These experiments show that by using a reference-dose procedure or a combination of reference-dose and standard procedures, one can demonstrate graded dose effects in the place-conditioning task. Moreover, in contrast to previous studies reported by Barr et al. (1985)
and Bevins (2005)
, these studies illustrate the advantage of using the reference-dose procedure for making comparisons to doses both above and below the reference dose. If one focuses only on findings from groups that received the standard procedure (i.e., ethanol vs. saline), the conclusion from our studies would be that ethanol doses of 1.5, 2 and 4 g/kg yielded similar levels of CPP, whereas 0.5 g/kg did not produce significant CPP, suggesting that the former doses are equally rewarding while the latter dose has no appreciable rewarding effect. However, by using the reference-dose procedure, we were able to extend our understanding of the ethanol dose-effect function in two important ways. First, our finding that the reference-dose groups that received 0.5 g/kg ethanol in combination with intermediate ethanol doses (i.e., Group 2-vs.−0.5 g/kg in Exp. 1 and Group 1.5-vs.−0.5 g/kg in Exp. 3) developed significantly weaker preference than standard-procedure groups that received only the higher dose (i.e., Groups 2-vs.−0 g/kg and 1.5-vs.−0 g/kg, respectively) implies that mice distinguished between the effects of 0.5 g/kg ethanol and saline, a finding that was not apparent from the standard procedure alone. Second, the finding of significant preference in Group 1.5-vs.−4 g/kg shows that the reference-dose procedure was able to detect a difference between an intermediate and high ethanol dose that was not detected in the standard procedure.
Our studies support the following conclusions about the relative rewarding efficacy of various ethanol doses in DBA/2J mice as indexed by the CPP procedure. Starting at the low end of the dose-effect curve, we conclude that 0.5 g/kg is more rewarding than saline. The lack of conditioning in Group 1.5-vs.−0.5 g/kg suggests that the rewarding effects of 0.5 and 1.5 g/kg ethanol cannot be distinguished. However, the significant conditioning effect in Group 2-vs.−0.5 g/kg indicates that DBA/2J mice can distinguish between the rewarding effects of 0.5 and 2 g/kg ethanol. Finally, at the high end of the dose-effect curve, the lack of significant conditioning in Group 2-vs.−4 g/kg suggests difficulty in distinguishing between the rewarding effects of 2 and 4 g/kg, whereas the significant conditioned effect in Group 1.5-vs.−4 g/kg indicates that DBA/2J mice are able to distinguish between the effects of 1.5 and 4 g/kg.
By comparing the results of multiple standard and reference-dose procedures, the current experiments were able to more fully characterize ethanol’s effects across a wide range of doses. These experiments also illustrate the importance of examining more than one reference dose to characterize CPP dose effects. For example, the combined results of Exps. 1 and 2 showed that comparisons to a reference dose of 2 g/kg could detect a difference between the reference dose and a lower comparison dose (0.5 g/kg), but not a higher comparison dose (4 g/kg). In contrast, by slightly reducing the reference dose to 1.5 g/kg in Exp. 3, we were able to detect a difference between the reference dose and a higher comparison dose (4 g/kg), but not a lower comparison dose (0.5 g/kg). Nevertheless, in both cases, by combining the results from reference-dose and standard procedures, we were able to infer that 0.5 g/kg was more rewarding than saline, even though the standard procedure with 0.5 g/kg failed to yield significant CPP.
One issue that must be considered in the interpretation of these studies is whether the greater sensitivity of the reference dose procedure was due, in part, to the higher test session activity produced by the reference-dose procedure. For example, one might argue that the lower CPP in the reference-dose group in Exp. 1 (Group 2-vs.−0.5 g/kg) relative to the 2 g/kg standard procedure group (Group 2-vs.−0 g/kg) was a byproduct of the greater test session activity level in the reference-dose group, a possibility that is generally consistent with recent studies showing an inverse relationship between test session activity and CPP magnitude (Gremel and Cunningham 2007
). Although a possible contribution of test activity cannot be dismissed, several findings argue against this interpretation. For instance, the ability of 0.5 g/kg to offset the rewarding effect of a 1.5-g/kg injection in the reference dose procedure (Group 1.5-vs.−0.5 g/kg compared to Group 1.5-vs.− 0 g/kg in Exp. 3) was not accompanied by a significant group difference in test activity. Moreover, the reference-dose procedure that successfully detected a difference between 1.5 and 4 g/kg (Group 1.5-vs.−4 g/kg in Exp. 3) produced a test activity rate that was similar or greater than that produced in standard procedures with either dose alone, an outcome contrary to predictions based simply on group differences in test session activity.
