Deciphering the relationship between vulnerability toethanol-induced behavioral sensitization and ethanolconsumption in outbred mice
Rémi Legastelois*, Béatrice Botia*, Fabien Coune, Jérôme Jeanblanc & Mickaël NaassilaUniversité de Picardie Jules Verne, UFR Pharmacie, INSERM Unit ERi 24 GRAP, France
ABSTRACT
Ethanol (EtOH)-induced behavioral sensitization (EIBS) is proposed to play a role in early and recurring steps of alcoholdependence, but its impact on alcohol abuse is not clear. EIBS development is dependent upon animal species, strainand also individual factors. We proposed here to decipher the co-expression of EIBS and EtOH intake in individualanimals among outbred Swiss mice, which exhibit heterogeneity that parallels what may occur in humans. To do so,mice were exposed to a two-bottle choice with free access to water or 10% EtOH for 6 days just before and immediatelyafter chronic intraperitoneal 2.5 g/kg ethanol injections once a day for 10 consecutive days. Based on theirsensitization scores, mice were split into resistant and sensitized animals. First, we showed that individual susceptibilityto EIBS is inversely correlated with voluntary EtOH consumption. Exposure to repeated EtOH during EIBS developmentincreased subsequent EtOH intake among the entire population. Very interestingly, subsequent analyses suggested thatthe less the mice are sensitized the more they increase their EtOH intake; however, resistant mice were sensitive to EtOHadulteration with quinine, whereas sensitized ones maintained their EtOH intake levels, therefore exhibiting acompulsive-like drinking pattern. In addition, we showed that resistant mice do not exhibit a weaker sensitivity to theaversive properties of EtOH that may contribute to their higher level of EtOH intake compared to sensitized mice. Thisstudy confirms and extends previous data showing a deep relationship between propensity for EtOH consumption andsusceptibility to EIBS in Swiss mice.
Correspondence to: Mickaël Naassila, INSERM ERI 24, GRAP, Université de Picardie Jules Verne, UFR de Pharmacie, 1 rue des Louvels, Amiens, France.E-mail: [email protected]
INTRODUCTION
Drug-induced behavioral sensitization is defined inrodents as an enhancement of locomotor activity follow-ing repeated drug administrations. About 20 years ago,(Robinson & Berridge 1993) put forward the hypothesisthat behavioral sensitization may underlie some com-ponents of addiction. Indeed, the incentive saliencesensitization theory states that repeated exposure todrugs of abuse causes hypersensitivity to drugs and drug-associated stimuli of the neuronal circuits mediatingexcessive incentive salience characterized by a pathologi-cal ‘wanting’ to take drugs. This incentive salienceprocess leads to compulsive patterns of drug-seeking
behavior (Robinson & Berridge 1993, 2001). Hence, thisparadigm has been largely used worldwide to study long-term neuroadaptations associated with addiction todrugs of abuse in rodents.
Considerable evidence points to individual variabilityin the response to ethanol (EtOH)-induced behavioralsensitization (EIBS) between species, strains and individu-als. For example, some mice exhibit substantial signs ofsensitization (sensitized mice) and others, characterizedas non-sensitized or resistant, display no or minimalenhancement of locomotor activity following chronicEtOH injections (Quadros et al. 2002a,b; Abrahao,Quadros & Souza-Formigoni 2009, 2011; Botia et al.2012). Regarding these differential responses, numerous
studies have examined the relationship between vulner-ability to EIBS and voluntary EtOH consumption. Forinstance, it has been repeatedly shown that mice withhigh EtOH preference (C57BL/6J) exhibit no or minimalEIBS, but DBA/2J mice, a strain with extreme EtOH avoid-ance, display high levels of sensitization (Belknap, Crabbe& Young 1993; Phillips, Dickinson & Burkhart-Kasch1994; Phillips et al. 1995; Yoneyama et al. 2008; Melon& Boehm 2011). A negative correlation also has beenobserved between EIBS levels and 3% EtOH (but not 10%EtOH) consumption in mice derived from DBA/2J andC57BL/6J genitors (Phillips et al. 1995), suggesting theexistence of a genetic determinism in vulnerability toEIBS. Most of the studies focusing on the relationshipbetween EtOH consumption and EIBS have used inbredmice, and particularly the DBA/2J and C57BL/6J strains;however, working with outbred Swiss mice is consider-ably helpful in understanding inter-individual variabilitybecause of their genetic and phenotypic differences thatmodel a heterogeneous population.
To date, the relationship between EIBS and volun-tary EtOH intake remains unclear. In general, datasupport that pre-exposure to chronic EtOH injectionsincreases subsequent EtOH consumption, but the resultsare still unclear. For example, EtOH consumption wasmarginally increased in C57BL/6J, but not in DBA/2J,after sensitization development (Lessov et al. 2001).Moreover, EtOH consumption can be enhanced by EtOHpre-exposure in both C57BL/6J and DBA/2J mice evenwhen no sensitization is detected (Camarini & Hodge2004).
