These experiments are the first to show that pretreatment with ethanol 5 min before each CS+ conditioning trial interferes with development of a conditioned place preference induced by ethanol given immediately before CS+ exposure. Experiment 1 suggested that this interference effect was unique to the 5-min pre-exposure interval since pre-exposure at 15 min did not interfere with place preference when compared to a group that was matched for overall ethanol exposure, but received the extra ethanol injection 60 min after each trial. Although the place preference observed after pretreatment with ethanol 30 min before the trial was statistically marginal, the general conclusion of no interference at pretreatment intervals longer than 5 min is certainly consistent with the previous report of no interference with ethanol pretreatment at 65 min (Cunningham et al., 2002
). The finding that pre-exposure to a saline injection 5 min before each CS+ trial had no effect on place preference (Experiment 2) argues against an interpretation of the interference effect that simply appeals to stress induced by handling and injection. Rather, the overall pattern of findings suggests that the interference was caused by the overlap of ethanol’s early pharmacological effects (i.e., effects that occur 5–10 min after injection) with the ethanol-CS+ pairing.
Because all groups in Experiment 1 were matched for overall exposure to ethanol, the interference effect observed in Group −5 is not readily attributed to differences in chronic ethanol tolerance. However, one might argue that two closely spaced ethanol injections (i.e., 5 min apart) induce greater chronic tolerance than the same two injections given at longer intervals (e.g., 15–60 min). In other words, due to temporal summation, giving two injections 5 min apart might be functionally equivalent to giving a larger single dose, which would be expected to induce greater chronic tolerance. Although such summation is certainly possible, previous studies involving chronic pre-exposure to a larger ethanol dose (4 g/kg) have failed to show any evidence of tolerance to ethanol’s rewarding effect in the place conditioning procedure (Cunningham et al., 2002
). Thus, it seems unlikely that the present findings were due to greater chronic tolerance in the 5-min ethanol pre-exposure groups.
Athough an explanation based on chronic tolerance is not compelling, one might derive an account of the time delay effect based on the phenomenon of acute functional tolerance, which refers to neuroadaptations that occur over time during a single drug experience (e.g., Radcliffe et al., 2006
). For example, one might argue that groups given ethanol pretreatment at longer delays will develop greater acute tolerance to ethanol-induced sensory-motor disturbances induced by a second ethanol dose that is given before the previous dose has been eliminated. Assuming that such disturbances would interfere with development of an association between ethanol’s rewarding effect and novel cues that signal the second dose, the acute tolerance analysis predicts that interference should be greatest with a short delay. The minute-by-minute analysis of activity on the first ethanol conditioning trial provides some support for this interpretation, showing a less rapid suppression of activity in Groups −15 and −30 (during Minute 2 after the second injection) compared to Group −5. If acute tolerance to ethanol’s activity suppressing effects is correlated with acute tolerance to sensory disturbances that interfere with learning, one might expect less interference with place conditioning in Groups −15 and −30 groups than in Group −5.
The present studies do not support the predicted summation of ethanol’s rewarding effects based on the temporal proximity of the pre-exposure injection to the US injection. This prediction was based on the previous observation that injection of an ethanol US 30 min before CS exposure produces a conditioned place preference (Cunningham et al., 1997
). Given that outcome, one might have expected the combination of a pretreatment injection 30 min (or less) before the US injection to produce an enhanced conditioned place preference. The failure to see temporal summation in the present situation is at odds with the previous observation of summation with two closely spaced ethanol injections when the first injection occurred just before CS exposure and the second injection was given 5 min later (Cunningham et al., 2003b
). In that case, magnitude of place preference was enhanced, as would be expected when the CS is paired with a larger ethanol dose (Cunningham et al., 1992
; Risinger & Oakes, 1996
). Thus, the present findings suggest that temporal summation of rewarding effects does not benefit a CS that is uniquely paired with the second in a series of closely spaced ethanol exposures. Moreover, when considered together with previous findings, the present studies suggest that environmental cues associated with the onset
of a lengthy drug experience may be more strongly associated with the drug’s rewarding effects than cues that signal additional drug exposure later within the same drug experience.
In contrast to the place preference data, activity data recorded during the conditioning trials provide clear evidence of temporal summation. That is, activation produced by the ethanol US injection was substantially reduced in mice that received ethanol pretreatment injections, as would be expected in DBA/2J mice that received a functionally higher dose of ethanol (Cunningham et al., 1992
; Dudek & Phillips, 1990
). At the same time, the general similarity of the activity effect across all three pretreatment times suggests that interference with place preference in the 5-min group was unrelated to processes involved in temporal summation of ethanol’s activating effects.
In both experiments, test session activity was lowest in the group that had received ethanol pretreatment 5 min before each CS+ trial, raising the possibility that differences in expression of conditioned place preference were secondary to effects on general test activity. However, this interpretation is not consistent with previous studies showing an inverse relationship between test session activity and magnitude of conditioned place preference (e.g., Gremel & Cunningham, submitted; Cunningham, 1995
; Vezina & Stewart, 1987
). That is, based on previous findings, one would expect that a reduced level of test session activity would be associated with stronger expression of conditioned preference, not weaker. Thus, it seems unlikely that the interference effect observed here was due to reduced test activity in the 5-min groups. One potentially interesting interpretation of that reduced activity is that it reflects a conditioned suppression of activity controlled by general contextual cues of the apparatus. In other words, based on the temporal contiguity of contextual cues with two closely spaced ethanol injections, those cues might have acquired the ability to elicit a conditioned suppression of activity. If true, this interpretation suggests that mice in the 5-min group were able to form an association that influenced general activity, but were unable to form the association needed to express a preference for the ethanol-associated floor.