As in previous CPP dose-effect studies with other abused drugs (e.g., cocaine: Bevins 2005
; morphine: Barr et al. 1985
), our study showed a monotonic dose-effect curve for ethanol-induced CPP. These findings contrast with the biphasic dose-effect relationship typically reported for self-administration of ethanol (e.g., Meisch and Thompson 1974
) and other abused drugs (e.g., Yokel 1987
). The decrease in rate of self-administration that occurs at higher doses has been explained in several ways, including satiation, drug-induced interference with responding and aversive effects of high drug doses (Wise 1987
; Yokel 1987
). Although CPP has been shown to be sensitive to high dose aversive drug effects (e.g., methamphetamine: Cunningham and Noble 1992
), it is presumably less sensitive to satiation and response interference because animals are tested in the absence of drug.
One potential advantage of the reference-dose procedure over the standard CPP procedure is that the reference-dose procedure may be able to detect small changes in the rewarding effects of ethanol produced by pharmacological manipulations. For example, if a treatment drug co-administered with an ethanol dose of 2 g/kg produced only a small decrease in ethanol reward, a standard CPP procedure might be insensitive to this manipulation, suggesting that the treatment drug had no effect. However, if the same treatment drug were co-administered with a low comparison dose of ethanol in a reference-dose procedure (e.g., 2 g/kg + vehicle vs. 0.5 g/kg + treatment drug), a small drug-induced decrease in ethanol reward might have a greater likelihood of being detected. In this case, one would actually expect the treatment drug to increase CPP (by reducing the effect of the low comparison dose) relative to a reference-dose group given vehicle treatment (e.g., 2 g/kg + vehicle vs. 0.5 g/kg + vehicle).
A variation on this strategy would be to co-administer the treatment drug with a comparison dose that is identical to the reference dose (e.g., 2 g/kg + vehicle vs. 2 g/kg + treatment drug). Because no CPP is expected in the control condition (2 g/kg + vehicle vs. 2 g/kg + vehicle), a treatment drug effect would be manifest either by development of preference for the compartment paired with the reference dose (indicating that the treatment drug had reduced the rewarding effect of the comparison ethanol dose) or aversion for that compartment (indicating that the treatment drug had enhanced the rewarding effect of the comparison ethanol dose). In fact, this approach was recently used successfully to characterize a treatment that reduced ethanol reward. Specifically, Font et al. (2006)
reported that Swiss mice developed a significant preference for a tactile cue previously paired with an ethanol reference dose of 2 g/kg relative to a cue that had been paired with the same ethanol comparison dose combined with D-penicillamine, a drug that reduces the ethanol metabolite acetaldehyde.
Theoretical Analysis of Dose Effects on Performance of CPP
One theoretical approach for understanding why the standard and reference-dose procedures might yield different conclusions is illustrated in , which depicts hypothetical values of the stimulus-ethanol association (Associative Strength, V) as a function of increasing numbers of conditioning trials for each ethanol dose used in the standard procedure (solid lines). A key assumption in this analysis is that dose-effects on performance in the standard CPP procedure may be obscured either by a response floor or response ceiling, represented by the shaded regions in . The potential problem can be seen most clearly after a relatively large number of trials (C). Because asymptotic associative strength (V) exceeds the response ceiling, doses of 1.5 g/kg and higher are all assumed to produce the same (maximal) effect on CPP performance. In contrast, because the asymptotic V value produced by 0.5 g/kg is assumed to be near the response floor, CPP performance is expected to be marginal or non-significant.
Figure 2 This figure depicts hypothesized strength (V) of the stimulus-ethanol association as a function of number of conditioning trials. Solid lines represent associative strength conditioned to the CS+ by increasing doses of ethanol (0.5, 1.5, 2.0 and 4.0 g/kg) (more ...)