In light of these data and considering the heterogene-ity in EtOH responsiveness, it becomes of intense interestto examine more precisely the correlation between vul-nerability to EIBS and propensity to drink EtOH in indi-vidual animals within outbred mice. Interestingly, theoutbred Swiss mice shows sensitization to repeated EtOHinjections similarly to the DBA/2J mice (Correa et al.2004; Didone et al. 2008; Faria et al. 2008) and exhibitrelatively high levels of EtOH intake (Correia et al. 2009;Ribeiro et al. 2012). Here, we use Swiss mice (1) to inves-tigate if the level of EtOH intake is correlated to the vul-nerability to develop EIBS and if the latter is associatedwith a change in EtOH intake, (2) to assess a putativecompulsive-like EtOH consumption using an adulterationof the EtOH solution with quinine (Hopf et al. 2010) and(3) to study variations in the sensitivity to the aversivepharmacological properties of EtOH in a CTA paradigm.This study aims at providing important insights onthe consequences of inter-individual variability of EtOH-sensitization development on subsequent EtOH con-sumption, compulsive EtOH intake and alterations in sen-sitivity to EtOH’s aversive effects commonly observed inalcohol-dependent patients.
MATERIALS AND METHODS
Subjects
Adult female Swiss mice were purchased from Janvier (LeGenest Saint Isle, France) and kept in a temperature-(21 ± 0.5°C) and humidity-controlled (55 ± 10%) envi-ronment under an established photoperiod (07.00–19.00 hours). In the present study, we used femalesbecause numerous studies have found that female ratsdevelop a more robust sensitization to psychostimulantsthan males (Robinson 1984; Hu & Becker 2003;McDougall et al. 2007). Taking into account thatsensitization to various drugs of abuse shares similarneurocircuits, it is likely that female rodents exhibit anenhanced sensitization to EtOH compared to males(Itzhak & Martin 1999; Lessov & Phillips 2003). Besides,we have already published data on EIBS using femalemice and showed that estrous levels were similar betweencontrol and sensitized mice (Legastelois, Botia & Naassila2013). Moreover, EIBS in female Swiss mice has been wellcharacterized in terms of EtOH dose and patterns of EtOHinjections (Didone et al. 2008). Throughout voluntaryEtOH consumption experiments, mice were individuallyhoused with free access to food. During the EIBS proce-dure, mice were housed in groups of 10 in clear plasticcages (44 cm × 44 cm × 34.5 cm) with softwood fibersbedding and with free access to food and tap water. Thenumber of animals was kept to a minimum, and all effortswere made to avoid making the animals suffer. Experi-ments were carried out in strict accordance with both theguidelines for Care and Use of Laboratory Animals(National Institutes of Health) and the European Com-munity regulations for animal use in research (CEE no.86/609) and the procedures also were approved by thelocal ethics committee (Comité Régional d’Ethique enMatière d’Expérimentation Animale de Picardie).
Drugs
EtOH (96%, v/v) was obtained from Prolabo(Fontenay-sous-Bois, France). For the EIBS experiment,EtOH was diluted to 20% (v/v) in 0.9% saline solution.All injections were made intraperitoneally (i.p.) involumes of 1.25 ml per 100 g body weight. Quininehydrochloride and sucrose were obtained from Sigma-Aldrich (Saint-Louis, MO, USA) and diluted in tap wateror 20% EtOH solution.
Locomotor activity
Locomotor activity was assessed in the LE 8811 IR motoractivity monitor (Bioseb, Vitrolles, France). Mice wereplaced in a 40 × 40 × 30 cm open field with opaqueacrylic walls, transected with infrared photocell beams2 cm above the floor at 16 sites along each side. The test
room was shielded from external noise and equipped witha ventilation system, which served to create the samewhite noise as all the other rooms of the animal care unit.Locomotor chambers were illuminated with indirectwhite light (20 lux). Horizontal locomotion was meas-ured from photocell beam interruptions using ActiTracksoftware (Bioseb).
Experimental design
We performed two separate experiments. For experiment1, we used 70 mice in a two-bottle choice paradigm atdifferent EtOH concentrations (3, 6, 10 and 20%;n = 16–18 per concentration). For experiment 2, we used90 mice divided into four groups: water/Rep-S (n = 10);water/Rep-E (n = 30); EtOH/Rep-S (n = 10) and EtOH/Rep-E (n = 40) (Rep-S: repeated saline; Rep-E: repeatedEtOH). In the experiment 2 (summarized in Table 1), wefirst assessed the individual levels of fluid intake (water,10% EtOH). The mice were then submitted to repeatedsaline or 2.5 g/kg EtOH treatment for 10 days and asensitization score was attributed to each mouse (fordetails see the EIBS procedure section below). Individuallevels of fluid intake (water, 10% EtOH) were againassessed after sensitization procedure. We examined thecorrelations between both water and EtOH consumptionpreceding EIBS and sensitization score, and more inter-estingly, between sensitization score and the changes inwater or EtOH consumption: (consumption after EIBS)—(consumption before EIBS). Since EtOH is known for itssweet and bitter tastes, we also examined sensitivity tosucrose and quinine. Finally, we investigated sensitivity toquinine-adulterated EtOH, which may reflect maladap-tive or compulsive-like intake.
EIBS procedure
The behavioral sensitization procedure was similar to thatwe previously described (Simon O’Brien et al. 2011; Botiaet al. 2012). On the first two days of the sensitization pro-cedure (habituation, day 7–day 8), all mice were injectedwith saline (12.5 ml/kg i.p.) and immediately placed inthe center of an open field. Locomotor activity wasrecorded for the next 5 minutes, as has been done inseveral other studies (Didone et al. 2008; Faria et al. 2008;Botia et al. 2012; Carrara-Nascimento, Olive & Camarini2012). Mice were then divided into several groups thatwere equated in terms of horizontal locomotion at day 8.The next day, the sensitization procedure started. Duringthe subsequent 10 days (day 9–day 18), mice received onedaily, i.p. injection of saline (water/Rep-S and EtOH/Rep-Sgroups; n = 10 per group) or 2.5 g/kg EtOH (water/Rep-Eand EtOH/Rep-E groups; n = 30 and 40, respectively)(Table 1). At day 18, the sensitization score was calculatedas (locomotor activity at day 18)—(locomotor activity at Ta
day 9). EtOH-treated mice were considered sensitized iftheir increase in locomotor activity exceeded the coeffi-cient of variance of the water/Rep-S group, as previouslydescribed by Boudreau & Wolf (2005). At day 54, micewere challenged with an i.p. injection of saline or 2.5 g/kgEtOH as described in Table 1.
