An analysis of mortality showed that for the PD 1–5 regimen, MDMA caused a significant increase in mortality (). No significant MDMA-induced increase in mortality was found for the other 3 treatment regimens. All mortality in the experiment occurred either during the drug exposure period or shortly thereafter with one exception. The exception was 1 animal that died after weaning. The increased mortality observed in the PD 1–5 regimen was dose-dependent: increasing mortality with increasing MDMA dose. With the exception of a single animal in the PD 1–5 regimen, the saline controls were unaffected. Accordingly, the PD 1–5 mortality was the result of drug treatment and not early handling, maternal separation during dosing, or the stress associated with subcutaneous injections. Despite the increased mortality in the PD 1–5 regimen, the assignment of extra litters to the PD 1–5 regimen compensated for the losses sufficiently such that there were enough survivors to test behaviorally.
Offspring mortality from neonatal MDMA treatment
Analyses of body weights during treatment for the 4 different dosing regimens (10, 15, 20, and 25 mg/kg = groups MDMA-10, 15, 20, and 25, respectively) showed effects of drug treatment, sex, day, and drug treatment × day (all p < 0.0001). Slice effect ANOVA for the PD 1–5 regimen showed no treatment differences on PD 1 or 2; differences were significant on PD 3–5. On PD 3, the MDMA-25 and MDMA-20 groups were significantly lighter than saline controls, whereas on PD 4–5, all MDMA groups were lighter than controls.
Similarly for PD 6–10 body weights, slice ANOVA showed no drug treatment effects on PD 6, but all MDMA groups were lighter than saline controls on PD 7–10.
For PD 11–16 body weights, slice ANOVA showed no drug treatment effects on PD 11 and significant differences on PD 12–15 with all MDMA-treated groups lighter than saline controls.
The same pattern was seen for PD 16–20 body weights, with slice ANOVA showing no differences on PD 16 and significant differences on PD 17–20 with all MDMA-treated groups weighing less than saline controls.
In order to determine if body weight reductions seen during dosing recovered, offspring were weighed weekly from the end of treatment to the end of behavioral testing (PD 21–98; ). A repeated-measure ANOVA on weekly weights showed a significant drug treatment effect [F(4,757) = 16.39, p < 0.0001], but no drug treatment × week interaction. All MDMA-treated groups had lower body weights than the saline-treated group. Sex (males weighed more than females) and week effects were significant as was the sex × week interaction (all p < 0.0001). Regimen was not a significant factor alone, but did interact with week (p < 0.0005). However, slice effect ANOVA performed each week failed to show any week as having a significant regimen effect. The week that came closest was week 12 (PD 98; p = 0.057).
Offspring body weights (in grams)
Elevated Zero Maze
No significant effects of drug treatment (either main effects or interactions) were found on time in open, number of open entries, latency to first open entry, or number of head dips. There were no effects of sex on time in open, latency to first open entry, or head dips. There was a significant effect of sex on number of open entries (p < 0.0001) reflecting that females were more active than males. There was a regimen effect on all 4 measures. Across all treatment groups, animals in the PD 11–15 regimen spent more time in the open, made more open entries, had shorter latencies to first open entry, and had more head dips than animals in the other regimens. This was followed closely by the PD 6–10 regimen, suggesting that handling/injection stress during these ages made the animals less anxious as adults. At the other end of the continuum, the PD 1–5 regimen had the least time in open, fewest open entries, and fewest head dips, suggesting they were more anxious. Data for time in open and number of open entries are shown in .
Elevated zero maze: principal measures of performance are shown (time in open and number of open zone entries)
All measures showed similar patterns of significant drug treatment effects. For the measures of horizontal distance, drug treatment effects were evident (d.f. = 4, 554: total distance F = 3.46, p < 0.01, peripheral distance F = 3.03, p < 0.02, and central distance F = 2.70, p < 0.03). Sex was significant for total and peripheral distance (p < 0.001), but not central distance. In addition, there were drug treatment × interval interactions (d .f. = 20, 3,035: total distance F = 2 .42, p < 0.0 0 04, peripheral distance F = 1.80, p < 0.02, central distance F = 2.62, p < 0.0001). One other factor was significant only for central distance and this was the drug treatment × regimen × interval interaction [F(60, 3,195) = 1.44, p < 0.02]. For vertical movement (rears), the drug treatment main effect was not significant, but sex (p < 0.0001) and the drug treatment × interval interaction [F(20, 3,090) = 2.15, p < 0.01] were. For illustrative purposes, the distance measures are shown in . Pair-wise group comparisons comparing each MDMA group to control showed that for total distance the 3 highest-dose MDMA groups were less active than saline controls, while the MDMA-10 group did not differ significantly. This pattern was repeated for both peripheral and central distances, with no apparent differential peripheral versus central differences in the direction or magnitude of the MDMA effects.
