Abstract
Rationale
Although the distinction between "biased" and "unbiased" is generally recognized as an important methodological issue in place conditioning, previous studies have not adequately addressed the distinction between a biased/unbiased apparatus and a biased/unbiased stimulus assignment procedure. Moreover, a review of the recent literature indicates that many reports (70% of 76 papers published in 2001) fail to provide adequate information about apparatus bias. This issue is important because the mechanisms underlying a drug's effect in the place-conditioning procedure may differ depending on whether the apparatus is biased or unbiased.
Objectives
The present studies were designed to assess the impact of apparatus bias and stimulus assignment procedure on ethanol-induced place conditioning in mice (DBA/2 J). A secondary goal was to compare various dependent variables commonly used to index conditioned place preference.
Methods
Apparatus bias was manipulated by varying the combination of tactile (floor) cues available during preference tests. Experiment 1 used an unbiased apparatus in which the stimulus alternatives were equally preferred during a pre-test as indicated by the group average. Experiment 2 used a biased apparatus in which one of the stimuli was strongly preferred by most mice (mean % time on cue = 67%) during the pre-test. In both studies, the stimulus paired with drug (CS+) was assigned randomly (i.e., an "unbiased" stimulus assignment procedure). Experimental mice received four pairings of CS+ with ethanol (2 g/kg, i.p.) and four pairings of the alternative stimulus (CS−) with saline; control mice received saline on both types of trial. Each experiment concluded with a 60-min choice test.
Results
With the unbiased apparatus (experiment 1), significant place conditioning was obtained regardless of whether drug was paired with the subject's initially preferred or non-preferred stimulus. However, with the biased apparatus (experiment 2), place conditioning was apparent only when ethanol was paired with the initially non-preferred cue, and not when it was paired with the initially preferred cue. These conclusions held regardless of which dependent variable was used to index place conditioning, but only if the counterbalancing factor was included in statistical analyses.
Conclusions
These studies indicate that apparatus bias plays a major role in determining whether biased assignment of an ethanol-paired stimulus affects ability to demonstrate conditioned place preference. Ethanol's ability to produce conditioned place preference in an unbiased apparatus, regardless of the direction of the initial cue bias, supports previous studies that interpret such findings as evidence of a primary rewarding drug effect. Moreover, these studies suggest that the asymmetrical outcome observed in the biased apparatus is most likely due to a measurement problem (e.g., ceiling effect) rather than to an interaction between the drug's effect and an unconditioned motivational response (e.g., "anxiety") to the initially non-preferred stimulus. More generally, these findings illustrate the importance of providing clear information on apparatus bias in all place-conditioning studies.
Notes
The fact that an apparatus is labeled "unbiased" as defined by the average performance of a group of untreated subjects does not preclude the possibility that individual subjects may show a marked preference for one of the stimulus alternatives. As with most behavioral measures, every random sample of untrained subjects is expected to yield a relatively normal distribution of scores centered around the sample mean (Fig. 1). Thus, individuals at the tails of the distribution might be described as having a bias, even though the average response of the sample is not biased. Some investigators have attempted to "correct" this situation by discarding subjects who show an initial preference or aversion that exceeds some arbitrary criterion. However, such subject selection strategies raise interpretative problems, especially if the population response to the apparatus has not been well characterized or if the experimental manipulations produce group differences in the numbers of discarded subjects (e.g., targeted gene mutation)
The procedure of exposing animals to only tactile cues in an "open" apparatus during conditioning trials instead of confining them to separate compartments was derived from morphine place-conditioning studies in rats originally reported by Vezina and Stewart (1987a, 1987b). Those studies showed that conditioned place preference was generally stronger using this procedure, in part because animals were required to make direct contact with tactile cues in order to experience conditioned reinforcing effects. Furthermore, animals confined to a smaller portion of the apparatus during conditioning expressed a weaker conditioned preference than those given access to the entire apparatus, an effect that was attributed to the greater novelty of the (open) test environment for previously confined animals. Their finding of significantly higher levels of test session activity in previously confined animals supported this interpretation and the suggestion that increased activity may have directly interfered with expression of conditioned place preference. Because the literature offers relatively few direct comparisons between "open" versus "confined" place conditioning procedures, it is difficult to know whether use of the open apparatus procedure in the current studies limits the generality of these findings. Based on the studies of Vezina and Stewart, one might speculate that magnitude of conditioned place preference would generally be stronger in the present studies than those that used a confinement procedure. However, the literature offers no reason to expect that apparatus bias or stimulus assignment bias would have fundamentally different effects in these two procedures
The use of relatively long test sessions (e.g., 60 min) is more common in mouse place-conditioning studies than in rat place-conditioning studies because mice, unlike rats, typically remain active throughout such tests. The value of using longer test durations is underscored by previous studies showing that important effects of some manipulations would have been completely missed had a short test duration been used (Cunningham et al. 1995, 1998). However, under training and test conditions similar to those used here, previous studies have shown that strength of conditioned place preference is relatively constant over a 60-min test in DBA/2 mice (Cunningham et al. 1995, 1998). Moreover, average time scores during the first 30 min of the final test in the present studies were highly correlated with time scores averaged over the entire 60 min both in experiment 1 (r=0.97, n=92, P<0.0001) and in experiment 2 (r=0.96, n=92, P<0.0001). Thus, there is no reason to believe that effects of bias reported here are an artifact of using a long test duration
It is strongly recommended that apparatus bias be reported as the amount (or percentage) of time spent in contact with each of the stimulus alternatives by vehicle-treated animals that have received the same total number of exposures to each stimulus as drug-treated animals. This approach is preferred to the use of pre-test preferences in naive animals because bias may change over time within an experiment simply as a result of repeated handling, injection and exposure to the stimulus alternatives (see Bardo and Bevins 2000 for a similar comment). Moreover, this information should be reported for each of the specific alternative environments (e.g., time in black compartment versus time in white compartment), not according to assignment as CS+ or CS−. The importance of the latter recommendation is illustrated by noting that in experiment 2, because of the counterbalancing, there was no difference in the percentage of the pre-test spent in contact with the cues later assigned as CS+ (51%) or CS− (49%), even though the apparatus was clearly quite biased (see lower panel in Fig. 1)
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Acknowledgements
This research was supported by NIAAA grants AA07702 and AA07468. Thanks are extended to Carly Petersen for assistance in data analysis and to Anita Bechtholt and Rick Bevins for comments and suggestions. We also thank Rachel Smith for help in constructing floors.
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Cunningham, C.L., Ferree, N.K. & Howard, M.A. Apparatus bias and place conditioning with ethanol in mice. Psychopharmacology 170, 409–422 (2003). https://doi.org/10.1007/s00213-003-1559-y
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DOI: https://doi.org/10.1007/s00213-003-1559-y