Drinking experiments
In experiment 1, mice were exposed to a continuoustwo-bottle free choice paradigm (n = 70) with free accessto tap water and a 3, 6, 10 or 20% EtOH solution(n = 16–18 per group). In experiment 2 (summarized inTable 1), mice from the water/Rep-S and water/Rep-Egroups had access only to tap water during drinkingexperiments. Mice from the EtOH/Rep-S and EtOH/Rep-Egroups were exposed to a continuous two-bottle choiceparadigm with free access to tap water and 10 or 20%EtOH solution. Quinine adulteration was tested using0.15 g/l quinine in tap water or 20% EtOH. Sucroseintake was tested using concentrations of 0.03 and 0.1%in tap water. In both experiments, fluid intake (g/kg bodyweight/24 hour), preference (EtOH intake/total fluidintake) and body weight were measured. The placementof each solution was alternated after each volumetricmeasurement to control for side preference. Fluid con-sumption volumes were recorded in the morning andindividual levels of fluid intake or preference wereobtained by calculating the mean values from 6 consecu-tive days. During all drinking experiments, 6–8 ‘dummy’bottles were used to estimate liquid spillage due to miceactivity and bottle manipulation.
Conditioned taste aversion (CTA)
Another cohort of mice was first exposed to an EIBSprocedure according to the procedure described above(resistant n = 16; sensitized n = 13). Then, mice wereindividually housed for 3 days for a CTA experimentaccording to a procedure previously described (Houchiet al. 2008). Mice were submitted to daily, 2-hourrestricted water access (water between 1300 and 1500hour) for 7 days. The next day, mice were exposed to a0.2 M NaCl solution for 1 hour (1300 to 1400 hour).Two days after and every other day for 7 days, all micereceived a 1-hour access period to NaCl immediately fol-lowed by an i.p. saline injection (pre-conditioning days1–4). Two days after and every other day for 7 days, micereceived a 1-hour access period to NaCl solution immedi-ately followed by a 2.5 g/kg EtOH (resistant and sensitizedgroups) i.p. injection (conditioning days 1–4). The salineintake baseline was calculated as the mean of salineintake on pre-conditioning day 4 and conditioningday 1. Noteworthy, on conditioning day 1, the EtOH injec-tion occurred after the drinking session and therefore
had no outcome on saline intake. Throughout the CTAprocedure, mice were offered restricted access to tapwater for 2 hours on intervening days. To prevent possibledehydration, all mice had access to tap water for 30minutes, 5 hours after the NaCl-solution access period.
Blood EtOH concentration
On the challenge day (day 54), mice were euthanized bydecapitation 5 or 30 minutes after a 2.5 g/kg EtOH injec-tion and blood was collected. Blood EtOH concentrationswere measured in plasma with the AM1 Alcohol Ana-lyser (Analox Instruments, IMLAB, Lille, France). Theprecision of this assay is 1–2%, sensitivity is 0.1 mg/100 ml, and the curve is linear up to 400 mg/100 ml.
Statistical analysis
All data analyses were conducted using SigmaStat2.0software. Data were analyzed with one- or two-wayanalyses of variance (ANOVA) and with repeated-measures (RM) ANOVA when appropriate. ANOVAs werefollowed by Tukey’s post hoc tests. Linear regressionanalyses were performed to investigate correlations.
RESULTS
Experiment 1
Dose—dependent increase in EtOH intake
Different groups of mice (n = 16–18) were exposed to atwo-bottle free choice paradigm with EtOH (3, 6, 10 or20%) and tap water (Supporting Information Fig. S1). Aone-way ANOVA showed an effect of concentration forEtOH intake (F3,66 = 24.650; P < 0.001) and preference(F3,66 = 12.957; P < 0.001). Post hoc tests revealed a dose-dependent increase in EtOH intake and preference(P < 0.001 versus 3% and 6%; Supporting InformationFig. S1a,b). Because EtOH intake levels were very low atthe 3 and 6% concentrations (under 1 g/kg/24 hour andabout 10% preference), we used 10 and 20% EtOH toanalyze correlations between vulnerability to EIBS andEtOH drinking in experiment 2.
Experiment 2
EtOH pre—exposure does not changesensitization development
Figure 1a shows changes in locomotor activity of micerepeatedly treated with saline or 2.5 g/kg EtOH afterwater or 10% EtOH consumption. A two-way RM-ANOVA on the induction phase (day 9–day 18) revealedan effect of group (F3,896 = 14.991; P < 0.001), an effectof day (F9,769 = 2.590; P < 0.01) and a significant inter-action (F27,769 = 2.071; P < 0.001). Post hoc tests revealed
an increase in locomotor activity for the EtOH/Rep-E andwater/Rep-E groups at day 18 (P < 0.001 versus D9).No differences were detected between EtOH/Rep-E andwater/Rep-E groups or between EtOH/Rep-S and water/Rep-S groups throughout the induction phase ofsensitization (D9–D18) (Fig. 1a).
