Although DCS did not enhance the rate of extinction, there was evidence that DCS enhanced the persistence of extinction, i.e., reconditioning of the initial place preference was impaired in DCS-treated mice. The lack of a facilitating effect of DCS on the rate of extinction was consistent across two doses (30 and 60 mg/kg) and two experiments that manipulated the strength of initial place preference by varying the number of conditioning trials. In both studies, however, administration of DCS prior to each extinction trial impaired subsequent reconditioning of the extinguished place preference. This effect on reconditioning was not due to unconditioned effects of DCS because neither dose conditioned a place aversion (Experiment 1) or had any effect on activity levels. Furthermore, the effects of DCS on reconditioning were not due to a nonspecific effect of chronic DCS exposure on learning because DCS pre-exposure before initial conditioning did not impair development of ethanol CPP (Experiment 4).
In Experiment 2, the strongly conditioned place preference resulted in a relatively slow rate of extinction, which should have allowed any extinction-facilitating effects of DCS to be observed. However, because extinction took such a long time in this experiment, it was hypothesized that DCS might be more effective in facilitating the extinction of a weaker, more susceptible, place preference. Because previous work from our laboratory had shown that testing after only two CS+ and two CS− conditioning trials produced a weaker CPP than that seen after four CS+ and four CS− trials (Cunningham et al., 2002
), Experiment 3 examined DCS effects on extinction of a weaker ethanol-induced CPP. The results of that experiment showed that the weaker CPP extinguished more rapidly than CPP in Experiment 2 as indexed by the number of test trials required to reduce overall mean preference below 60% (6 tests in Experiment 3 vs. 12 tests in Experiment 2). However, despite beginning extinction with a weaker CPP, mice given DCS before each extinction test did not show an enhanced rate of extinction. Nevertheless, as in Experiment 2, DCS injections administered during extinction impaired subsequent reconditioning.
The results of Experiments 2 and 3 are not consistent with previous reports of the facilitating effects of DCS on extinction of conditioned fear and cocaine-induced CPP. In contrast to our experiments, those studies reported effects of DCS on rate of extinction (e.g., Botreau et al., 2006
; Walker et al., 2002
). Additionally, the effects of DCS on the reconditioning of an extinguished place preference in the current experiments are not in agreement with previous reports showing that DCS given during extinction blocks reinstatement, but not reconditioning, of conditioned fear (Ledgerwood et al. 2004
; Ledgerwood et al. 2005
). Interestingly, in contrast to the results of Ledgerwood et al. (2004)
, Kelley et al. (2007)
showed that extinction-specific DCS administration did not impair reinstatement of cocaine CPP. More recently, Paolone et al. (2008)
showed that rats treated with DCS during extinction of cocaine CPP showed an inability to exhibit cocaine-induced reinstatement. However, because the control group (Saline-treated animals) showed no significant reinstatement of cocaine CPP (as indicated by a signficant increase in preference following the cocaine-priming injection), the interpretation of this effect is limited. Thus, considering the current reconditioning effects of DCS, as well as the data from Kelley et al. (2007)
, it appears that both reconditioning and reinstatement of drug-induced CPP may involve different mechanisms than those involved in the conditioned fear procedures used by Ledgerwood et al. (2004
). However, a more systematic examination of the different methods of reinstatement and reconditioning within each of these behavioral procedures is required to clarify these discrepancies.
In contrast to most of the previously published reports of the extinction-facilitating effects of DCS, the current set of experiments involved examination of two doses of DCS (30 and 60 mg/kg) in the DBA/2J mouse strain. In previous reports, effective doses for the extinction-facilitating effects of DCS in rats have ranged from 5 to 30 mg/kg with the majority of experiments using 15 mg/kg, whereas in mice, effective doses have ranged from 15 to 30 mg/kg (e.g., Kelley et al., 2007
; Tomilenko & Dubrovina, 2007
). Interestingly, doses of 15 and 30 mg/kg DCS have been shown to equally enhance extinction of a food-associated operant behavior in C57Bl/6 mice (Shaw et al., 2008
). However, because at high doses, DCS can exhibit antagonist-like characteristics (for review see Lanthorn, 1994
) as well as the fact that the cognitive enhancing effects of DCS are eliminated at both very low (i.e., 2.5 mg/kg) and high DCS doses (i.e., 50 mg/kg) in mice (Flood et al., 1992
), we hypothesized that the extinction-facilitating effects of DCS in DBA/2J mice would be greatest at a dose of 30 mg/kg. Therefore, although Experiment 1 revealed no detectable stimulus properties or locomotor effects of either 30 or 60 mg/kg DCS, we decided to examine only the lower of these two doses in Experiment 2. Although this dose resulted in no effect on extinction of ethanol CPP (Experiment 2), it did significantly impair subsequent reconditioning and, as such, we are confident that DCS did reach biologically relevant levels. This is further supported by previous reports showing that an even lower dose of 20 mg/kg DCS has anticonvulsant effects against audiogenic seizures in DBA/2 mice (De Sarro et al., 2000
). Therefore, we feel strongly that the doses used in Experiments 2 and 3 should have been sufficient to produce biologically relevant, potentially extinction-facilitating, levels of DCS in the brains of the DBA/2J mice used in these studies. It has been suggested, however, that the cognitive-enhancing effects of DCS may be strain dependent (Sunyer et al., 2008
), and therefore, it is possible that DBA/2J mice, although adept at expressing ethanol CPP, may not be as susceptible to the extinction-facilitating effects of DCS as other mouse strains and/or species.
