A drug of abuse, Foxy or Methoxy Foxy gained popularity among recreational users as an alternative to MDMA (Ecstasy). Considerable research into the consequences of MDMA use is available, yet much remains unknown about the neurobiological consequences of Foxy use. In addition, research into the long-term neuropsychological repercussions associated with these two compounds remains incomplete. The goal of the present research was to explore the effects of MDMA or Foxy on cognitive processes associated with adolescent exposure considered over much of the lifespan. Here we investigated whether the reported effects following adolescent exposure resolved in early adulthood or continued throughout life. The protocol involved repeated doses of either MDMA or Foxy during the period defined as mid-adolescence (postnatal days 34 - 46) in rats, followed by the use of four series of learning and memory tasks repeated at different points in the rodent lifespan. At four time points in adulthood, the animals were trained and tested on a on a series of spatial and non-spatial memory tasks designed to assess the impact and severity of Foxy and MDMA. Oddly, MDMA-treated rats were impaired on a step down passive avoidance task. The performance of the drug-treated rats was markedly inferior to that of the control animals on more demanding water maze tasks, with some results suggesting a lack of flexibility in adapting to changing task demands. MDMA rats were the most impaired. While some persistent cognitive deficits were found, no significant group differences in serotonin or dopamine levels were found in any of the measured regions of the brain changes, cortical or subcortical. These results provide evidence for compromised neurocognition that continues long after drug exposure in the absence of any discernable changes in neurotransmitter levels. Several possible physiological and neurochemical mechanisms associated with these compounds requiring further study are also outlined.
While adolescence is time of considerable neurodevelopmental change, it is also characterized as a period marked with significant risk-taking behavior [
The recreational use of MDMA is associated with reported effects including euphoria [
Indolealkylamines (IAA) are a class of chemical derivatives of 5-hydroxy- tryptamine (5-HT, serotonin) [
Research into the effects of Foxy with rodent models have led to reports of Foxy-associated deficits [
Although the available evidence is considerable, most of the measures of behavioral performance occur within the confines of a short-term investigation or cross-sectional comparison [
Rat adolescence is defined as the period from the 21st postnatal day (PND) until about PND 60 [
Developmental investigations of the effects MDMA, suggest the age of exposure to be a critical variable [
Despite efforts to reduce the use MDMA, including the introduction of more effective restrictions in precursor control [
While the studies discussed here are suggestive, only limited data exists on the long-term consequences associated with MDMA or Foxy use, with little information available that longitudinally tracks the effects of these compounds across an extended period of time. Generally, the use of assessments having occurred at a specific time point in the life of the research subject, and using different methodologies, make age-related progression of deterioration associated with these compounds, if any, a challenge. Although there is evidence of persistent deficits following adolescent exposure of these two compounds [
Twenty-one male Long-Evans rats Wistar rats (~100 g at first injection) were used to assess the long-term effects of MDMA or Foxy. The rats were individually housed in standard stainless-steel cages under standard laboratory conditions of lighting (07:00-19:00 h), with an ambient temperature (19˚C - 21˚C) and humidity between 45% and 50%. Food and water were freely available throughout the investigation. After a one week facility acclimation period and prior to the beginning of the experiment, the animals were randomly assigned to one of two drug treatment conditions or a saline control group. From 35 to 45 days of age, the rats were exposed to MDMA, Foxy, or a corresponding volume of saline. Behavioral testing occurred in adulthood when the rats were 111 days old and had been drug free for 66 days. Behavioral testing included four multi-day assessment periods beginning at PNDs 111, 219, 327, and 435. Data collection was performed during the light period (12:00-15:00 h). All procedures were conducted according to the guidelines approved by the Institutional Animal Care and Use Committee of Palm Beach Atlantic University utilizing standards outlined in the Guide for the Care and Use of Laboratory Animals [
Drug exposure began when the rats were in the mid-adolescent period of development (i.e., 35 days old). The rats received a total of six injections of MDMA (5 mg/kg; Sigma-Aldrich, St. Louis, MO), Foxy (5 mg/kg; Biosynth International, Naperville, IL), or a corresponding injection volume of isotonic saline. Drugs were injected intraperitoneally (IP) at a constant volume of 2 ml/kg. The injections were delivered at a rate of one injection session every 48 hours. Initial behavioral testing occurred in adulthood when the rats were 111 days old and had been drug free for 66 days.