Individual sensitization levels are stable throughoutthe experiments
At day 54, mice received a single i.p. injection of saline(water/Rep-S group) or 2.5 g/kg EtOH (water/Rep-E,EtOH/Rep-S and EtOH/Rep-E groups) (Table 1). A two-way RM-ANOVA focused on day 9, day 18 and day 54showed an effect of group (F3,85 = 17.075; P < 0.001), aneffect of day (F2,162 = 4.340; P < 0.05) and a significantinteraction (F6,162 = 3.362; P < 0.01). Post hoc testsshowed that the global level of sensitization was main-tained until day 54 (i.e. 5 weeks after the end of EIBSinduction), because the locomotor activity of micerepeatedly treated with EtOH (i.e. water/Rep-E and EtOH/Rep-E groups) was not different between day 18 and day54 (P > 0.05; Fig. 1a). Moreover, at day 54, the locomo-tor activity of mice repeatedly treated with EtOH andchallenged with 2.5 g/kg EtOH was significantly higherthan the locomotor activity of mice repeatedly treatedwith saline and challenged with 2.5 g/kg EtOH (EtOH/Rep-S group; P < 0.001; Fig. 1a). Importantly, linearregression analysis of locomotor activity of groupsrepeatedly treated with EtOH revealed a positive correla-tion between distance travelled at day 18 and day 54(R = 0.73; P < 0.001; Supporting Information Fig. S2).These latter results show that the individual levels ofsensitization were likely to be stable until day 54, andthus, across the diverse drinking protocols.
Swiss mice display inter—individual differences in EIBS
At the end of EIBS induction, mice from the EtOH/Rep-Egroup were retrospectively split into resistant and sensi-tized groups based on their sensitization score (seemethods section). A two-way RM-ANOVA on day 9, day18 and day 54 showed an effect of group (F1,36 = 12.850;P < 0.001), an effect of day (F2,70 = 18.630; P < 0.001)and a significant interaction (F2,70 = 34.307; P < 0.001).Post hoc tests failed to detect a difference in the acute EtOHresponse (day 9) between resistant and sensitized animals(P > 0.05). It revealed a significant increase in locomotoractivity between day 9 and day 18 for the sensitized(P < 0.001) but not for the resistant groups (P > 0.05).At day 54, the challenge day, the locomotor activity ofsensitized mice was still higher than locomotor activity atday 9 (P < 0.001), although it was reduced to someextent when compared at day 18 (P < 0.05). Sensitizedmice exhibited a higher locomotor activity at day 18
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Figure 1 Pre-exposure to 10% EtOH consumption does notchange EtOH-induced behavioral sensitization (EIBS) development.(a) On the two first days of the experiment (habituation days, day7–day 8), all mice were i.p. injected with saline solution.Then, fromday 9 to day 18, mice received 10 consecutive once-daily i.p. injec-tions of saline (EtOH/Rep-S and water/Rep-S groups, n = 10 for eachgroup) or 2.5 g/kg EtOH (EtOH/Rep-E and water/Rep-E groups,n = 40 and 30, respectively) immediately followed by a 5 minute-longlocomotor activity recording session. At day 54, challenge day, micereceived a single i.p. injection of saline (EtOH/Rep-S group) or2.5 g/kg EtOH (water/Rep-S, EtOH/Rep-E and water/Rep-E groups).(b) Mice from the EtOH/Rep-E group were split into resistant(n = 22) and sensitized (n = 18) groups based on their sensitizationscore. Data represent the mean value (± SEM) of distance traveled/5minutes. ***P < 0.001 indicates a difference between the distancetraveled in EtOH/Rep-E or water/Rep-E groups and water/Rep-Sgroup at D54; †††P < 0.001 indicates a difference of distance traveledbetween day 9 and day 18; #P < 0.05, ###P < 0.001 indicate a differ-ence between the distance travelled in resistant and sensitized miceat the same day
(P < 0.001) and day 54 (P < 0.05) when compared withthat of resistant mice (Fig. 1b).
Individual differences in EtOH metabolism are not involvedin vulnerability to EIBS
Blood ethanol concentrations (BECs) were analyzed onday 54, 30 minutes after a 2.5 g/kg EtOH challenge. Aone-way ANOVA failed to detect an effect of groupbetween water/Rep-E (n = 30), EtOH/Rep-S (n = 10) andEtOH/Rep-E (n = 37) groups (F2,74 = 0.198; P > 0.05;Supporting Information Fig. S3a). Moreover, statisticalanalysis failed to detect a correlation between BECs andsensitization scores in the EtOH/Rep-E group (R = 0.14;P > 0.05; Supporting Information Fig. S3b). To evaluatewhether the behavioral response between resistant andsensitized mice could potentially be derived from variabil-ity in the EtOH absorption, BECs were assessed 5 minutesafter a 2.5 g/kg ethanol injection on the challenge day.The statistical analysis failed to detect any difference inBECs (P = 0.911) between resistant (330 ± 8 md/dl) andsensitized (321 ± 7 md/dl) mice (Supporting InformationFig. S3c).
Vulnerability to EIBS is negatively correlatedwith EtOH intake
Mice from the EtOH/Rep-E group were exposed to a two-bottle free choice paradigm of 10% EtOH and waterbefore (day 1–day 6) and after (day 19–day 24) the EIBSprocedure. First, linear regression analyses revealednegative correlations between the sensitization score and10% EtOH intake (R = 0.41; P < 0.01; Fig. 2a) andbetween the sensitization score and 10% EtOH preferencebefore EIBS (day 1–day 6; R = 0.40; P < 0.01; Fig. 2d),suggesting that mice drinking more EtOH are less sensi-tive to EIBS. These correlations were maintained after thesensitization procedure (day 19–day 24) for both 10%EtOH intake (R = 0.51, P < 0.001; Fig. 2b) and prefer-ence (R = 0.50; P < 0.01; Fig. 2e). Daily ethanol intakevalues both before (10% ethanol for 6 days) and after(10% ethanol for 6 days and 20% ethanol for 6 days) EIBSare shown on Supporting Information Fig. S4).
