3.1 Effect of trial duration on extinction of EtOH-CPP (Exp 1)
Experiment 1 was performed in order to determine the optimal extinction parameters in our procedure by varying extinction-trial durations. shows that on Test 1 following conditioning, all four groups showed significant preference for the EtOH-paired floor. However, after extinction (Test 2) only the group that had received the 30 min trial duration (Ext-30 min) showed significant extinction of EtOH-CPP. A two-way ANOVA revealed a significant Test × Group interaction [F(3,67) = 4.8, p < .01] and subsequent paired t-tests for each group revealed that only the Ext-30 min group showed significant extinction [t(16) = 4.0 p < .005]. As shows, including the Conditioning Subgroups (G+ and G−) in the analysis further confirmed these findings. Therefore, Experiment 1 revealed that a trial duration of 30 min is necessary to completely extinguish EtOH-CPP. As such, all subsequent extinction experiments utilized a 30-min trial duration.
Extinction of EtOH-CPP was determined by trial duration
3.2 Effect of systemic administration of the MEK inhibitor, SL327, on extinction of EtOH-CPP (Exps 2 & 3)
In order to determine the involvement of ERK activity in extinction of EtOH-CPP, the MEK inhibitor SL327 was administered prior to the 30-min, non-reinforced CS+ cue exposures during each extinction trial. As shows, SL327 did not impair extinction of EtOH-CPP—that is, all groups that underwent extinction showed a significant decrease in preference on Test 2. These findings were supported by a significant Test × Group interaction [F(3,67) = 12.4, p < .001] and significant main effects of Test [F(1,67) = 88.4, p < .001] and Group [F(3,67) = 5.5, p < .005]. Paired t-tests comparing preference on Tests 1 and 2 revealed significant extinction in all but the No Extinction group (p’s < .05). Further analysis revealed a simple main effect of Group only on Test 2 [F(3,67) = 16.5, p < .001]. Post-hoc comparisons of Test 2 preferences showed that the SL-50, but not SL-30, group differed significantly from the Vehicle group (p < .05). Therefore, the highest dose of SL327 appeared to be aversive and, when administered 30 min before cue exposure, resulted in a decrease in preference below the indifference point (50% preference) on Test 2. This finding suggested that a dose of 50 mg/kg SL327 possessed aversive properties that may have actually counter-conditioned the initial EtOH-CPP, resulting in a significant avoidance of, not just an indifference for, the previously EtOH-paired cue.
As shows, CS+-trial locomotor activity decreased over the course of extinction (significant main effect of Trial [F(3,144) = 34.5, p < .001]), but did not differ between drug treatment groups (no significant Group × Trial interaction, p > .05).
Because the SL-50 group in Experiment 2 developed a place aversion during extinction, the parameters of SL327 administration in Experiment 3 were changed in hopes of eliminating this effect. By extending the pre-trial interval to 90 min, we hoped to reduce the aversive properties of SL327 and/or weaken the ability of any aversive properties to enter into an association with the CS+ cue during extinction (i.e., prevent counter-conditioning a place aversion). The vehicle was also changed to 50% DMSO in order to better reflect the SL327 administration parameters used in previous experiments (e.g. Mouledous et al., 2007
). As seen in , the results showed that although these manipulations eliminated the place aversion seen in Experiment 2, neither dose of SL327 interfered with normal extinction of EtOH-CPP. Analysis revealed a significant Test × Group interaction [F
(3,67) = 4.6, p
< .01] as well as main effects of both Test [F
(1,67) = 31.0, p
< .001] and Group [F
(3,67) = 4.4, p
< .01]. Paired t-tests comparing preference on Tests 1 and 2 revealed significant extinction in all but the No Extinction group (p
’s < .05). Further analysis revealed a simple main effect of Group only on Test 2 [F
(3,67) = 8.9, p
< .001]. Subsequent post-hoc comparisons of Test 2 preferences revealed that neither the Ext-30 nor Ext-50 group differed from the Vehicle group (p’s > .05). Therefore, when administered at 30- or 90-min pre-trial intervals, neither dose of SL327 impaired extinction of EtOH-CPP in mice.
SL327, administered 90-min prior to trials, did not impair extinction of EtOH-CPP
In contrast to Experiment 2, however, both doses of SL327 reduced locomotor activity during extinction trials when compared to vehicle-treated animals (). A one-way ANOVA revealed significant main effects of Group [F(2,50) = 8.3, p < .005] and Trial [F(3,150) = 127.8, p < .001]. Subsequent post-hoc analysis showed that the Vehicle group exhibited higher locomotor activity than both the SL327 groups (p’s < .01). These data suggest that although CS+-trial locomotor activity decreased over the course of extinction, both doses of SL327 caused a general reduction in activity.
The combined results of Experiments 2 and 3 showed that inhibition of ERK-signaling with the systemic MEK inhibitor, SL327, did not impair extinction learning. This effect was evident at two doses that were administered in two vehicles, at two pre-trial intervals. However, despite failing to prevent extinction, these doses of SL327 did reduce locomotor activity when administered 90 min before the non-reinforced extinction trials.
