There was no mortality in the Saline (0/21), MA10 (0/21) or FEN (0/21) groups. However, 3 animals in the MA12.5 group died (3/21), leaving 18 for testing. In the AMPH group 5 died (5/21), leaving 16 for testing.
The effects of the drugs on body temperature are shown in . As can be seen, all 4 drugs affected body temperature. There was a significant Treatment main effect, F(5,117) = 53.9, p < 0.0001, a Time main effect, F(32,3343) = 3.6, p < 0.0001, and a Treatment × Time interaction, F(160,3444) = 3.0, p < 0.0001. Slice-effect ANOVAs at each time showed Treatment effects at all intervals (p < 0.001). A posteriori comparisons of each drug group to Saline controls showed that the MA10, MA12.5, and AMPH groups were hyperthermic from the second interval to the last (bracketed range at top of ). MDMA also induced hyperthermia, but with a delayed onset that was significant from the second dose (2 h) onward (bracketed range in middle of ). FEN, on the other hand, induced hypothermia that was significant from 30–300 min and at two later intervals (bracketed range at bottom of plus two later intervals shown by asterisks).
Figure 1 Body temperatures: Mean ± SEM body temperature recordings from subcutaneously implanted temperature transponders in rats treated with Saline, MA, AMPH, MDMA, or FEN every 2 h. The arrows denote times when drug injections were delivered. Average (more ...)
Note that the hyperthermic pattern, in terms of rise, peak, and duration seen in the AMPH group followed that of the MA12.5 group after the first and second doses, but decreased compared to the MA12.5 and MA10 groups at and after the third and fourth doses.
In order to understand the impact of the cooling intervention, we tallied the number of animals that received cooling interventions, which occurred when a body temperature reached or exceeded 40.2°C. The number of animals cooled per group was as follows: Saline = 0/21, MA10 = 10/20 (1 animal had a nonfunctioning temperature transponder), MA12.5 = 11/18, AMPH = 8/16, MDMA = 7/21, FEN = 0/21. We then compared whether the cooled vs. un-cooled subgroups differed significantly in maze performance (see below).
Body weight data are shown in . Body weight was measured prior to treatment, at 8 and 72 h after the first dose, and at one week intervals thereafter. An ANOVA on pre-treatment body weights showed no significant differences. At 8 h after the first dose (i.e., 2 h after the last dose), there was a significant Treatment effect, F(5,474) = 2.57, p < 0.05, however, a posteriori comparisons between the Saline group and each drug group failed to reach significance (not shown). The Treatment effect was also significant 72 h post-treatment, F(5,466) = 5.58, p < 0.0001. A posteriori comparisons at this time point showed that each group differed from Saline (). A repeated measure ANOVA on weekly body weight showed no significant Treatment main effect, however the Treatment × Week interaction was significant, F(15,336) = 3.02, p < 0.0001, as was the main effect of Week, F(3,336) = 402.32, p <.0001. Slice ANOVAs performed on each week showed no significant treatment effect on any week, hence, the interaction was not the product of any one week difference but rather the change over weeks between groups. For example, the Saline group did not lose weight following treatment and between weeks 1 and 4 gained an additional 41 g on average. By contrast, the drug groups each lost weight after treatment and the groups that lost the largest amount, also gained the largest amount between weeks 1 and 4. For example, the MA10, FEN and MDMA groups all lost about the same amount of weight following treatment and each gained 52–56 g between week 1 and 4. This as compared to groups MA12.5 and AMPH which lost about 10 g more than the other drug groups following treatment and gained more (69–71 g) between week 1 and 4. The net result was that all groups ended statistically equal in body weight but the rate of weight gain was different among the groups. Body weights at 2 and 4 weeks are shown. The 2 week point is presented because it is the day on which CWM testing began and 4 week point is shown because it is within 24 h of the last day of testing.
Body weight (g) of rats immediately prior to treatment and at 72 h, 2 and 4 weeks post-treatment
Straight Channel Swimming
There was no Treatment main effect on straight channel swimming times. The main effect of Trial was significant, F(3,336) = 46.2, p < 0.0001. There was also a significant Treatment × Trial interaction, F(15,336) = 1.7, p < 0.05. Slice-effect ANOVAs showed a significant effect on trial-1 (p < 0.001) but not on trials 2-4 (F-values <1). This effect was attributable to the FEN group. On Trial-1 the FEN group swam faster (shorter times) than the Saline group: Trial-1 mean ± SEM for Saline = 35.9 ± 3.1 s vs. FEN = 23.0 ± 3.0 s. Although the reason for this isolated effect is not known, it clearly does not reflect a deficit in swimming ability and disappeared after trial-1.