Previously, we suggested two possible explanations for interference produced by same drug pre-exposure in the place conditioning procedure (Cunningham et al., 2002
). One explanation was based on the assumption that drug pretreatment somehow interfered directly with the drug’s motivational effect. For example, we suggested that ethanol pre-exposure reduced conditioned place aversion produced by post-CS ethanol injection because it reduced the aversiveness of the rapid transition from the sober to the intoxicated state. However, an explanation of the present results based on direct interference with ethanol’s rewarding effect seems unlikely given the previous finding of reward summation across two injections given 5 min apart (Cunningham et al., 2003b
). Rather, the present findings seem more reasonably explained by assuming that ethanol pretreatment directly interfered with the formation of an association between the CS and ethanol US 5 min later.
The exact mechanism(s) underlying this hypothesized associative interference are unknown. As noted earlier, acute tolerance to sensory-motor disturbances that interfere with learning is one possibility. However, the Pavlovian conditioning literature on proximal US pre-exposure offers several other possibilities (Domjan, 1980
; Domjan & Best, 1977
). One possible explanation, derived from Rescorla’s contingency analysis of Pavlovian conditioning (Rescorla, 1967
), is that ethanol pretreatment 5 min before the conditioning trial degraded the excitatory CS-US contingency because ethanol pretreatment increased the probability of the US in the absence of the CS. However, this interpretation is complicated by the fact that all
groups in Experiment 1 received an extra ethanol injection in the absence of the US (i.e., before or after the CS-US pairing) on each trial. Thus, the excitatory contingency was equally degraded in all groups, which leads to the (incorrect) prediction of no differences among groups. Because contingency is based only on the relative probabilities of the US in the presence and absence of the CS, this analysis cannot explain the present findings unless one modifies the definition of contingency by restricting the time window in which relative probabilities are calculated. For example, one might assume that an extra US will degrade the excitatory contingency only if it occurs within a narrow time period before CS exposure (e.g., Cannon et al., 1975
The conditioning literature offers several other theoretical explanations that consider the time interval between the proximal US and a conditioning trial US. For example, according to opponent-process theory (Solomon, 1977
), proximal ethanol exposure should initiate a time-dependent primary affective process (presumably rewarding) that evokes a delayed opponent affective process (presumably aversive) that counteracts the primary process and persists after the primary process has dissipated. Opponent-process theory can explain the time-delay effects seen in Experiment 1 only by assuming that the strength of the residual opponent process evoked by ethanol exposure at −5 min exceeded the strength of the opponent process remaining from ethanol exposure at −15 or −30 min. In that case, the net affective response to the conditioning trial US would be smallest in Group −5, consistent with the reduced conditioning seen in that group. However, this analysis is not supported by previous data suggesting that the primary affective (rewarding) process induced by a 2 g/kg ethanol injection persists for at least 30 min after injection as indicated by its ability to induce preference for a delayed CS (Cunningham et al., 1997
Consistent with the outcome of Experiment 1, Wagner’s SOP theory (Wagner, 1975
) predicts that interference produced by a proximal US will be inversely related to the time delay between US exposures. According to SOP theory, ethanol pretreatment at the short time delay would reduce the effectiveness of the ethanol US through a memory “priming” mechanism (i.e., processing of the conditioning trial US would be reduced by the recent and still ongoing processing of the proximal US). Another possible interpretation is that strong sensory experiences produced by the proximal US injection might have reduced learning about the CS due to attentional or associative competition (Best & Domjan, 1979
). For example, internal stimuli produced by the pretreatment ethanol injection may have overshadowed the target CS, preventing its association with the ethanol UR. Alternatively, pretreatment ethanol may have directly prevented memory formation due to its hypothesized amnesic properties (Ryabinin, 1998
). Although all of these theories offer a general explanation of the findings observed here, none of them specifies the precise form of that temporal gradient, which presumably depends on the nature, duration and intensity of the US. Regardless of the interpretation, the unique contribution of these experiments lies in showing that, in the case of ethanol conditioned place preference, the interference mechanism was optimally engaged at the 5-min pretreatment interval, but was not a factor at other pretreatment times. Additional research is needed to address these mechanisms.
The overall pattern of findings reported here is generally similar to that previously reported in studies examining effects of proximal US exposure on development of taste aversion conditioned by injection of lithium chloride in rats. For example, Domjan and Best (1977)
found that interference was inversely related to the time interval between the proximal US and conditioning trial, showing a substantial reduction in conditioned taste aversion by drug pretreatment at −30 min, weaker interference at −360 min, but no interference at −24 hrs. Although the specific time course of the proximal US exposure effect on lithium-chloride induced conditioned taste aversion in rats differs from that of the proximal US exposure effect on ethanol-induced conditioned place preference in mice, the general similarity of these phenomena suggests generality of the proximal US exposure effect across species, drug and response system. Moreover, this similarity extends to the paradoxical finding that proximal US exposure produces interference during a pre-trial time interval in which a backward US-CS pairing produces conditioned taste aversion (Domjan & Best, 1977
) or conditioned place preference (Cunningham et al., 1997
Although the mechanisms underlying the interference effect are unknown, the present findings also have potentially important implications for interpreting effects of putative ethanol agonists on acquisition of ethanol-induced conditioned place preference. More specifically, these studies suggest that an ethanol agonist should interfere with acquisition, but only if administered within a relatively narrow time window just before each CS-ethanol pairing. Moreover, these studies suggest that the agonist’s effect on ethanol-induced activation during conditioning might not be predictive of its impact on conditioned place preference. Thus, in cases where a putative agonist is found to have no effect at one pretreatment interval, it would be prudent to consider other pretreatment intervals, especially relatively short ones.