Fig. 1 Locomotor activity: horizontal distance (least-squares means ± SEM in centimeters ) summed over a 1-hour test session in 10-min intervals for each treatment regimen. Group sizes, summed across regimens and sexes (male/female), were: saline = 78/79, (more ...)
For all variables, the drug treatment × interval interaction was further analyzed using slice effect ANOVA for each interval. Drug treatment (irrespective of treatment regimen) was significant for all variables for interval 1 (analyzed in 10-min intervals). In interval 1, the MDMA groups were less active than saline controls. For central distance, the 3-way interaction of drug treatment × regimen × interval was further analyzed by slice effect ANOVA for each interval of each regimen. For all 4 regimens, drug treatment effects were found only for interval 1. Pairwise group comparisons are shown in . For the PD 1–5 and PD 6–10 regimens, all four MDMA-treated groups were less active; however, for the PD 11–15 regimen, the effect was less pronounced with only the MDMA-15 and MDMA-25 groups showing significant reductions in central distance. For the PD 16–20 regimen, no MDMA-related hypoactivity during interval 1 was observed, and the MDMA-25 group showed a small but significant increase.
Fig. 2 Locomotor Activity: horizontal distance (least-squares means ± SEM in centimeters) in the center of the arena during the first 10-min interval of the 60-min test shown separately for each regimen with males and females combined. Group sizes, summed (more ...)
Novel Object Recognition
An analysis of time spent investigating the 2 identical objects during familiarization showed no drug treatment or regimen effects. Similarly, during retention testing with 1 familiar and 1 novel object, no significant drug treatment or regimen effects were obtained (not shown).
There were no significant effects of drug treatment (main effects or interactions) or regimen on straight channel swimming times, indicating that neither MDMA nor age of treatment had any effect on basic swimming ability or motivation to escape (not shown).
Cincinnati Water Maze
Analyses of CWM performance showed no drug treatment effects (either main effects or interactions) for errors, latency to escape, or start returns (not shown). In addition, there were no significant effects of regimen on any of these measures. There were significant effects of sex for errors (p < 0.0001), latency (p < 0.0001), and start returns (p < 0.0001). On this test, as we have found previously, females (regardless of treatment group) performed significantly better than males (i.e. females had fewer errors, shorter latencies, and fewer start returns).
MWM – Phase 1
Analyses on learning trials focused on latency, path length and cumulative distance from the goal during all 3 test phases. There was good agreement among these variables, and we have previously shown them to be highly correlated among themselves [Vorhees and Williams, 2006
]; therefore, data for path length are reported to illustrate the findings.
MDMA treatment significantly impaired learning to find the hidden platform as measured by path length during phase 1 [acquisition; F(4, 634) = 4.22, p < 0.003]. Other significant factors were sex (p < 0.0003), regimen (p < 0.03), day (p < 0.0001) and regimen × day (p < 0.004). When further analyzed, the regimen × day effect was primarily attributable to a regimen effect on day 1 (p < 0.001). The day-1 regimen effect, averaged across groups, is illustrated in . As can been seen, the PD 6–10 and PD 11–15 regimens had longer path lengths than those in the PD 1–5 regimen and the PD 11–15 regimen had longer path lengths than the PD 16–20 regimen. The sex effect was caused by the fact that across all groups and regimens, males performed better than females (ordinary mean ± SEM averaged across days: males = 740.0 ± 12.8 cm, females = 841.2 ± 17.2 cm). In terms of the hypothesis under investigation, there were no drug × regimen, drug × regimen × sex, drug × regimen × day, or 4 -way drug × regimen × sex × day effects. The significant main effect of drug treatment, averaged across regimens, is illustrated in . As can be seen, during phase 1, all four MDMA-treated groups had significantly increased path lengths compared to saline controls by approximately 15%.