3.3 Effect of SL327 on acquisition of EtOH-CPP (Exp 4)
Although SL327 did not prevent extinction of EtOH-CPP, previously published experiments have reported that SL327 impairs acquisition of CPP (e.g., Valjent et al., 2000
). Thus, Experiment 4 was performed to extend these previous findings by examining the effects of SL327 on acquisition of EtOH-CPP in mice. The results of Experiment 4 showed that SL327 (50 mg/kg) administered 90 min prior to CS+ conditioning trials did not impair acquisition of EtOH-CPP (, Test 1). In order to assess the persistence of CPP, both groups received five additional drug-free preference tests. Analysis of the preference across all six tests revealed no group differences in EtOH-CPP. This was confirmed by the absence of either a Test × Group interaction or main effect of Group (p
’s > .05). These data showed that SL327 was unable to impair either EtOH reward or the memory formation necessary for acquisition of EtOH-CPP in mice. Furthermore, this finding persisted across multiple, drug-free preference tests.
SL327 had no effect on acquisition of EtOH-CPP
Analysis of the locomotor activity during conditioning revealed that, similar to Experiment 3, SL327 reduced locomotor activity. However, SL327 did not prevent the development of EtOH-induced sensitization that normally occurs during EtOH-CPP conditioning trials (). These findings were supported by significant interactions of Trial × Trial Type [F(1,46) = 111.5, p < .001] and Group × Trial Type [F(1,46) = 25.0, p < .001] as well as significant main effects of Group [F(1,46) = 23.4, p < .001], Trial [F(1,46) = 34.2, p < .001], and Trial Type [F(1,46) = 578.7, p < .001], but no significant Group × Trial × Trial Type interaction (p > .05). Paired t-tests comparing EtOH-induced activity levels on Trials 1 and 2 revealed significant increases for both the Vehicle [t(23) = 6.5, p < .001] and SL-50 [t(23) = 7.6, p < .001] groups. Furthermore, the Vehicle group showed significantly greater levels of EtOH-induced activity than the SL-50 group on both Trial 1 [t(46) = 5.2, p < .001] and Trial 2 [t(46) = 4.5, p < .001]. These data showed that although SL327 reduced locomotor activity, it did not interfere with development of EtOH-induced sensitization.
3.4 Effect of SL327 on expression of EtOH-CPP following 2- and 4-trial conditioning procedures (Exps 5 & 6)
Experiments 5 and 6 were performed in order to assess the effect of SL327 on expression of EtOH-CPP following normal 2-trial, and extended 4-trial, conditioning. The results of Experiment 5 showed that both the Vehicle- and SL327-treated groups showed similar levels of preference for the EtOH-paired floor as confirmed by an independent t-test (p > .05). SL327 did, however, significantly reduce test activity [t(45) = 6.8, p < .001]. These effects were replicated when animals that had received extended CPP conditioning (4 trials) were tested in that SL327 had no effect on expression of EtOH-CPP (p > .05) while significantly reducing test activity [t(46) = 5.5, p < .001]. These results suggest that inhibiting ERK activity with SL327 does not interfere with the conditioned motivational effects of EtOH or its retrieval from memory as assessed during a drug-free CPP test.
3.5 Effect of SL327 on pERK levels in the dorsal striatum and motor cortex (Exp 7)
Experiment 7 was performed in order to confirm that SL327 had crossed the blood-brain-barrier and was reducing ERK activity. Western immunoblot analysis of pERK protein levels in the dorsal striatum and motor cortex were examined as these regions have shown EtOH-induced activation (Asyyed et al., 2006
) while the dorsal striatum has repeatedly been used to assess MEK-inhibition by SL327 in previous CPP experiments (Valjent et al., 2000
; Salzmann et al., 2003
). As shown in , although levels of pERK were not significantly increased 5 min after an EtOH injection, pre-treatment with SL327 caused a significant reduction in both the dorsal striatum and motor cortex. Because the initial Pre-treatment × Injection ANOVA for each region revealed only a significant main effect of Pre-treatment (Dorsal Striatum: [F
(1,20) = 5.1, p
< .05], Motor Cortex: [F
(1,20) = 6.3, p
< .05]) and no significant interaction or main effect of EtOH injection (p
’s > .05), the EtOH and Saline injection groups were collapsed for further analysis of the SL327 effect. Subsequent independent t-tests comparing SL327 and Vehicle pre-treated animals revealed that SL327 significantly reduced pERK levels in both the dorsal striatum [t
(22) = 2.3, p
< .05] and motor cortex [t
(22) = 2.7, p
< .05] (, insets). Specifically, SL327 administration resulted in a reduction of approximately 40% of the pERK levels in both brain regions. Therefore, despite having no effect on learning behavior in Experiments 2–4, or on expression of CPP in Experiments 5–6, SL327 was, in fact, crossing the blood-brain-barrier and significantly reducing pERK levels in multiple brain regions.
SL327 significantly reduced pERK levels in the motor cortex and dorsal striatum