3.1 General Characteristics
The experimental plan was for 20 litters per treatment regimen however, an extra litter was added to the P1−10 and P6−15 regimens because of increased mortality in the MA25 dose. In the P11−20 regimen an assignment error occurred, leaving 19 litters. Hence the final number of litters and surviving offspring are shown in . also shows offspring mortality. As can been seen, the MA25 treatment increased offspring mortality compared to Sal and MA10 groups. This effect was greatest in the P1−10 treatment regimen, second largest in the P6−15 regimen, and least severe in the P11−20 regimen. No significant increase in offspring mortality occurred in the MA10 treated group for any regimen relative to the Sal groups. Because of the extra pair of offspring/litter assigned to the MA25 treatment, there were adequate numbers of offspring remaining for behavioral testing in all the MP25 groups, and in fact there were more than needed to meet the design of the experiment; hence, at weaning extra offspring (i.e., anything above 1 male and 1 female/litter) were not weighed or tested but only used as cagemates so that all animals were pair-housed. Although animals in the MA25 group were small for their age by the end of treatment they were not otherwise outwardly compromised.
Entries represent number of litters in the experiment, offspring mortality (percent dying), and number surviving as a function of treatment regimen and MA dose.
Offspring body weights during dosing were analyzed separately for each regimen. shows representative body weights at the beginning and end of dosing. Groups did not differ on the first day of treatment for any treatment regimen. Significant body weight decreases emerged on the second or third day of treatment and remained lower throughout the period of drug treatment for both the MA10 and MA25 groups compared to Sal (see ).
Offspring average body weight (g) shown on the first and last day of dosing for each treatment regimen (see for the number tested). Data are Least Square (LS) mean (± LS SEM) of males and females.
Offspring body weights after dosing were analyzed separately for preweaning and postweaning ages. Representative body weights are shown in with the total number of animals tested. Group differences in body weight were significant on P21 () and P28 (not shown) with both the MA10 and MA25 groups weighing significantly less than Sal controls. By P56, the last weight taken before behavioral testing began on P60, the MA25-treated group males and females weighed significantly less in all regimens except for the females in the P11−20 regimen. For the MA10 group males, body weights were reduced in the P1−10 and P6−15 regimens but not in the P11−20 regimen. For MA10 females, no regimen showed significantly reduced body weight on P56. By the end of testing on P98, both MA treatment groups had body weights comparable to Sal controls except for the MA25 males and females in the P1−10 and P6−15 regimens ().
Table 3 Offspring body weight expressed as LS mean (g) ± LS-SEM (percent reduction) from P21−98. P56 was chosen to show body weight shortly before the start of behavioral testing. P98 was chosen to show body weight at the conclusion of behavioral (more ...)
3.2 Locomotor Activity
There were significant main effects of group (F(2,266) = 21.67, p < 0.0001) and sex (F(1,261) = 4.3, p < 0.05; females were more active than males) on horizontal activity, as well as a main effect of interval (p < 0.0001) and a group × interval interaction (F(22,3154) = 2.31, p < 0.001), but no regimen main effect or interactions of regimen and group. The MA25 group was less active during the exploratory, habituation, and baseline phases. This effect diminished but did not entirely disappear as the animals reached asymptotic baseline levels. The MA10 group showed less of an effect compared to the MA25 group, but they were still significantly less active during the 10 and 15 min intervals with a trend for hypoactivity at the 5 min interval relative to the Sal group (). The treatment main effect is illustrated in the inset). Analyses of other indices of activity (total, central, and peripheral distance traveled, and rearing) showed the same pattern of differences; the data for these measures showed no indication of differential spatial distribution of activity differences (central vs. peripheral changes) and no indication that the MA-induced reduction in horizontal activity was shifted to increased rearing; in fact both MA-treated groups showed significantly reduced rearing, paralleling the changes horizontal movements.
Fig. 1 Locomotor Activity averaged across exposure regimens: Rats were tested for 1 h during the light phase of the light/dark cycle. Ordinate shows horizontal activity (total number of photobeam interruptions per 5 min interval). Data are LS mean (± (more ...)
3.3 Acoustic Startle/PPI
Maximum startle amplitude was analyzed by prepulse using a group × regimen × sex × prepulse ANOVA (). The main effect of group was significant (F(2,245) = 2.6, p < 0.03; (inset)). The main effect of prepulse was also significant (p < 0.0001). Neither sex nor regimen were significant nor were any of the interactions with the exception of the sex × prepulse interaction (p < 0.01) in which males showed larger responses and greater change in the presence of prepulses. ANCOVA with the no-prepulse trials used as the covariate showed no significant effect of MA treatment on PPI as an index of sensory gating.
