The purpose of the present series of experiments was to examine in greater detail several different measures of ethanol reinforcement and relapse in WSP and WSR mice. Hence, multiple phenotypes were investigated in an attempt to determine whether the segregation of alleles related to ethanol withdrawal severity conferred line differences in a subset of behaviors related to ethanol reinforcement, more generally across all measures, or whether withdrawal severity and reinforcement appeared to be genetically independent. In general, the present results indicate that there were subtle line differences in several aspects of ethanol self-administration, which depended on the reinforcer solution presented and the reinforcement schedule imposed. The results are summarized in .
Summary of findings in WSP-1 and WSR-1 mice.
The first set of studies examined operant ethanol self-administration under different reinforcement schedules. Importantly, we were able to successfully train all WSR mice (19/19) and most WSP mice (13/16) to respond for access to ethanol on a FR schedule of reinforcement. When responding on a FR4 schedule of reinforcement, WSR mice exhibited a higher overall level of responding on both levers throughout the sucrose fading procedure than the WSP mice (). This result is consistent with early work demonstrating that operant responding maintained by a sucrose-milk solution was significantly higher in male WSR-2 than in WSP-2 mice (Balster et al., 1993
While the responding maintained by sucrose/ethanol combinations was unaltered in WSR-1 mice as the ethanol was introduced and subsequently increased in a stepwise fashion (), the introduction of ethanol into the sucrose solution did alter responding in the WSP-1 mice. The near significant line × solution interaction on active lever responding (P=0.06) and the significant line × solution interaction for the number of reinforcers was most likely attributable to the changes in reinforcers earned by WSP-1 mice as the ethanol content of the solution was altered. It should be noted that the current procedure implemented a procedure in which the quantity of the acquired reinforcer was not fixed (i.e., as is the case with a dipper cup or a set infusion volume). Instead, mice were presented with 30-sec sipper access periods during which the rate and quantity of consumption was entirely self-regulated by the animal. Therefore, while one interpretation of the small changes in responding upon introduction of ethanol could be that ethanol exhibited little reinforcing efficacy, an equally plausible explanation could be that mice simply consumed more or less ethanol per reinforcer earned. The waxing and waning of ethanol intake in WSP-1 mice across the sucrose/ethanol solutions offered () would favor the latter interpretation, and would suggest that WSP-1 mice were more attentive to changes in the sucrose and ethanol content of the solutions presented during fading. However, the lines did not differ in their self-administration (volume or dose) of unadulterated 10E.
There are several possible explanations for the line differences in the initiation phase of operant 10E self-administration. First, WSP mice may find sweetened ethanol solutions more rewarding when responding in an operant setting, as they self-administered doses of ethanol that are presumed to be pharmacologically active (e.g., 1.2 – 2.0 g/kg with intake of the 10S/5E, 10S/10E, and 5S/10E solutions). Consistent with this idea, WSP (but not WSR) mice of both replicates exhibited a conditioned place preference to ethanol (Crabbe et al., 1992
), which suggests that they are more sensitive to the rewarding effects of ethanol in the place conditioning procedure. Second, the higher intake in WSP versus WSR mice could reflect a line difference in overall fluid intake. While an earlier home-cage drinking study reported that overall fluid intake was consistently higher in WSP than in WSR mice (Kosobud et al., 1988
) there was no line difference in total fluid intake during any phase of our home cage drinking study. Third, WSR mice exhibited a gradual decline in ethanol intake throughout the sucrose fading procedure (i.e., beginning with the initial introduction of 2E). It is possible that WSR-1 mice are more sensitive than WSP-1 mice to ethanol's chemosensory properties, which were revealed in a limited access setting. This suggestion also would be consistent with the transient development of a greater conditioned taste aversion to ethanol in both replicates of WSR versus WSP mice (Chester et al., 1998
), and would suggest that WSR -1 may be slightly more sensitive than WSP-1 mice to ethanol's aversive effects. Another possibility is that WSR mice simply did not consume ethanol from the sipper upon each presentation when responding on the FR4 schedule. We recently observed that C57BL/6 mice, which exhibit high ethanol preference and self-administration, only approached the sipper approximately 50% of the time when they were responding on a FR4 schedule (Ford et al., 2007a
). A preliminary examination of sipper contacts during the initial sipper presentation of 10E in the current study suggested that both WSP and WSR mice were not consistently sampling during the 30 sec of sipper access. Thus, inconsistent consumption from the sipper on a FR4 schedule may partially contribute to the decreased consumption throughout sucrose fading in WSR mice.