All variations of spatial and nonspatial assessments took place in a circular white acrylic plastic swimming pool 183 cm in diameter. Depending on the assessment, different extra-maze cues and escape parameters were employed. 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 an opaque white color using a nontoxic water-based paint (Sargant Art, Hazelton, PA). The swimming pool and associated cues were located in a quiet testing room approximately 36.88 square meters in size. The number of external stimuli available to aid navigation when viewed from the surface of the pool was limited by the use of white curtain panels that surrounded the pool and obscured distal cues on two of four walls. Except on probe trial tests, a flat white escape platform (15 cm × 15 cm) was used throughout all phases of training and testing. The platform was located 18 cm from the wall of the swimming pool, thus necessitating that the rat swim away from the swimming pool wall in order to locate the platform. For the cued water maze task described below, the platform projected 15 mm above the surface of the water. For all other phases of the experiment, the escape platform was submerged to a depth of 15 mm below the surface of the water.
Rodent general levels of activity were measured across two 5 min periods (one per day) in a 60.96 cm × 60.96 cm chamber consisting of alternating black and white 10.16 cm squares. Activity measures included the number of squares crossed during the measurement period and the number of times the rats reared onto their hind legs. Lastly, motivational and sensorimotor deficits were assessed using a cued version of the MWM task described below.
Step-down passive avoidance testing took place in a standard operant chamber (21-cm × 28-cm; Lafayette Model 84,022) with a stainless steel electrified grid floor. A 10.14-cm × 10.14 cm platform was located in the center of the chamber. Whenever the rat left the platform, physically touching the grid floor, a 4 mA current of foot shock was delivered.
Water maze protocols were employed to assess rodent learning and memory. For the cued place learning task, the platform was 15 mm above the water’s surface; For the rest of the tasks, the platform was submerged to a depth of 15 mm below the surface of the water (the place & spatial learning set tasks). On a given trial during the spatial segments of the experiment, the rat was gently released into the pool at one of four compass points, labeled west, east, north, or south, and allowed a maximum of 60 sec per trial to reach the escape platform. The platform location was positioned at one of four compass positions―southwest, southeast, northeast, or northwest. Escape times to the platform were recorded with a stopwatch and errors, operationally defined as crossing one of four quadrants associated with the four cardinal compass points, were recorded.
1) Simple (Cued) Place Learning
The cued place learning MWM navigation task was administered the day after step-down passive avoidance testing. Using a visible escape platform, this phase allowed for the assessment of nonassociative influences―general swimming ability, motivational deficiencies, and nondeclarative memory ability―that could influence performance during the spatial place and learning set tasks. The task included two days with 10 trials per day with the escape platform located in one of four possible locations. After successfully navigating to the platform, the rats were allowed to rest on it for about 15 sec before the next trial.
2) Spatial Water Maze Tasks
The next two phases of the protocol were tests of spatial reference memory that varied in difficulty. The tasks involved learning the location of a submerged platform that remained constant across all trials within a given phase of the experiment. Because often only minor deficits are typically seen using the standard (high cue) version of this test [
A more simple (high cue) version of the place-learning task lasted two days and consisted of training the rats for 10 trials per day. As earlier, the rats were allowed to remain on the platform for 15 sec at the completion of each trial. Further, a test of retention was conducted through the use of a probe trial on the second day. This assessment consisted of removing the escape platform and testing the subject for a 60 sec “free swim” not less than two hours after the last place learning trial. Time spent swimming in the target quadrant and the number of crossings over the former platform location were recorded.
The subsequent phase began the following day. A low-cue version of the place-learning task, considered to be more difficult, was accomplished through a reduction in the availability of extra-maze cues to aid navigation. The rats were trained during four consecutive trials per day Task difficulty was increased by placing a white curtain around the water maze as well as a single 60-watt red light bulb, located beyond the curtain and below the horizon of the pool, approximately three meters from the water maze. This effectively left the rat with few visual cues to aid navigation. After successfully locating the platform, the rats were allowed to remain and rest for 15 sec. Lastly, a daily probe trial was administered not less than two hours after the last trial of the daily four-trial series.
3) Spatial Learning Set
A final phase of testing, learning set acquisition, required the animals to learn a new escape platform location daily for five consecutive days. All animals received four consecutive trials per day. Since this task requires the animal to recall its response on the immediately preceding trial, the averaged performance on Trial 2 of each day was used as an index of working (short-term) memory. As before, the rats were allowed to sit on the platform for 15 sec at the completion of each trial.