Fig. 2 Prepulse Inhibition of Acoustic Startle: LS mean (± LS-SEM) acoustic startle amplitude (Vmax = maximum amplitude recorded 100 ms post-stimulus onset measured in arbitrary units of voltage change (mV)) verses prepulse intensity (dB, SPL). PP = (more ...)
3.4 Straight Channel
No group main effect or group-related interactions were found on straight channel swimming latencies. The only significant effect was trial (p < 0.0001) which was attributable to the progressive improvement of all groups in traversing the channel across successive trials.
3.5 Cincinnati Water Maze (CWM)
For errors in the CWM, there were multiple significant effects related to group. There was a significant group main effect (F(2,291) = 16.43, p < 0.0001), as well as group × regimen (F(4,290) = 3.58, p < 0.01) and group × day (F(28,4059) = 1.79, p < 0.01) interactions. Other effects were sex (p < 0.0001; males committed more errors than females), regimen (p < 0.01; regimen P11−20 committed more errors than regimens P1−10 or P6−15), day (p < 0.0001; errors decreased across days), and sex × day (p < 0.0001). The group (inset) and group × regimen (main panel) effects on errors are shown in . A posteriori group comparisons showed no differences among groups for the P1−10 regimen. For regimens P6−15 and P11−20, both MA groups committed more errors than Sal controls with the effect in the P6−15 regimen showing the largest dose-dependent effects ().
Fig. 3 Performance in the Cincinnati water maze (CWM): Data are LS mean (± LS-SEM) averaged across days (15), trials (2 trials/day), and sexes. A, errors, B, latency to escape, C, start returns. Main panels: performance by treatment regimen. Insets: (more ...)
Fig. 4 Cincinnati water maze (CWM) errors by day: Data are LS mean (± LS-SEM) for each day averaged across trials (2 trials/day) and sexes. Data are shown separately for each treatment regimen (P1−10, 6−15, and 11−20). See tables (more ...)
For latency, the pattern was the same. There was a significant group main effect (F(2,292) = 14.6, p < 0.0001; (inset)), as well as group × regimen (F(4,291) = 3.76, p < 0.01; ) and group × day (F(28,4059) = 1.83, p < 0.005) interactions. As can be seen, the pattern was similar to that for errors except that the MA25 group in the P11−20 regimen showed a non-significant trend toward longer latency.
For start returns, a similar pattern was seen but without a group × regimen interaction. Hence, there was a significant group main effect (F(2,273) = 15.75, p < 0.0001; (inset)), and a group × day (F(28,4056) = 1.49, p < 0.05) interaction but the group × regimen interaction was not significant (F(4,272) = 1.5, p > 0.2). The pattern appeared similar to that for errors and latency, but the small absolute number of start returns was insufficient to obtain a significant group × regimen interaction, yet the overall pattern of MA treatment effects was the same as for the other measures ().
3.6 Morris Water Maze (MWM)
Latency to find the platform, path length and cumulative distance from the platform on learning trials all showed similar effects during all three phases of testing (acquisition, reversal and shift); hence, only latency data are presented. For acquisition, there was a significant group main effect (F2,269) = 17.51, p < 0.0001) and a group × regimen × sex interaction (F(4,261) = 2.96, p < 0.02), however, group × regimen was not significant (F(4,268) = 1.96, p = 0.10). Sex × regimen (p < 0.002) and day (p < 0.0001) effects were also obtained. Overall, both MA-treated groups had significantly longer latencies than Sal controls ( top panels). Learning curves for all three phases are illustrated in . The group × regimen × sex interaction is shown in . MA-dependent treatment effects differed by sex such that for males, MA impaired performance for regimens P6−15 and P11−20, but for females, regimens P1−10 and P11−20 were impaired.
Fig. 5 Morris water maze (MWM) platform trials by treatment regimen: Data are LS mean (± LS-SEM) latency (s) to find the platform averaged across trials (4 trials/day), day (5 days), and sex. Left panels: latency as a function of treatment regimen. Right (more ...)
Fig. 6 Morris water maze (MWM) latency (s) by day for platform trials: Data are LS mean (± LS-SEM) to find the platform (4 trials/day) averaged across sex for each regimen. Upper panel: acquisition (10 cm platform); middle panel: reversal (7 cm platform); (more ...)
