Research reportUnconditioned and conditioned factors contribute to the ‘reinstatement’ of cocaine place conditioning following extinction in C57BL/6 mice
Introduction
Drug addiction is a chronic neuropsychiatric disorder characterized by drug craving and relapse to compulsive drug-seeking behavior [14], [19]. A well-documented major contributing factor to drug craving and relapse during abstinence is the ability of drug-associated contextual cues (e.g. drug paraphernalia etc.) to elicit drug craving and -seeking. By virtue of being present while the individual is experiencing the rewarding and reinforcing effects of the drug, contextual cues gain secondary motivational significance or conditioned reward value [6], [7], [8], [18], [27], [28], [39]. Place conditioning is one animal model to study the influence of drug-associated contextual stimuli on drug-seeking [2], [3], [15]. In this model, a drug is differentially paired with either one or the other of two distinctive sets of contextual stimuli or environments. Following conditioning, the animals are allowed unrestricted access to both environments in the absence of the drug. An increase in the amount of time the animal spends in the presence of the drug-paired environment indicates that the context has acquired secondary or conditioned incentive properties, presumably via pairing with the rewarding effects of the drug [2]. According to this procedure, animals are tested in a drug-free state, enabling an investigation of the appetitiveness of drug-associated contextual stimuli without the confounding influence of consummatory variables [2]. Moreover, the place conditioning paradigm is a useful non-invasive model for studying simultaneously both the rewarding and motor-activating effects of primary reinforcers/rewards [2], [3], [6], [15].
Recent place conditioning studies in rats [20], [25], [33] demonstrate that extinguished place conditioning can be reinstated by injections of the conditioning drug (the unconditioned stimulus). These results are consistent with the self-administration model and indicate that re-exposure to the drug elicits relapse to drug-seeking behavior [9], [14], [19], [21], [39]. As such, the reinstatement (RI) of place conditioning may provide an animal model for investigating the influence of both drug and contextual cues upon drug-seeking behavior. Furthermore, extinguished place conditioning can be reinstated by drug doses lower than those used to produce conditioning as reported for rats [20], [25], [33], [46], and recently for Swiss Webster mice [17]. These results suggest that a sensitization process [20] might contribute to the RI by drug primes, an observation also consistent with results of self-administration studies [9], [14], [19], [21], [39]. There is currently only one published report examining the extinction and RI of drug-induced place conditioning in mice. This recently published report indicated that extinguished cocaine (COC) place conditioning in Swiss Webster mice could be reinstated by priming injections of COC and other stimulant drugs [17]. Given that the unconditioned motor-activating [24], [37], [41], [42] and reinforcing/rewarding [4], [24] effects of COC in mouse can be strain-specific and that it is the primary species used for the genetic analysis of drug effects and addiction, it is important to extend this work to other mouse strains and variations in procedure.
Thus, in the present report, C57BL/6 (B6) mice were used to explore several issues related to the effects of COC on the RI of extinguished COC-induced place conditioning. The B6 mouse was selected for study for two principal reasons. First, the B6 strain is the primary background strain used in generating transgenic and gene knock-out mice, thus the data derived from studying this strain might be more predictive of behavior expressed by genetically manipulated mice. Second, compared to other strains (e.g. DBA/2J or BALB/c), B6 mice are more sensitive to the conditioned rewarding effects of COC [4] but are less sensitive to the motor-activating effects of this drug [38]. Thus, it is hypothesized that any observed effects of COC administration upon reward should be dissociable from non-specific motor effects of COC in these mice.
To explore several issues related to the RI of extinguished COC-induced place conditioning in mice, we conducted three experiments. Experiment 1 provides a dose-response function for COC-induced RI of place conditioning following extinction. Experiment 2 addressed issues related to potential unconditioned effects of COC administration on the expression of place conditioning. Two major issues faced by laboratories studying behavioral genetics are the availability and cost of transgenic and gene knockout mice. This is particularly true for studies of drug-related behavior where the variability in the data can be high and relatively large samples (10–20 animals) are required in order to achieve adequate statistical power. For these reasons, it may be necessary to study older mice with an experimental history. Thus, Experiment 3 determined whether the results of Experiment 1 could be generalized to older mice with an extensive experimental history.
Section snippets
Subjects
Mice for all experiments were male B6 mice obtained from The Jackson Laboratories (Bar Harbor, MN). They were maintained in polyethylene cages in an AAALAC-accredited colony room, controlled for temperature (25 °C) and humidity (71%), under a 12-h day–12-h night cycle (lights off: 18:00 h). Food and water were available ad libitum throughout the experiments. Mice for Experiments 1 and 2 were 8 weeks of age (20–25 g), were housed individually throughout the experiment, and were allowed 7 days to
Experiment 1: Dose-response function for COC RI of place conditioning
The times spent in the non-preferred compartment during the different phases of testing are summarized in Fig. 1(A and B), as are the activity scores in Fig. 1(C and D). ANOVAs indicated that both the time spent and the motor activity in the non-preferred compartment varied across Test Session [Test Session Effect for occupancy time: F(12, 324)=12.97, P<0.0001; for motor activity: F(12, 324)=3.09, P<0.0004]. However, a comparison of Fig. 1A with C indicates that the change across tests differed
COC place conditioning, extinction, and RI in mice and rats
The present study using B6 mice indicates that repeated pairing of COC with the non-preferred compartment of a two-compartment place conditioning apparatus increases its positive characteristics resulting in approach behavior (i.e. increased time in the COC-paired environment) when mice are tested in a drug-free state. These observations confirm previous reports of COC place conditioning in mice [10], [17] and rats [2], [3], [12], [15], [25], [43]. Concomitant activity measures indicate that
Conclusions
The present study demonstrates that following the repeated pairing of a moderate dose of COC with a particular context, place conditioning can be observed in mid-aged male B6 mice with extensive experimental histories, as well as young adult experimentally naı̈ve male B6 mice. As reported for rats [12], [25] and Swiss Webster mice [17], COC place conditioning in B6 mice is enduring, lasting 7–10 days. Further, the expression place conditioning was unrelated to the expression of locomotor
Acknowledgements
The authors would like to thank Drs R.A. Fuchs and T.E. Kippin for their helpful insights and comments in the preparation of this manuscript. Funding for this study was provided by NIDA grants DA14185-01 (LDM) and DA-03906 (Dr P.W. Kalivas). KKS is supported by a fellowship from the CIHR.
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