One week after the completion of the last behavioral testing phase of the experiment, the animals were sacrificed by cervical fracture. Immediately thereafter, the brains were removed, the hippocampus, prefrontal cortex and striatum were dissected from 1 mm coronal sections, frozen on dry ice and stored at −80˚C. Subsequently, the tissue samples were homogenized in cold 0.2 N perchloric acid (Fisher Scientific) with the resulting homogenates centrifuged for 5 minutes at 14,000 rpm. In an analysis of biogenic amines, the supernatant was divided into aliquots for the analysis of biogenic amines dopamine and 5-HT. The aliquots of the supernatant were injected onto a C-18 reverse phase column connected to an LC amperometric detector (Bioanalytical Systems, West Lafayette, IN) fitted with a glassy carbon target electrode. The mobile phase for the separation of dopamine and 5-HT consisted of the following―100 mM citric acid, 75 mM sodium phosphate, 50 mg/l disodium ethylenediamine tetraacetate, 176 mg/l octane sulfonic acid sodium salt, 15% methanol (pH 4.2), pumped at a flow rate of 0.8 ml/min. Peak height determinations were recorded with an integrator, with calculations of the quantities of dopamine and 5-HT determined on the basis of known standards. Statistical analysis of tissue dopamine and 5-HT concentrations were considered in units expressed in ng/mg.
For the step-down passive avoidance task, step-down latencies were analyzed for the two-day period. For all MWM tasks, escape latencies and navigation errors were the two primary measures of performance. Owning to the fact that optimal swim path distances differed depending on the various start and escape locations, the recorded escape latencies for the four start locations were normalized. Normalization was accomplished by computing the ratio of the minimum swim distance in cm for each of the two longer swim paths to the escape platform (e.g., a south start location and a northeast 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. In order to meet the ANOVA assumptions, the time data were transformed using the reciprocal transformation. Post-hoc analyses were performed using TukeyHSD or paired t-tests with a Bonferroni correction to control for multiple comparisons. A priori alpha level for acceptance was set at p < 0.05 and the data analyzed using SPSS [
Assessment of the activity data revealed no group differences in the number of squares traversed or in the number of rearings during the measurement period.
The step-down passive avoidance data for each measurement period is presented in
With a few exceptions, the results in the second test period were similar to that in the first period. Once again, main effects of drug group, F(1, 18) = 43.67, p < 0.001, η p 2 = 0.708, and day of testing were found, F(2, 18) = 5.49, p < 0.025, η p 2 = 0.379, but post hoc examination of the drug group means indicated that only the MDMA latencies differed from those of the saline-treated rats. Finally, the drug group × day interaction was significant, F(2, 18) = 5.60, p < 0.025, η p 2 = 0.379. Post hoc examination of the means (see
When the animals were assessed for the 3rd period, as before, the main effects of days, F(1, 18) = 186.19, p < 0.001, η p 2 = 0.912, and drug groups, F(2, 18) = 17.56, p < 0.001, η p 2 = 0.661, as well as drug group X days interaction, F(2, 18) = 20.80, p < 0.001, η p 2 = 0.698, were all significant. Simply, stepdown latencies were longer on day 2 and stepdown latencies of the MDMA-treated rats were significantly different from that of both the Foxy- and saline-treated animals, with the latter two not significantly different. However, unlike at earlier assessment periods, all three groups remained on the escape platform significantly longer on day 2, although, as suggested by the interaction, escape latencies on this day were significantly longer for the saline- and Foxy-treated animals than rats in the MDMA group (see
It should be noted that three rats, one from each group had died during the period between the 3rd and 4th assessment periods. As is evidenced by the group and drug group × days interaction, both nonsignificant, no drug associated effects were found at the fourth measurement point. Only the main effect of days was significant, with stepdown latencies consistently longer on day 2 for all groups, F(1, 15) = 228.24, p < 0.001, η p 2 = 0.927.
For the cued place learning data, the escape latencies were analyzed using 3 (drug groups) × 2 (blocks of trials) × days (of testing) mixed ANOVAs, with the latter two factors as within-subjects effects. An examination of Period 1 revealed a main effect of blocks, F(1, 18) = 34.72, p < 0.001, η p 2 = 0.659, with latencies improving by the second block of training. Similarly, the main effect of days was
significant, F(1, 18) = 51.23, p < 0.001, η p 2 = 0.740, as was the drug group X days, F(2, 18) = 11.57, p < 0.01, η p 2 = 0.562. The remaining interactions were nonsignificant. Closer examination of the group X days interaction revealed that latencies between the saline- and Foxy-treated rats differed on day two of the first assessment period.