The current understanding of conditioned responding for ethanol under a non-food restricted condition suggests that ethanol is a weak behavioral reinforcer in mice. As described by Ford et al. (2007a)
, oral self-administration of ethanol supports a much lower response rate than that observed for intravenous infusions of psychostimulants such as cocaine (e.g., Little, 2000
). The difficulty in assessing the reinforcing properties of orally administered ethanol in animal models is due in part to the delayed onset of pharmacological effects (Meisch, 2001
) and the quantity of fluid volume necessary to support a behaviorally-relevant dose. Both of these procedural challenges are problematic for operant conditioning procedures that incorporate a FR schedule of responding. This potential difficulty may be overcome with recent modifications in operant conditioning procedures in the rat, where a “sipper” model procedurally separated the appetitive and consummatory phases of ethanol self-administration (Samson et al. 1998
). By providing continuous access to ethanol (typically 30 min) following the completion of a single response requirement (RR), this experimental procedure permits the animal to regulate its own consumption (and hence the onset of ethanol pharmacology) rather than having intake dictated by intermittent access following repeated response demands that are associated with a FR schedule of reinforcement. It also removes the possibility that intoxication will gradually interfere with FR performance during later parts of the session. With this in mind, we determined whether the two schedules of reinforcement would detect line differences in the maintenance of ethanol self-administration.
An examination of the maintenance phase of ethanol self-administration revealed that there was no line difference in 10E intake when mice were responding on a FR4 schedule. However, when a schedule was imposed (RR8) that allowed mice to regulate their own rate of 10E consumption for 30 uninterrupted minutes, WSR mice significantly increased their 10E intake by 76% over the intake in WSP mice (). Notably, the enhancement of drinking in WSR mice following a schedule manipulation from FR4 to RR8 is similar to our recent results in male C57BL/6 mice, where transition from a FR4 to a RR4 schedule of reinforcement was associated with a doubling of the ethanol dose consumed during a 30 min session (Ford et al., 2007a
). Although BECs were not measured, WSR mice were consuming a dose of ethanol (0.8 g/kg) within 30 minutes that has previously been shown to produce BECs ≥ 50 mg/dl (or 10.9 mM; e.g., Elmer et al., 1987
; Czachowski et al., 2002
). As reviewed by Spanagel (2009)
, the subjective effects of ethanol can be detected by social drinkers experiencing BECs of 30 mg/dl and the function of ion channels and receptors can be inhibited by concentrations of ethanol in the range of 10 – 20 mM. These findings suggest that WSR mice consumed a pharmacologically active dose of ethanol and that they did find ethanol to be reinforcing when responding on the RR schedule. Additionally, comparison of the results on the FR4 versus RR8 schedule would suggest that there are line differences in the manner by which WSP and WSR self-regulate their 10E intake.