Importantly, our results showed an increase in 10%EtOH intake (+90%; Supporting Information Fig. S5a)and preference (+63%; Supporting Information Fig. S5b)between day 1–day 6 and day 19–day 24 in the EtOH/Rep-E group. The difference in 10% ethanol intakebetween experiment 1 and experiment 2 (SupportingInformation Fig. S1 and S5, respectively) may come froma shorter housing habituation period in experiment 1that could have induce stress-mediated EtOH drinking(Sillaber et al. 2002). To determine the consequences ofEIBS development on EtOH consumption at the indivi-dual level, we calculated changes in drinking as follows:
(consumption after EIBS, day 19–24)—(consumptionbefore EIBS, day 1–6]. Thus, we assessed changes in10% EtOH intake, preference and total fluid intake.Linear regression analyses showed negative correlationsbetween the sensitization score and changes in 10% EtOHintake (R = 0.35; P < 0.05; Fig. 2c) and between thesensitization score and changes in 10% EtOH preference(R = 0.37; P < 0.05; Fig. 2f) but not between thesensitization score and changes in total fluid intake(R = 0.20, P > 0.05; data not shown). This effect wasspecific to EtOH because the sensitization score was notcorrelated with the change in water intake in the water/Rep-E group (R = 0.10, P > 0.05; Supporting Informa-tion Fig. S6). Moreover, repeated saline injections hadno effect on EtOH consumption as we detected nochanges in EtOH intake in the EtOH/Rep-S group (Sup-porting Information Fig. S7a) and preference afterchronic saline treatment (Supporting InformationFig. S7b) (P > 0.05).
A two-way RM-ANOVA on 10% EtOH intake inresistant and sensitized groups before and after EIBS(Fig. 2g) showed an effect of group (F1,38 = 17.008;P < 0.001), treatment (F1,36 = 12.06127; P < 0.001),but no interaction (F1,36 = 3.24; P > 0.05). A two-wayRM-ANOVA on EtOH preference in resistant and sen-sitized groups (Fig. 2h) showed an effect of group(F1,38 = 11.684; P < 0.01), treatment (F1,36 = 9.669;P < 0.01), but no interaction (F1,36 = 2.842; P > 0.05).Our results suggest a higher increase in EtOH intake andpreference in the resistant group (+98% and +69%,respectively) compared to the sensitized group (+69% and+45%, respectively) after EIBS (Fig. 2g, h). Based on thecorrelations, it is likely that the increase in 10% EtOHintake is displayed by the resistant subpopulation of mice,but not the sensitized group. Therefore, we performedfocused correlations within the resistant and sensitizedsubpopulations of mice. We found that the sensitizationscore was correlated to 10% EtOH intake (R = 0.40;P < 0.05; Supporting Information Fig. S8b) and to thechange in 10% EtOH intake (R = 0.41; P < 0.01; Sup-porting Information Fig. S8c) in the resistant but not sen-sitized group of mice, suggesting that only resistant miceexhibit an increase in EtOH consumption after an EIBSprocedure.
Resistant mice do not display compulsive—like EtOH intake
First, we confirmed the aversive properties of quinine inboth resistant and sensitized mice by exposing mice fromthe EtOH/Rep-E group to a two-bottle free choice proce-dure, with one bottle containing 0.15 g/l quinine dis-solved in tap water and the other one containing tapwater for 6 days (day 31–day 36) (Table 1). This quinineconcentration was chosen because it considerablyreduces intake without totally inhibiting the response.
We thus showed here an aversive-like effect of quininefor most mice (mean preference = 19.3 ± 2% for thequinine + water solution) in accordance with a recentstudy showing an aversive-like effect of quinine in
C57BL/6J mice at concentrations of >250 μM (Lesscher,van Kerkhof & Vanderschuren 2010). No correlationwas detected between quinine preference and thesensitization score (Supporting Information Fig. S9a). In
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Figure 2 Vulnerability to EtOH-induced behavioral sensitization (EIBS) is negatively correlated with 10% EtOH consumption. Mice from theEtOH/Rep-E (n = 40) group were submitted to a two-bottle choice paradigm (10% EtOH and tap water) before (day 1–day 6) and after(day 19–day 24) EIBS. Changes in EtOH drinking (EtOH intake and preference) were calculated as (10% EtOH drinking after EIBS)—(10%EtOH drinking before EIBS). Correlations were investigated between the sensitization score and changes in (1) 10% EtOH intake (a) andpreference (d) before EIBS, (2) 10% EtOH intake (b) and preference (e) after EIBS and (3) 10% EtOH intake (c) and preference (f). Mice weresplit into resistant and sensitized groups based on their sensitization score (n = 18–22). Each histogram represents the mean value (± SEM) ofEtOH intake (g) and preference (h) before and after EIBS. ***P < 0.001 indicates a difference of drinking between before and after EIBS;#P < 0.05, ###P < 0.001 indicate a difference between resistant and sensitized groups at the same time point
addition, a one-way ANOVA failed to detect a differencefor quinine solution preference between resistant andsensitized groups (P > 0.05; Supporting InformationFig. S9b).