By the 2nd assessment period, all differences among the three groups and evidenced by the lack of either a drug main effect or interaction (i.e., all ps > 0.05). As the main effect of blocks, smallest F(1, 15) = 12.97, p < 0.01, η p 2 = 0.464, and days, smallest F(1, 15) = 17.96, p < 0.001, η p 2 = 0.545, suggested, the animals generally performed better on latter trials and days of training.
When the easy place learning data were considered, drug effects, if any, resolved over assessment periods. During the first phase of testing, only the main effects of days, F(1, 18) = 19.59, p < 0.001, η p 2 = 0.521, and blocks, F(1, 18) = 27.22, p < 0.001, η p 2 = 0.602 were significant, each suggestive of improvements as a function of the number of trials. However, assessment of the probe trial data revealed a significant drug effect, F(2, 18) = 10.28, p < 0.01, η p 2 = 0.533. Subsequent TukeyHSD tests revealed that the saline rats spent significantly more time in the target quadrant (M = 33.15 sec) than either of the drug groups both of which spent similar amounts of time in the target quadrant (Ms = 21.99, Foxy & 22.59, MDMA).
Reviewing the 2nd assessment phase, with the exception of a significant effect of drug group, F(2, 18) = 14.38, p < 0.001, η p 2 = 0.615, no additional main effects or interactions were found. Interestingly, consideration of the main effect revealed that escape times were significantly higher in the Foxy group (M = 3.91 sec) than in the MDMA and saline groups. Here, the escape times of the latter two groups were comparable (Ms = 2.82 & 2.68). Nonetheless, probe trial assessment of time spent in the target quadrant differed by drug group, F(2, 18) = 27.64, p < 0.001, η p 2 = 0.754 and, once again, TukeyHSD tests revealed that the saline rats spent significantly longer in former escape platform quadrant (M = 38.01) than either drug group (Ms = 24.21 & 21.26, MDMA & Foxy).
By the 3rd assessment period, all group differences associated with either main effects or interactions were absent. In addition, as evidenced by similar times spent in the target quadrant, probe trial performances were comparable. This pattern persisted in the 4th assessment period as well.
Data from the four hard place learning assessment periods are presented in
an improvement in escape latencies across the assessment period. In addition, escape latencies were comparable on the probe trials.
A drug-related divergence in performance began to emerge in the 2nd assessment period. Here, the main effect of days was significant, F(4, 72) = 7.80, p < 0.001, η p 2 = 0.302, as was the main effect of drug group, F(2, 18) = 7.58, p < 0.01, η p 2 = 0.457. As seen in
As seen in
Although drug effects were detected in the 2nd and 3rd assessment periods, no drug associated effects were found in the 4th assessment period and only the main effect of days was significant, F(4, 56) = 5.76, p < 0.01, η p 2 = 0.291, (see
The results associated with the four assessment periods of Learning set performance are presented in
Considering the 1st assessment period, although the main effect of drug group was nonsignificant, a main effect of trials, F(1, 18) = 56.15, p < 0.001, η p 2 = 0.757, and a group X trials interaction, F(2, 18) = 4.50, p < 0.05, η p 2 = 0.333, were found. Thus, the rats typically found the platform more quickly on trial 2 than trial 1; but when considered within each trial, escape latencies differed across the groups. Post hoc examination of the groups revealed that on trial 2, the saline-treated rats found the platform significantly faster than the MDMA- but not the Foxy-treated rats.
The second assessment period revealed a somewhat similar pattern to that of the first assessment. While the main effect of drug group was nonsignificant, a main effect of trials was found, F(1, 18) = 18.94, p < 0.001, η p 2 = 0.513. More important, the group X trials interaction remained significant during this assessment phase, F(2, 18) = 6.00, p < 0.025, η p 2 = 0.400. Subsequent TukeyHSD tests revealed that the saline-treated rats located the platform faster on trial 2 than on trial 1. The performance of these animals was superior to that of the two drug-treated groups on this trial, whereas the trial 2 performances were comparable (see
Consideration of the 3rd assessment period data revealed the following: Main effects of both drug groups, F(1, 18) = 5.80, p < 0.025, η p 2 = 0.392, and trials, F(1, 18) = 23.01, p < 0.001, η p 2 = 0.561, were found. Here, escape latencies among the saline-treated rats were superior to the drug-treated rats; but the latter two did not differ, and escape latencies where faster on trial 2. As before, the group × trials interaction was significant, F(2, 18) = 8.30, p < 0.01, η p 2 = 0.480. Decomposition of the interaction revealed that only the saline-treated rats found the escape platform faster on trial 2 than on trial 1. In addition, the trial 2 performance of the saline -treated rats was superior to that of both drug groups with the latter two again performing similarly.