The present studies also utilized the reinstatement procedure, where non-contingent exposure to drug, non-drug stimuli or stress after extinction can cause an animal to resume a previous drug-reinforced behavior, to determine whether there were line differences in this model of drug craving (see reviews by Lê & Shaham, 2002
; Shaham et al., 2003
; Stewart, 2003
; Epstein et al., 2006
). When responding under non-reinforced conditions, both lines achieved the extinction criterion of ≤ 30% of baseline responding prior to the reinstatement tests (even though overall responding was higher in WSR than WSP mice). Importantly, both selected lines exhibited significant levels of reinstatement following either the presentation of the light cue (WSP only) or the combination of light cue and oral ethanol priming (WSP and WSR) when compared to their respective baselines (). These findings are consistent with recent work, which demonstrated that conditioned stimuli (CS) and contexts could reinstate ethanol seeking behavior in C57BL/6 mice (Tsiang & Janak, 2006
; Finn et al., 2008
In earlier work with rats (Bäckström & Hyytiä, 2004
), an oral ethanol prime paired with a CS (i.e., discriminating odor plus light cue), robustly reinstated ethanol-seeking behavior to levels greater than that seen with the CS alone (approximately 60% versus 40% of pre-extinction baseline levels of responding, respectively). We chose to use an oral ethanol prime, as we reasoned that it would be a more potent stimulus (i.e., it incorporated smell, taste and pharmacological onset just as when the mice were self-administering during reinforced sessions) than an ethanol injection. However, the oral ethanol prime did not promote reinstatement in either WSP or WSR mice, and it only tended to increase non-reinforced responding when paired with the light CS in WSR mice. These results are not entirely surprising, given the difficulty in promoting ethanol seeking with an ethanol priming injection (e.g., Lê & Shaham, 2002
; Nie & Janak, 2003
; Finn et al., 2008
) and the potential aversive chemosensory properties of ethanol in WSP and WSR mice. Nonetheless, pairing the light CS with an oral ethanol prime produced a non-significant increase in non-reinforced responding on the active lever over that following light cue exposure only in WSR mice, an effect that was comparable in magnitude to that previously reported in rats (65% versus 43% of pre-extinction response levels; Bäckström & Hyytiä, 2004
). The absence of a similar effect in WSP mice may be explained by the near-maximal response of this selected line to the light CS alone (65% of pre-extinction responding). In general, the present results suggest that non-drug stimuli are highly effective at promoting ethanol-seeking in WSP and WSR mice, but that there may be line differences in the relative salience of the CS components.
Home cage drinking of a sweetened ethanol solution (5S/10E) did not differ in male WSP-1 and WSR-1 mice. This result is similar to the early report by Kosobud et al. (1988)
, where female WSP-1 and WSR-1 mice did not differ in their consumption of 10E at the end of a preference test, although the WSR-1 mice drank more at lower concentrations. It was anticipated that intake of a sweetened ethanol solution would be higher than for unsweetened ethanol, but a comparison of the present work with that of Kosobud et al. (1988)
suggests that this may not be the case (for both studies: ethanol dose ~ 3.5 g/kg; ethanol preference ratio ≤ 0.2). A direct, contemporaneous comparison of intakes of sweetened versus unsweetened ethanol, each versus water, would be needed to confirm the effect of sweetening. However, across 22 inbred mouse strains, there was no strain for which addition of sweetener did not significantly increase ethanol intake (Yoneyama et al, 2008
). The lines did, however, differ in their intake of, and preference for, the 5S solution. Specifically, sucrose intake and preference was significantly higher in WSR versus WSP mice. Thus, the lack of line difference in intake of the sweetened ethanol solutions suggests that chemosensory factors such as the taste and smell of alcohol might be more important determinants of sweetened ethanol intake than an innate line difference in preference for sucrose.