To better characterize EtOH drinking patterns, wesought to evaluate compulsive EtOH drinking by adul-teration of the EtOH solution with quinine, as previouslydescribed (Sanchis-Segura & Spanagel 2006; Hopf et al.2010). Mice from the EtOH/Rep-E group were exposed toa two-bottle free choice paradigm of 20% EtOH and waterin the absence (day 25–day 30) or presence (day 37–day42) of 0.15 g/l quinine (Table 1). Here, we used the EtOHconcentration that induces the highest EtOH intakeand preference (i.e. 20% EtOH; Supporting InformationFig. S1) to detect more accurately inter-individual differ-ences in EtOH-intake decreases due to quinine adultera-tion. Linear regression analyses of data from day 25 today 30 revealed negative correlations between thesensitization score and 20% EtOH intake (R = 0.59;P < 0.001; Fig. 3a) and between the sensitization scoreand 20% EtOH preference (R = 0.53; P < 0.001; Fig. 3d),in line with results obtained with 10% EtOH consump-tion before and after EIBS (Fig. 2a, b, d and e). Analyses ofdata from day 37 to day 42 also revealed negative corre-lations between the sensitization score and quinine +20% EtOH intake (R = 0.44; P < 0.01; Fig. 3b) andbetween the sensitization score and quinine + 20% EtOHpreference (R = 0.36; P < 0.05; Fig. 3e). We then calcu-lated changes in drinking as follows: (consumption withquinine, day 37–42)—(consumption without quinine,day 25–30). We thus obtained changes in 20% EtOHintake, preference and total fluid intake. Statisticalanalysis showed a positive correlation between thesensitization score and changes in quinine + 20%EtOH intake (R = 0.37, P < 0.05; Fig. 3c), preference(R = 0.28, P < 0.05; Fig. 3f), but not total fluid intake(R = 0.05, P > 0.05; data not shown). Moreover, focusedcorrelations within the resistant and sensitized sub-populations of mice revealed that the sensitization scorewas correlated to the change in quinine + 20% EtOHintake (R = 0.49; P < 0.05; Supporting InformationFig. S10c), specifically in the resistant group of mice.
A two-way RM-ANOVA on 20% EtOH intakein resistant and sensitized groups in the absenceand presence of quinine showed an effect of group(F1,38 = 15.405; P < 0.001), treatment (F1,36 = 26.127;P < 0.001) and an interaction (F1,36 = 9.59; P < 0.01).A two-way RM-ANOVA on EtOH preference in resistantand sensitized groups showed an effect of group (F1,38 =12.544; P < 0.001), treatment (F1,36 = 27.587; P <0.001) and an interaction (F1,36 = 6.96; P < 0.05). Posthoc tests revealed a difference in 20% EtOH intake(+125%; Fig. 3g) and preference (+90%; Fig. 3h) inresistant compared with sensitized mice (P < 0.001).
They also showed a decrease in EtOH intake (–44%;Fig. 3g) and preference (–46%; Fig. 3h) in the resistant,but not the sensitized group, after quinine addition(P < 0.001). These results suggest that sensitized miceare less sensitive to EtOH adulteration with quininecompared to resistant mice.
Lack of relationship between vulnerability to EIBS andsucrose intake
At day 43 and day 45, mice were given a two-bottle freechoice between one bottle of 0.3 or 1% sucrose dissolvedin tap water and one bottle of tap water. Data analysesfailed to demonstrate a correlation between the sensiti-zation score and 0.3% sucrose solution (R = 0.10;P > 0.05; Fig. 4a) or 1% sucrose solution (R = 0.06;P > 0.05; Fig. 4b) preference. In addition, a two-wayRM-ANOVA failed to detect a difference in sucrose prefer-ence between resistant and sensitized mice (P > 0.05;Fig. 4c).
Experiment 3
Lack of difference in ethanol—induced conditionedtaste aversion
Sensitized and resistant mice were exposed to a CTAprocedure (Fig. 5). A two-way RM-ANOVA showed aneffect of day (F3,116 = 23.811; P < 0.001), no effects ofgroup (F1,29 = 2.455; P > 0.05), and no interaction(F6,116 = 1.263; P > 0.05). These results reveal that thereis no statistical difference between the two groups in theirsensitivity to the aversive properties of ethanol.
DISCUSSION
We proposed here to analyze the co-expression of EIBSand EtOH intake in individual animals among an outbredpopulation of Swiss mice that exhibit levels of inter-individual variation similar to those that may occur inhuman populations (Rice & O’Brien 1980). The mainfinding of the current study is the demonstration thatvoluntary EtOH intake after repeated EtOH treatment isinversely correlated to the development of EIBS; however,EtOH adulteration with quinine altered drinking morestrongly in EIBS resistant mice, thus indicating that theincrease in EtOH drinking does not seem to be associatedwith a compulsive motivation for EtOH.
