Drug Concentrations and Stability
Drug solutions of both compounds were prepared and sent to ABC Laboratories for analytical chemistry to determine drug concentrations by HPLC analysis (Methods; M-1736-000 and USP 30 Paroxetine HCl). Each sample was analyzed in triplicate. The first set of samples was taken from prepared test solutions for concentration verification. Mean concentrations of the test solutions were within 3% of target concentrations.
Stability Assessment: Fluoxetine hydrochloride is known to be stable in solution ≥ 7 days but to determine the stability of paroxetine we tested it at the low and high concentrations at three time points: immediately after preparation and at 3 and 7 days. The paroxetine solutions showed no decrease in active drug content over the 7-day period.
End of Study Drug Purity: After the completion of the experiment, crystalline samples of both drugs were sent to ABC Laboratories to determine drug purity. Both paroxetine HCl and fluoxetine HCl were unchanged (mean percent potency of 101%).
General Characteristics
There were no deaths; 100% of the animals enrolled in the experiment survived and were tested. Group sizes varied slightly from the design because of experimenter error or equipment malfunction. The exact N for each test is provided in the figure captions.
Body weight data were analyzed by age. Once offspring were assigned to treatment groups, their body weights from P1-28 were analyzed to ensure that no preexisting differences occurred. This analysis showed no differences as a function of later assignment to the groups (F(5,200) = 1.09, p > 0.36). The effect of age was significant (F(4,960) = 23327.9, p < 0.0001), demonstrating time-dependent growth. The group × week effect was not significant (F(20,960) = 0.50, p > 0.96).
Body weights during drug treatment (P33-62) were analyzed with Groups A and B combined. There was a significant effect of treatment (F(5,227) = 4.84, p = 0.0003), day (F(29,6922) = 1481.86, p < 0.0001), and treatment × day (F(145,6931) = 1.49, p = 0.0001). Slice-effect ANOVAs on each day showed no treatment effects on P33-43, however significant effects were obtained on days P44-62. Since there were no significant group differences before P44, only the P44-62 body weight data are illustrated in Figure 1. Body weights on the last day of treatment (P62) were analyzed separately for Groups A and B and are shown in Figure 2. As can be seen, for Group A, the Flu10 and Par 17 groups weighed less than Control. For Group B, only the Flu10 group weighed less than Control. The Par17 for Group B was not significantly different from Control (p = 0.12).
Group A animals completed testing by P62, hence there are no body weight data for this group after P62. For Group B, body weights were recorded on P63 and weekly thereafter until the end of testing on P126. In order to map body weight recovery after the end of treatment, data for the first two weeks after the end of treatment (P63-77) were analyzed separately. There was no significant treatment main effect (F(5,95) = 1.94, p < 0.10). The day/week factor in the analysis was significant (F(2,228) = 4256.37 = p < 0.0001), indicating time-dependent growth, and there was a significant treatment × week interaction (F(10,228) = 9.59, p < 0.0001). Slice-effect ANOVAs on each week showed differences on P63 (p < 0.01) but not on P70 or P77, indicating that recovery was rapid (< 1 week). This pattern is illustrated in Figure 3. As can be seen, only the Flu10 group weighed significantly less on P63 and no differences remained on P70, P77, or thereafter (i.e., between P84-126 (not shown)).
Elevated plus maze
For Group A there were no significant differences in time spent in the open arms, number of open arm entries, or latency to first open arm entry (Figure 4A-C, respectively). A significant treatment group effect was found on number of head dips (F(5,95) = 2.74, p < 0.03). Post hoc group comparisons showed that the Par17 group had fewer head dips than Controls (Figure 4D). No other group comparisons were significant. ES for time in open arms were = 0.15 (small), for arm entries = 0.13 (small), for latency = 0.12 (small), and for head dips = 0.27 (small).
For Group B there were no significant differences in time spent in the open arms, number of open arm entries, latency to first open arm entry, or head dips (Figure 4E-H, respectively). ES for time in open arms = 0.18 (small), for arm entries = 0.22 (small), for latency = 0.16 (small), and for head dips = 0.09 (small).