Studies of DCS on extinction of ethanol-induced learning are particularly interesting because ethanol has direct interactions with the NMDA receptor. Ethanol inhibits glutamatergic transmission of the NMDA receptor by both glycine-reversible and glycine-independent mechanisms (Buller et al. 1995
). Further, ethanol exposure can reduce the potency of glycine at its binding site, thereby inhibiting NMDA receptor transmission in rat cerebellar cells (Hoffman et al. 1994
). In a study examining the effects of DCS on extinction during withdrawal from ethanol, Bertotto et al. (2006)
showed that chronic ethanol exposure (14 days of a 6% v/v ethanol containing liquid diet) impaired the subsequent extinction of conditioned fear. Furthermore, the previous chronic exposure actually enhanced the extinction-facilitating effects of a sub-optimal dose of DCS. Similarily, in a recently published report, Vengeliene et al. (2008)
showed that a low dose of DCS (5 mg/kg), administered 60 minutes prior to the extinction trial, facilitated the extinction of an ethanol-paired operant behavior in rats following extensive ethanol exposure during saccharine-fading, training, and discrimination training. However, because in the current study, mice were administered only four or two injections of 2 g/kg ethanol over eight and four days (Experiments 2 and 3, respectively), it is unlikely that these sub-chronic ethanol exposures significantly altered NMDA-receptor function in such a way that impaired the effects of DCS during the initial extinction trials.
Because extinction lasted for 12 trials in Experiment 2 and six trials in Experiment 3, we were able to examine the effects of DCS with different amounts of extinction. We found that DCS had no effect on rate of extinction during the initial few trials, when preference was highest, nor did it have effects during later trials, when preference was lower. These findings suggest that the failure to observe effects of DCS on extinction were not due to ceiling or floor effects on preference. However, it is possible that the absence of an effect on the later extinction trials was due to previous exposure to DCS during initial extinction trials. Pre-exposure to DCS has been shown to reduce the learning-enhancing effects of DCS in the Porsolt Swim Test (Lopes et al. 1997
), a linear maze apparatus (Quartermain et al 1994
), and extinction of conditioned fear (Parnas et al. in 2005
). All of these reports hypothesized that pre-exposure to DCS caused a desensitization of the NMDA receptor at the glycine-binding site, thereby reducing the effects of subsequent DCS exposures. Therefore, in the current experiments, the effects of DCS, expected to be the greatest during the first few extinction trials when the greatest amount of learning should occur, were either non-existent or undetectable with our behavioral assay, whereas any effects of DCS injections later in the extinction phase were most likely hindered by NMDA-receptor desensitization caused by the initial DCS exposures.
The impairment of reconditioning by DCS, evident in both Experiments 2 and 3, may have resulted from an extinction-facilitating effect of DCS. Specifically, despite showing no effect on extinction behavior, DCS may have deepened the extinction learning, thereby impairing the subsequent reconditioning process. This hypothesis is supported, in part, by the finding that repeated exposure to DCS in Experiment 4 before conditioning had no affect on the new learning that occurs during initial ethanol CPP conditioning. Thus, it seems unlikely that the impaired reconditioning in the DCS groups was simply a result of NMDA-receptor desensitization caused by DCS exposure during extinction.
Given the complexity of the actions of DCS and the procedural sensitivity of the effects of DCS, it is not surprising that several reports have shown inconsistencies in the ability of DCS to enhance extinction. For example, extinction enhancing effects of DCS have not been observed in a variety of behavioral disorders including mild arachnophobia (Guastella et al., 2006) and obsessive-compulsive disorder (Storch et al., 2007
). Further, in studies of rodents, DCS effects on extinction may sometimes be limited to low anxiety animals (Tomilenko & Dubrovina, 2007
) or to animals that show large amounts of extinction within a session (e.g., Weber et al., 2007
). In a recent report, Woods and Bouton (2006)
demonstrated that DCS (30 mg/kg) enhanced the rate of fear extinction in rats, but did not weaken contextual renewal of conditioned fear. This effect, however, was not significant at a lower dose of 15 mg/kg. Moreover, when the authors attempted to replicate the significant findings using 30 and 60 mg/kg, they found no effect of either DCS dose.
In a more recent follow-up report, these authors showed that DCS did, in fact, have slight extinction-enhancing effects but only in animals that showed the greatest amount of overall extinction learning. Specifically, Bouton et al. reported that when analyzing only the rats that showed above-median extinction levels, DCS (30 mg/kg) showed signficant facilitation of extinction (Bouton et al., 2008
). However, despite using multiple variants of this “median-split” technique to analyze our current set of experimental data, we were unable to detect any extinction-enhancing effects of DCS at any dose. In fact, we performed median-split analyses using three different measures of extinction including preference on the last test of extinction (Tests 12 and 6 for Experiments 2 and 3, respectively), decrease in preference over the course of extinction (calculated as a preference difference score from the first to the last test of extinction), and finally, preference on the second test of extinction (Test 2) for animals that showed above-and below-median within-session extinction on Test 1. This latter analysis was performed after considering previously published reports suggesting that the extinction-facilitating effects of DCS are greatest during the first few extinction trials. All in all, none of these median-split analyses revealed a signficant effect of DCS on extinction of ethanol CPP (data not shown).
In conclusion, these experiments demonstrated that DCS did not facilitate the rate of extinction of ethanol-induced CPP, regardless of the strength of the initial preference. Nevertheless, administration of DCS during extinction impaired subsequent reconditioning of ethanol-induced CPP, demonstrating that DCS did enhance some aspect of the extinction experience. This was further supported by the findings of Experiment 4 that revealed no effect of chronic exposure to multiple doses of DCS on the initial development and expression of ethanol CPP. These findings emphasize the general importance of using multiple measures of learning to assess pharmacological effects on extinction. Additionally, these findings suggest that DCS may not be capable of facilitating the behavioral therapy used in the rehabilitation of alcoholic patients, though it may provide a means by which to reduce the potential for relapse to alcohol-seeking behavior.