone cue that coterminated with a 1-second foot shock) with a 1-minute interval between pairings. For testing, each animal was returned to the dark side of the box, and freezing was measured for 5-minute without any light/tone or foot shock presentation. In this study, young adult male Lister Hooded rats were given a single injection of 10 mg/kg i.p. MDMA on experiment days 1, 2, and 8. Rats were trained on experiment day 8 immediately prior to MDMA treatment and tested 24 hours later. MDMA-treated rats and saline controls did not significantly differ in freezing time during the test. These results suggest that treatment with repeated doses of 10 mg/kg MDMA prior to and following memory acquisition has no effect on long-term context memory.

Johansson et al. (2015) performed another variation of the contextual FC task, contextual fear discrimination. This task took place in two contexts, Context A and Context B, which differed by a variety of sensory modalities (different floor/walls, noise, illumination, and scent). Training took place in Context A, and each animal completed one training trial per day for 3 days. For each trial, the animal was introduced to Context A, and after a 3-minute baseline period they received a 2-second foot shock and then remained in the context for an additional 15 seconds. Testing began 3 days later, and each animal was exposed to both Context A and Context B on all 12 days of testing (random order of exposure with a 1.5- to 2-hour interval between each exposure). The trials in Context A were identical to training (3-minute baseline + 2-second foot shock + 15-second postshock period), and the trials in Context B were 3 minutes in duration with no foot shock. In this study, adult male ICR mice were given two injections of 20 mg/kg i.p. MDMA at a 2-hour interval, 4 days prior to training. The freezing behavior of saline controls increased significantly in Context A and decreased significantly in Context B over the 12 days of testing and overall was significantly greater in Context A than in Context B on the last 8 days of testing. Conversely, the freezing behavior of MDMA-treated rats remained constant over the 12 days of testing and did not significantly differ between Context A and Context B. These results suggest that pretreatment with two doses of 20 mg/kg MDMA leads to later deficits in learning to discriminate between two contexts.