Fig. 7 Morris water maze acquisition by sex: Data are LS mean (± LS-SEM) latency (s) to find the platform as a function of treatment regimen and sex to illustrate the group × regimen × sex interaction. Left: males; Right: females. See (more ...)
For reversal, there were no interactions with regimen, but the group main effect was significant (F(2,268) = 6.94, p < 0.002). When sorted, only the MA25 group showed significantly longer latencies than Sal controls (, middle).
For shift, both the group main effect (F(2,264) = 6.56, p < 0.002) and the group × regimen interaction (F(4,262) = 3.88, p < 0.01) were significant. The group × regimen interaction was analyzed further. A posteriori group comparisons showed neither MA group differed significantly from the Sal group for the P1−10 regimen. For the P6−15 regimen, the MA25 group had longer latencies than the Sal group. For the P11−20 regimen, both MA-treated groups had significantly longer latencies than the Sal group (, bottom). The main effect of group showed that both the MA25 and MA10 groups had significantly longer latencies than Sal controls averaged across regimens.
Twenty-four h after the last day of platform trials for each phase of MWM testing, a single 30 s probe trial without the platform was given. For the acquisition probe trial (), significant group effects were obtained for number of crossovers (F(2,254) = 5.17, p < 0.01), average distance from the platform site (F(2,254) = 7.54, p < 0.001), initial heading error (F(2,254) = 3.28, p < 0.04), percent time in the target quadrant (F(2,254) = 5.51, p < 0.005), percent distance in the target quadrant (F(2,254) = 4.29, p < 0.02), and MSD (F(2,254) = 5.52, p < 0.005). There were no interactions with regimen or with sex. On most of these measures, both MA-treated groups differed from Sal, except for crossovers where only the MA25 group had significantly fewer crossovers than Sal controls.
Fig. 8 Morris water maze (MWM) acquisition probe trial: Data are LS mean (±LS-SEM). A, Crossovers; B, average distance to the platform site; C, initial heading error; D, percent time in the target quadrant; E, percent distance in the target quadrant; (more ...)
For the reversal probe trial, group × sex (F(2,253) = 3.16, p < 0.03) and group × regimen × sex (F(4,253) = 3.20, p < 0.02) interactions were significant for crossovers. No group effects or group-related interactions were found on average distance or percent time or distance in the target quadrant, or on MSD. For initial heading error there was a significant group × sex interaction (F(2,253) = 4.08, p < 0.02). For the crossover 3-way interaction, only one significant effect was found by pairwise groups comparisons and that was for females in the P6−15 regimen (F(2,253) = 7.92, p < 0.0005). This effect was that both MA groups differed significantly from Sal controls (p < 0.01); female P6−15 LS means (± LS SEM) were: Sal = 0.9 ± 0.2; MA10 = 0.1 ± 0.2; MA25 = 0.3 ± 0.2. For initial heading error, slice ANOVAs showed a significant effect for males (p < 0.02) but not for females. Both the MA10 (p < 0.003) and MA25 (p < 0.05) group males were more off-course than Sal males; male LS means (± LS SEM) were: Sal = 71.8 ± 3.7, MA10 = 84.5 ± 3.8; MA25 = 80.1 ± 4.3.
For the shift probe trial, no significant effects were obtained.
In order to determine if any of the differences seen on platform trials were attributable to differences in swimming speed, analyses of swim speed were conducted. No significant group or group-related effects were found during the platform trials for any phase. On probe trials, analyses of swimming speed showed no group effects on the acquisition probe trial, but speed differences did appear on reversal and shift probe trials. For reversal there was a group main effect (p < 0.03) which when analyzed further revealed that the MA25 group swam faster than Sal controls. For the shift probe trial, there was a significant group × sex interaction (p < 0.05) for swimming speed which when further analyzed was only in the females (p < 0.02). Among females, the MA25 group swam faster than Sal females. Therefore, both of these probe trial speed effects showed no swimming impairment, but rather increases in swim speed in the MA25 group.
Following the three phases of hidden platform testing in the MWM the animals were given two days of cued (visible) platform trials (4 trials/day) with the start and platform locations randomized on each trial and with curtains closed around the maze to obscure extramaze cues. These trials were designed to ensure that all groups could locate the platform without relying upon spatial cues. No significant group main effects or group-related interactions were obtained during cued testing (not shown).