Experiment 1 showed that Swiss mice exhibit adose-dependent increase in EtOH consumption, as hasbeen observed in other strains (Thiele et al. 1998; Wandet al. 2001; Naassila, Ledent & Daoust 2002). No differ-ences in EIBS levels were detected between water/Rep-Eand EtOH/Rep-E groups and sensitization levels werestable throughout the experiments. Whereas one study
Figure 3 Resistant mice do not display compulsive-like EtOH intake. Mice from the EtOH/Rep-E group were submitted to a two-bottlechoice (20% EtOH and tap water) between day 25 and day 30 (n = 40). They were then submitted to another two-bottle choiceparadigm with one bottle containing tap water and the other one 0.15 g/l quinine in 20% EtOH between day 37 and day 42. Changes inEtOH drinking (EtOH intake and preference) were calculated as (quinine + 20% EtOH drinking)—(20% EtOH drinking). Correlations wereinvestigated between the sensitization score and changes in (1) 20% EtOH intake (a) and preference (d) in absence of quinine, (2) 20% EtOHintake (b) and preference (e) in presence of 0.15 g/l quinine and (3) 20% EtOH intake (c) and preference (f ). Mice were split into resistantand sensitized groups based on their sensitization score (n = 18–22). Each histogram represents the mean value (± SEM) of EtOH intake(g) and preference (h) in absence and presence of 0.15 g/l quinine. ***P < 0.001 indicates a decrease of EtOH drinking after quinineaddition; ###P < 0.001 indicates a difference between resistant and sensitized groups at the same time point. EIBS: EtOH-induced behavioralsensitization
demonstrated that EtOH drinking-in-the-dark may facili-tate the development of subsequent EIBS (Tarragon et al.2012), our results showed that EIBS is not altered by aperiod of previous access to EtOH. This divergence is likely
to be due to the differences in the durations of EtOHaccess each day (4 hours in the drinking-in-the-dark pro-cedure versus 24 hours in the continuous access proce-dure) and possibly to the quantity of EtOH consumed andthe peak of BECs achieved in each paradigm.
As previously described in preclinical models(Quadros et al. 2002a,b; Abrahao et al. 2009, 2012;Botia et al. 2012), we confirmed inter-individual differ-ences in response to EtOH administrations among theEtOH/Rep-E group. In addition, the present data showedan absence of relationship between acute EtOH hyper-locomotor response and vulnerability to EIBS as alreadysuggested in the literature (Masur & dos Santos 1988;Phillips et al. 1995). We noticed a reduction to someextent in the sensitization levels of the sensitized groupsat day 54 (compared to day 18), which could be attribut-able to the absence of EtOH treatment between the induc-tion and expression phases of EIBS and to the isolation ofmice during the drinking experience from day 18 to day54 (Araujo et al. 2005). We also provided evidence thatvariability in EIBS expression is not linked to alteration inEtOH absorption/metabolism. Therefore, addressing theheterogeneity in EtOH-responsiveness becomes of intenseinterest because it would provide information about vul-nerability to EtOH addiction.
Interestingly, we showed here that Swiss mice thatconsume more EtOH are less vulnerable to EIBS develop-ment. This observation has been made many times whencomparing different strains of mice (Belknap et al. 1993;Phillips et al. 1994, 1995; Yoneyama et al. 2008; Melon& Boehm 2011), but never within one specific strain. Thisinter-individual variability could be explained by anenhanced sensitivity to both positive (i.e. stimulant) andnegative (i.e. sedative) EtOH effects. We hypothesize that
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Figure 4 Lack of relationship between vulnerability to EIBS andsucrose intake. Mice from the EtOH/Rep-E group were submitted toa two-bottle free choice between 0.3% (day 43–day 44) or 1% (day45–day 46) sucrose solution and tap water (n = 40).Correlations wereinvestigated between the sensitization score and 0.3% (a) or 1% (b)sucrose solution preference. Mice were split into resistant and sensi-tized groups based on their sensitization score (n = 18–22). (c) Resultsfrom 0.3 and 1% sucrose preference are presented as mean ± SEM
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Figure 5 Lack of difference in ethanol-induced conditioned tasteaversion (CTA). Resistant and sensitized mice were submitted to aCTA procedure (n = 12–16 per group). Intake of saline solutionbefore (baseline) and after (conditioning days 2, 3 and 4) condition-ing in resistant and sensitized mice
increasing levels of response to EtOH stimulant effectsand to EIBS leads to a decrease in EtOH consumption,which could be a protective factor against excessive EtOHintake. Inversely, resistance to EIBS may represent a riskfactor in the vulnerability to consume EtOH.