A comparison of the operant and home cage drinking results revealed that the significant line difference in operant self-administration of most of the sweetened ethanol solutions did not correspond with the home cage drinking results where no overall line differences in consumption of 5S/10E were detected. The different results for intake of the 5S/10E solution in operant versus home-cage drinking procedures could be due to line differences in patterns of consumption that are only revealed under limited access conditions. This suggestion is consistent with the recent examination of 24 hr versus 2 hr limited access home cage drinking in knockout (KO) versus wild type (WT) mice with a null mutation in the 5α-reductase type 1 gene (Srd5a1
). Specifically, 24 hr 10E intake was significantly decreased in KO versus WT male mice, but 2 hr 10E intake was significantly increased in KO versus WT male mice (Nickel et al., 2006
We also measured the “alcohol deprivation effect” or ADE, which refers to the transient increase in ethanol consumption that occurs after abstinence, in WSP and WSR mice following over 30 days of consumption of sweetened ethanol. As described by Sanchis-Segura and Spanagel (2006)
, the ADE is considered a model of relapse, since the procedure allows the animal to self-administer alcohol after a period of protracted abstinence. However, it is unclear whether the ethanol itself is acting as a cue (i.e., taste and smell), as a priming stimulus, or both with regard to facilitating the transient increase in ethanol intake following deprivation. The present results indicate that neither WSP-1 nor WSR-1 mice developed an ADE, when the sweetened alcohol solution was presented after a 2 week period of abstinence. Ethanol intake was decreased in both lines, but the suppression was more persistent in WSR (4 days) than in WSP (2 days) mice. This result was not entirely surprising, since the increase in ethanol consumption is typically transient and requires repeated deprivations to extend beyond a single day of increased drinking (e.g., Rodd-Hendricks et al., 2000
; Bell et al., 2004
; discussed in more detail in Sanchis-Segura & Spanagel, 2006
). Another consideration is that the emergence of an ADE appears to be more robust and consistent in rat models, but it appears to be more sensitive to procedural variables and inconsistent in mouse genotypes. Specifically, a single 2-week deprivation (similar to that used in the present study) decreased daily ethanol intake in C57BL/6J mice (Melendez et al., 2006
), consistent with the present findings. A 6-day deprivation period with a single alcohol re-exposure per week was required to generate an ADE in C57BL/6J mice, but the emergence of this effect (week 1 vs. week 6) was quite variable between studies (Melendez et al., 2006
). Additionally, a 4-day deprivation produced varying effects in C57 substrains (ADE in C57BL/6NCr1, no effect in C57BL/6J), with evidence that repeated deprivations produced decreases in post-deprivation ethanol intake (Khisti et al., 2006
). Future studies will need to examine hourly intake in an attempt to capture the transient rise in drinking following abstinence and also employ multiple deprivations and procedural variations to determine whether an ADE would emerge differentially in WSP and WSR mice.
Two caveats regarding the current studies should be mentioned. Although the WSP/WSR replicate lines were derived from the same genetically heterogeneous stock, each pair of lines (WSP-1/WSR-1 versus WSP-2/WSR-2) was developed from a different set of 9 original mating pairs. Thus, there are many alleles that were excluded from these relatively small starting populations at the outset (this is called the “founder effect”). In consequence, the genes selected for in WSP-1 mice, for example, may be somewhat different from those selected for in the WSP-2 mice, even though the selection trait was identical. Thus, the strongest evidence for or against a correlated response to selection would be to find that the trait differed across both replicate pairs of WSP versus WSR selected lines. The second replicate lines were unavailable for the current studies. Further studies in the second replicate pair of lines could strengthen, or alter, the results we report here (for discussion, see Crabbe et al.,1990
). The second point relates to comparisons between these findings and reports from Kosobud and colleagues (1988)
. Mice had been selected for 17 or 19 generations when the earlier data were collected, but selection continued for another several generations (until S25). Continued selection undoubtedly produced genetic changes in all 4 selected lines. Thereafter, mice were randomly mated for an additional 85 generations (within each selected lines), and additional genetic change occurred during those years. The changes in gene frequency since S25 are due to the necessarily small population sizes, and are termed genetic drift. Thus, it is not unexpected to find differences between contemporary and archival data. That said, we did not test here two-bottle preference for ethanol versus water with unlimited access, so strict comparisons between these data and Kosobud and colleagues (1988)
are not possible.
In summary, the present series of experiments indicate that selected line differences in ethanol self-administration behavior depended on the qualities of the ethanol solution offered and the reinforcement schedule imposed. The strongest evidence supporting the negative genetic relationship between ethanol intake and withdrawal severity was provided by operant self-administration under an RR schedule of reinforcement. When animals were able to self-regulate their 10E consumption during 30 min of continuous ethanol access, ethanol intake was significantly higher in WSR-1 versus WSP-1 mice. However, the overall pattern of behavioral phenotypes examined reveal the absence of a consistent line difference in responses that reflect the rewarding or aversive effects of ethanol in WSP-1 and WSR-1 mice. Thus, the genes underlying ethanol self-administration and reinstatement behaviors do not consistently overlap with those which govern withdrawal severity in the WSP-1 and WSR-1 selected lines.