Fig. 3 MWM treatment regimen effects: path length (least-squares means ± SEM in centimeters) to find the submerged platform averaged across trials, sexes and treatment groups to illustrate the main effect of treatment regimen (age). a Regimen effects (more ...)
Fig. 4 MWM treatment effects: path length (LS means ± SEM in centimeters) during the 3 phases of MWM averaged across days, trials, and regimens. Group sizes averaged across days, trials, and regimens were: saline = 157, MDMA-10 = 155, MDMA-15 = 149, (more ...)
Variables analyzed on the probe trials were average distance from the platform site, percent time and distance in the target quadrant, number of site crossovers, and mean search distance (distance in the target quadrant minus distance in each of the other quadrants subtracted separately and divided by 3). Analyses of these variables for the phase-1 probe trial showed no significant effects of drug treatment or regimen. Sex was significant because males performed significantly better than females (average distance from platform site: males = 126.9 ± 1.7 cm, females = 139.0 ± 1.7 cm).
MWM – Phase 2
For phase 2 (reversal), the ANOVA on path length showed a drug treatment effect [F(4, 626) = 2.51, p < 0.04]. Other factors were a significant effect of sex (p < 0.0001), day (p < 0.001), regimen × day (p < 0.03), and a nearly significant effect of regimen (p < 0.052). In terms of the hypothesis, there were no significant interactions of drug treatment × regimen. Further analysis of the regimen × day effects showed that regimen was significant only on day 2 (p < 0.004). The day-2 regimen effect was that the PD 6–10 and PD 11–15 regimens had longer path lengths than the PD 16–20 regimen (). The drug treatment effect irrespective of regimen for phase 2 is shown in . As for phase 1, in phase 2 all four MDMA-treated groups had longer swim paths to the goal than saline controls.
For the probe trial conducted after phase-2 training, there were no drug treatment effects and no drug treatment × regimen interactions (not shown). Sex was significant as before.
MWM – Phase 3
The ANOVA on path length revealed an effect of drug treatment [F(4, 607) = 3.12, p < 0.015]. Sex was also significant (p < 0.0001); regimen was not. In terms of the hypothesis, there were no significant interactions between drug treatment × regimen. The effects of drug treatment, irrespective of regimen, are shown in . During this phase, the MDMA-20 and MDMA-25 groups differed significantly in path length to the goal compared to saline controls, whereas the MDMA-10 and MDMA-15 groups did not.
For the probe trial conducted after phase 3, there were no effects of drug treatment or drug treatment × regimen (not shown). Sex was significant as before.
Learning curves of path length for MWM phase 1 are shown in . Drug group differences emerged rapidly, and were already apparent in all MDMA groups on day 1. (inset) shows the emergence of these differences when plotted by trial for day 1. As can be seen, the MDMA-treated groups started out on trial 1 at performance levels no different from those of saline controls. However, thereafter saline controls showed greater reductions in path lengths than the MDMA-treated groups. These data reveal that the MDMA groups did not start the task with any preexisting swimming impairment that might explain their deficits in terms of performance effects. Rather, the MDMA-treated groups began the test comparably to controls, but failed to eliminate off-target swim paths to the goal as quickly as saline controls.
Fig. 5 MWM phase 1 (acquisition) learning curves. Main panel: mean ± SEM path lengths (cm) shown for each treatment group averaged across 4 daily trials, regimens, and sexes. Inset: path length shown by trial on day 1 averaged across regimens and sexes (more ...)
MWM – Swim Speed
On all 3 test phases, for both learning and probe trials, swimming speed was also analyzed. There were no drug treatment effects on swimming speed seen in any of these analyses (not shown). This further indicates that MDMA-treated animals do not have a performance deficit that would interfere with their ability to learn the task; rather, the data suggest that a central deficit led to the observed spatial navigation impairments.
MWM – Cued
There were no significant drug treatment impairments found on cued trials; in fact, the opposite was seen, and all the drug groups reached the visible platform more quickly than saline controls [F(4, 614) = 2.90, p < 0.03; saline = 14.2 ± 0.5, MDMA-10 = 12.7 ± 0.5, MDMA-15 = 12.7 ± 0.5, MDMA-20 = 13.2 ± 0.5, MDMA-25 = 12.2 ± 0.6 s]. This reinforces the evidence that MDMA-treatment did not impair swimming performance even though it impaired spatial navigation ability.