Cincinnati Water Maze
Multiple measures of CWM performance were recorded and intercorrelations among them were determined. An error in which an animal entered the left or right arm of a dead-end T was termed a T error, which is listed in as “T”; an error in which an animal entered the stem leading to a dead-end T was termed a stem error or “S” in ; an error in which an animal left and then returned to the start arm was termed as start return error or “R” in . Because we have collected data for a number of years combining T and R errors, we summed these as T+R in . Combining all three types of errors (T+S+R) was termed “total errors” (Tot err). Latency (s) was the time it took the animal to find the goal after being placed in the start position. Averaging each dependent measure across days provided an index of total performance on that variable, and Pearson correlation coefficients were calculated for each dependent variable to every other variable with the results shown in . On the first few days of testing, most animals fail to find the escape within the time limit but continue to search throughout the 5 min. shows the intercorrelations with scores corrected as described in Methods. shows the intercorrelations among variables with no correction for non-searching episodes.
Intercorrelation coefficients among dependent variables recorded in the CWM with correction for animals not finding the goal (A) and without correction for animals that not finding the goal (B)
As can be seen, no matter how the data are viewed, corrected or uncorrected, the correlations among the variables were high. In general, correlations were slightly higher with corrected scores than with uncorrected scores. Nevertheless, the correlations are sufficiently high that total error scores capture maze performance and it would be redundant to present each variable separately, therefore, only total error data are presented.
Total errors as a function of day are shown in and averaged across days in . Because of the number of groups, shows the Saline vs. AMPH group, and shows Saline vs. MA12.5 and MA10 groups, and shows Saline vs. the MDMA and FEN groups. There was a significant Treatment main effect on total errors, F(5,114) = 5.1, p < 0.001. The Day main effect was also significant, F(20,2044) = 117.6, p < 0.0001, whereas the Treatment × Day interaction was not, F(100,2142) = 1.2, p < 0.08. Since the Treatment × Day interaction was not significant, only the main effect of Treatment group was analyzed further by a posteriori pairwise comparisons (). Averaged across days, treatment group comparisons showed that the AMPH group made the most errors (p < 0.01). In addition, the MA12.5 and MA10 groups made significantly more errors than Saline controls (p < 0.05). The two MA groups did not differ from one another. Although both the FEN and MDMA groups made more errors than Saline controls, neither of these groups differed significantly from Saline controls.
Figure 2 Learning curves for the CWM: Data are mean ± SEM total errors per day. A = total errors in the Saline and AMPH groups; B = total errors in the Saline, MA10, and MA12.5 groups; C = total errors in the Saline, MDMA, and FEN groups. Significant differences (more ...)
CWM total errors: Data are mean ± SEM total errors per day plotted for each drug treatment group averaged across days of testing. Group sizes are as listed in . *P<0.05 vs Saline; ***P<0.001 vs. Saline.
Latency results showed the same pattern of group differences as did errors, i.e., there was a significant main effect of Treatment, F(5,113) = 4.7, p < 0.001. The main effect of Day was also significant, F(20,2056) = 122.5, p < 0.0001, whereas the Treatment × Day interaction was not. Treatment group averages (± SEM, s) were: SAL = 105.8 ± 8.8; AMPH = 164.8 ± 10.1; MA12.5 = 134.3 ± 9.5; MA10 = 133.3 ± 8.8; MDMA = 117.4 ± 8.8; and FEN = 116.6 ± 8.8.
3.5 Cooled Vs. Un-cooled Comparisons
Approximately half of the animals in the AMPH, MA10, MA12.5, and MDMA groups received cooling to prevent life-threatening hyperthermia. We conducted separate ANOVAs on CWM total errors for each of the four drug groups that induced hyperthermia with Cooling as the main effect. For AMPH, no significant main effect of Cooling (F < 1) or Cooling × Day interaction was found (p = 0.18). For MA12.5 and MA10, there was neither a significant main effect of Cooling (Fs < 1) nor Cooling × Day interaction (Fs < 1). Similarly, for MDMA, neither the main effect of Cooling (F < 1) nor the Cooling × Day interaction (F < 1) was significant. These data are summarized in .
Table 3 Cincinnati water maze total errors: Mean (±SEM) errors/day (N) across 21 days of testing (2 trials/day) for subsets of animals that reached a core temperature of 40.2°C and were cooled versus those that did not reach 40.2°C and (more ...)