Acoustic Startle/PPI
ASR-PPI data were analyzed two ways: (a) by prepulse intensity, and (b) using the 0 prepulse response amplitude as a covariate by ANCOVA in order to assess PPI after controlling for any possible differences in basal startle reactivity.
For the Group A startle response ANOVA, the treatment group main effect was not significant but showed a trend (F(5,98.2) = 1.95, p < 0.10). There was no significant treatment × prepulse interaction. Prepulse intensity was significant (F(3,354) = 100.32, p < 0.0001) showing that prepulse inhibition of ASR was obtained. In the ANCOVA analysis, no significant treatment main effect was seen (F(5,98) = 1.86, p = 0.11). The treatment × PPI interaction was not significant. The prepulse main effect was significant (F(2,236) = 68.01, p < 0.0001). The data are shown in Figure 5(top panel). ES for Group A = 0.27 (small).
An inspection of Figure 5 taken together with the treatment main effect trend (p < 0.10) led us to conduct two follow-up analyses. In both analyses we combined the two Flu groups' data and the three Par groups' data into single Flu and Par groups. In the first follow-up analysis, all prepulse trials were included in a treatment × prepulse ANOVA. The treatment main effect (F(2,39) = 7.13, p < 0.003) and prepulse main effect (F(3,177) = 88.43, p < 0.0001) were both significant and the treatment × prepulse interaction showed a trend (F(6,177) = 1.89, p < 0.09). Post hoc group comparisons (averaged across prepulse intensities) showed that both the Flu and Par groups differed from Control (mean ± SEM: Control: 149.0 ± 23.5; Flu: 224.4 ± 23.0; Par: 252.7 ± 23.0). This facilitated startle effect was most pronounced on the no-prepulse trials, therefore, the second follow-up ANOVA was with the combined dose groups but only on the no-prepulse trials. ES for the latter = 0.44 (medium). With no prepulses in the analysis, the ANOVA was a simple one-way analysis with 3 groups. The treatment effect was significant (F(2,39) = 6.55, p < 0.004). This effect is shown in Figure 6(left panel). Post hoc group comparisons revealed that both drug groups showed significantly increased startle amplitude compared with the Control group. ES for the latter = 0.42 (medium).
For Group B the treatment × prepulse ANOVA showed no significant main effect of treatment group or treatment × prepulse intensity interactions. Prepulse intensity was significant (F(3,339) = 92.60, p < 0.0001) showing that ASR was significantly modified by the PPI procedure (Figure 5, bottom panel). The ANCOVA analysis showed a similar outcome, no significant treatment main effect or treatment × prepulse interaction. The prepulse main effect was significant (F(2,226) = 48.20, p < 0.0001). ES = 0.18 (small).
As for Group A, follow-up analyses with dose-levels of each group combined were conducted for Group B, and there was no significant treatment main effect or treatment × prepulse interaction. ES = 0.06 (small). Prepulse was significant (F3,171) = 72.39, p < 0.0001). An additional analysis performed on the no-prepulse trials by one-way ANOVA showed no significant treatment effect (Figure 6, right panel). ES = 0.06 (small).
Porsolt FST
For Group A, there were no significant treatment group effects found on immobility time (Figure 7, top panel) or latency to immobility (not shown) in the FST. ES = 0.20 (small).
Similarly, for Group B there were no significant effects of treatment group found on immobility time (Figure 7, bottom panel) or latency to immobility (not shown) in the FST. ES = 0.21 (small).
Corticosterone
For Group A, all groups showed high corticosterone levels at the end of the 5 min FST compared with typical basal levels (~50 ng/ml) however there were no treatment differences among the groups (Figure 8, left panel).
For Group B, again all groups showed increased corticosterone levels in response to the stress at the end of the FST but there were no significant effects of treatment obtained (Figure 8, right panel).