By the 4th assessment period, a different pattern emerged (see
Examination of cortical or subcortical dopamine and serotonin levels revealed no significant differences between the saline and two drug groups or between the MDMA and Foxy-treated animals (all ps > 0.05).
While cost-effective, cross-sectional research designs comparing animals of different ages are limited by the possibility of flawed conclusions about changes that occur across time [
One unexpected result of the present experiment, was the persistent difference in day two stepdown latencies between the saline and MDMA-treated animals. While an improvement in the day two performance of the MDMA-treated animals was seen across assessment periods, the saline-treated animals remained on the platform significantly longer than the MDMA-treated rats. While speculative, a number of reports have implicated MDMA in long-term impairments to cognition, including cognitive impulsivity [
Mammalian adolescence is a period marked by widespread maturational changes in the brain [
When different time points in adolescent development are considered, there is convincing evidence that both neurotoxic and neurobehavioral changes normally associated with MDMA exposure are dependent on the age of exposure [
Including the present investigation, when compared to MDMA, less data is available that is concerned with the specific effects and possible health costs associated with the use of the drug Foxy. Certainly, previous reports about the consequences associated with its use by humans [
While there is support for the contention that adolescent Foxy exposure is capable of producing long-term cognitive changes [
One major finding of our study was a lack of group differences on any of our neurochemical measures. First, the drug effects do differ across species. In mice, MDMA has relatively selective neurotoxic effects on dopamine, while 5-HT concentrations are largely spared. Conversely, 5-HT neurotoxicity is commonly reported in studies employing the use of nonhuman primates or rats [
Consideration of a human longitudinal study [
In addition, although further research needs to be done, the age of drug exposure is instructive. Following MDMA exposure to rats from PNDs 35 to 60, Piper and Meyer [
In addition, García-Cabrerizo and García-Fuster reported that MDMA effects produce impairments of the GABAergic system and neurofilament proteins, at least in the hippocampus [
Collectively, the MDMA studies clearly point to learning and memory deficits, with many of the deficits reflective of problems in higher level cognition [
To reiterate, much less is known about the effects of Foxy [
Among the crucial variables that appear to drive the physiological effects associated these compounds, the dose of these compounds is a key consideration. While a discussion of this issue is beyond the scope of the present report, one investigation is illustrative. In this study, the effects of daily exposure of MDMA for four days over PNDs 38 to 41 were examined. An MDMA dose of 5 mg/kg had no effect on behavioral measures (e.g., anxiety, place conditioning) while a dose of 10 mg/kg dose produced a number of effects [
While issues associated with interspecies scaling are a concern [
In summary, when considered alongside of previous research [
Although the use of a longitudinal design in our study allowed for the examination of the long-term consequences of MDMA or Foxy following exposure in adolescence, there are still limitations that should be mentioned. First, the original sample size was less than optimal for a study lasting more than a year. Certainly, the loss of one animal in each group was a foreseeable yet undesirable consequence. Thus, the statistical power of the present study is limited. An additional limitation of the present study was the use only male rats. There continues to be concerns raised when including female animals such as an increase in data variability [
The present research was sponsored in part by a grant from the Palm Beach Atlantic University Faculty Research Committee to David M. Compton. The authors would like to thank L. Lander for her assistance with the collection of data and data entry and N. Hernandez for their assistance with the HPLC assessment of 5-HT and dopamine.
Compton, D.M., Dietrich, K.L., Esquivel, P. and Garcia, C. (2017) Longitudinal Examination of Learning and Memory in Rats Following Adolescent Exposure to 3,4-Methylenedioxy- methamphetamine or 5-Methoxy-N,N-Dii- sopropyltryptamine. Journal of Behavioral and Brain Science, 7, 371-398. https://doi.org/10.4236/jbbs.2017.79028