Predictions for the reference-dose groups (dashed lines in ) are based on the arithmetic differences in associative strength between cues that were paired with different combinations of ethanol doses in the reference dose procedure. It was assumed that the associative value for each cue was the same as that produced when the paired dose was used in a standard CPP procedure. Regardless of the amount of training, one can use this analysis to explain why the reference-dose procedure was able to detect effects of the lowest ethanol dose. More specifically, as shown in , the dashed lines for the reference dose groups that received 2-vs.−0.5 or 1.5-vs.−0.5 g/kg both fell below the response ceiling, which would explain why CPP performance in those groups was weaker than that seen in the groups that received the standard procedure with either of the higher doses alone. The finding of significant CPP in the 2-vs.−0.5 g/kg group is consistent with the placement of their dashed line in the middle of the performance range whereas the absence of CPP in the 1.5-vs.−0.5 group is consistent with the placement of their dashed line below the response floor. Moreover, the ability to detect a difference between 1.5-vs.−4 g/kg in the reference dose procedure is explained by the positioning of that dashed line below the response ceiling in the middle of the performance range.
Although the foregoing analysis is admittedly post hoc, it yields several predictions that could be tested in future studies. For example, the ability to detect differences between high ethanol doses in the standard procedure should be greater after a relatively low number of conditioning trials, before associative strength has reached the response ceiling (e.g., point A in ). Based on previous studies suggesting that CPP performance produced by 2 g/kg in the standard procedure is below asymptote after two CS+ conditioning trials (Cunningham et al. 1997
), our analysis would predict that a standard procedure should be more likely to reveal differences between high ethanol dose groups after only one or two trials. Nevertheless, because CPP learning is so rapid, it may be difficult to find the exact amount of training required to optimize the dose-effect curve in the standard procedure. An alternative strategy for assessing dose effects in the standard procedure might be to examine differences in rate of extinction between groups that have been trained to asymptotic levels of performance. In general, higher doses would be expected to produce greater resistance to extinction because more CS exposure would be needed to induce sufficient inhibitory learning to offset associative strength below the performance response ceiling. Because the rate of CPP extinction generally appears to be slower than the rate of CPP acquisition (e.g., Cunningham et al. 1998
), the extinction strategy might be better for revealing dose effects.
The theoretical analysis outlined here is based on the simplifying assumption that all other variables known to affect CPP magnitude are held constant. It should be noted, however, that variations in many other conditioning parameters can shift the CPP dose-effect curve. One such parameter is conditioning trial duration, which is inversely related to the strength of CPP conditioned by 2g/kg ethanol in the range between 5 and 30 min (Cunningham and Prather 1992
). In fact, an early ethanol dose-effect study that used 30-min trials showed significant CPP at 3 and 4 g/kg, but not at 1 or 2 g/kg (Cunningham et al. 1992
), suggesting that the ethanol dose-effect curve is shifted to the right when longer trial durations are used. Route of administration is another important variable that influences the dose-effect curve. For example, in contrast to the strong CPP produced at both 2 and 4 g/kg when ethanol was administered intraperitoneally in the present studies, only 4 g/kg was able to induce CPP when ethanol was infused intragastrically in a previous study (Cunningham et al. 2002a
), again suggesting a rightward shift in the dose-effect curve.
In summary, the present experiments have confirmed the utility of the reference-dose procedure for studying ethanol dose effects in the CPP procedure and have offered insight into alternative strategies for assessing the rewarding properties of ethanol, as well as other drugs of abuse, in a CPP framework. Standard CPP procedures showed that ethanol doses of 1.5, 2, and 4 g/kg elicited similar levels of preference, whereas a dose of 0.5 g/kg was unable to induce a significant preference. However, the reference-dose CPP procedure was able to distinguish between the rewarding effects of higher doses (1.5 vs. 4 g/kg) and, when used in combination with a standard procedure, revealed rewarding effects of a relatively low dose (0.5 g/kg). Thus, the reference-dose procedure provides a potentially more sensitive method for detecting small differences or changes in ethanol’s rewarding effect and may serve as an important alternative or supplement to the standard CPP procedure.