Our results showed an increase in 10% EtOH intakeand preference in mice exposed to EtOH but not tosaline injections during EIBS procedure, suggesting thatchronic EtOH treatment increases subsequent EtOHintake. Many studies argue that pre-exposure to drugs ofabuse predisposes rodents to self-administer the same orother drugs of abuse (Horger, Shelton & Schenk 1990;Horger, Giles & Schenk 1992; Valadez & Schenk 1994;Mendrek, Blaha & Phillips 1998; Lorrain, Arnold &Vezina 2000; Camarini & Hodge 2004). Regarding EtOH,the results are still unclear. While induction of robustEIBS in DBA/2J mice does not alter EtOH consumption,C57BL/6J mice exhibit an increase in EtOH consumptionafter a modest locomotor sensitization (Lessov et al.2001). In addition, C57BL/6 mice show increased EtOH(but not sucrose) consumption after chronic pre-exposure to high EtOH levels in inhalation chambers(Becker & Lopez 2004). These studies support the hypoth-esis that, whether or not EIBS development occurs,chronic EtOH exposure is likely to enhance subsequentvoluntary EtOH intake. Therefore, to examine the poten-tial relationship of changes in EtOH drinking with mag-nitude of EIBS, we performed linear regression analyses,using data for both traits from each individual. We founda negative correlation between the sensitization score andthe changes in EtOH drinking, and more specifically, weobserved an increase in EtOH drinking in mice that areless prone to EIBS development. In accordance with pre-vious studies (Lessov et al. 2001; Camarini & Hodge2004), we suggest that the enhancement of voluntaryEtOH drinking is greater in mice that exhibit no or modestEIBS after chronic EtOH treatment. Interestingly, previ-ous reports demonstrated that specific neurobiologicalfactors act in an opposite manner with respect to EtOHconsumption and sensitization. Indeed, neuropeptideY-deficient mice exhibit increased EtOH consumption andfail to display EIBS (Thiele et al. 1998; Hayes et al. 2012).Conversely, dopamine transporter knockout mice developstronger EIBS than their wild-type counterparts whereastheir EtOH consumption is decreased (Savelieva et al.2002; Morice et al. 2010). Altogether, these findingssuggest that the difference in phenotypes (i.e. resistantand sensitized) could be due to variations in the expres-sion of some neurobiological substrates that are eitherpre-existing and/or developed after chronic EtOH treat-ment. Our results raise an intriguing question as somegenetic studies have found a positive correlation betweenEIBS development and EtOH drinking, in contrast to ourpresent results. Indeed, a genetic selection for sensitivity
to EtOH-stimulated activity results in genetic differencesin EtOH drinking such that fast mice, which exhibitgreater susceptibility to EIBS, drink more EtOH comparedto slow mice (Phillips, Burkhart-Kasch & Crabbe 1991;Risinger et al. 1994). Moreover, high-alcohol preferringmice selected for their high EtOH preference exhibitstronger EIBS development compared with low-alcoholpreferring mice (Grahame et al. 2000; Chester et al.2001). It is noteworthy that a source of difference infindings across the literature between studies thatexamine genetics correlations and studies that examinephenotypic correlations between two traits, as in thepresent study, may come from the fact that the latter onesare influenced by both genetic and non-genetic factors.However, our results are in accordance with anothergenetic study highlighting a negative correlation betweenEIBS levels and EtOH drinking in mice derived fromDBA/2J and C57BL/6J genitors (Phillips et al. 1995).Altogether, these results clearly demonstrate that somegenetic and/or phenotypic factors may influence bothEIBS and EtOH consumption, supporting the direct linkbetween both behaviors and the role of ethanolsensitization in excessive alcohol drinking.
Another important result of the current study is themaintenance of EtOH intake levels in sensitized micedespite quinine addition. Indeed, after excluding indi-vidual differences in the perceived aversive propertiesof quinine, we showed that EtOH intake changed afterEtOH adulteration with quinine: (1) the changes in EtOHintake and preference were positively correlated withsensitization scores and (2) resistant mice significantlyreduced their EtOH intake levels whereas sensitizedmice maintained their EtOH intake levels, althoughwe cannot exclude a possible floor effect in sensitizedmice. Altogether, these data suggest that resistantmice greatly increased their EtOH consumption afterEIBS, but did not display compulsive-like intake. Interest-ingly, a recent study showed that Swiss mice previouslysensitized following i.p. EtOH injections showed higher10% EtOH intake when adulterated by quinine(Carrara-Nascimento et al. 2012). Thus, it would be ofinterest to study inter-individual differences in the ‘resist-ance’ to adulteration with quinine in mice repeatedlytreated with EtOH. Another result that should be noted inthe present study is the high amounts of 20% EtOH con-sumed by resistant mice. These data are consistent withrecent studies that already presented undoubtedly highEtOH consumption levels in Swiss mice (Correia et al.2009; Carrara-Nascimento et al. 2012; Ribeiro et al.2012) and confirm that mice that are more sensitive toEtOH adulteration are the ones that consume highestamounts of EtOH. In addition, because some theories alsopostulate the existence of a strong relationship betweenalcohol addiction and uncontrolled overconsumption
of natural reinforcers (Kampov-Polevoy, Garbutt &Janowsky 1997), we assessed sucrose palatability andexcluded a correlation between EtOH intake and sensitiv-ity toward sweetened solutions.
This increase in EtOH intake could be due to a lowsensitivity to the aversive properties of EtOH as indexedby CTA in rodents (Holstein, Spanos & Hodge 2011;Alaux-Cantin et al. 2013). Studies using a genetic corre-lational strategy have shown that strains displayingstronger taste aversion tended to show lower EtOH pref-erence (Broadbent, Muccino & Cunningham 2002). Inthe present study, our results suggest that the increase inEtOH drinking in mice that are more resistant to EIBSdevelopment is not associated with a low sensitivity to theaversive motivational effects of EtOH. Altogether theseresults could be consistent with the possibility that resist-ance to EIBS is a risk factor to the development of alcoholabuse and alcoholism.
In summary, we demonstrated that individual suscep-tibility to EIBS is inversely correlated with voluntary EtOHconsumption. Interestingly, pre-exposure to EtOH drink-ing did not affect EIBS levels. Exposure to repeated EtOHduring EIBS development increased subsequent EtOHintake only in more resistant mice. We also demonstratedthat resistant mice were sensitive to EtOH adulterationwith quinine whereas sensitized ones maintained theirEtOH intake levels. This study confirms and extends previ-ous data showing a strong relationship between propen-sity for EtOH consumption and susceptibility to EIBS inoutbred Swiss mice. Future studies will focus on the tran-sition between controlled and compulsive EtOH use toinvestigate more deeply the relationship between liabilityto EIBS and EtOH addiction.
Acknowledgements
This study was supported by the National Agency forResearch (SAMENTA 2011 grant N°ANR- 12-SAMA-008-01 SENSIBALCO), the Conseil Régional de Picardie(CRP), the Mission interministérielle de lutte contre ladrogue et la toxicomanie (Mildt)—Inserm—Institute ofCancer (Contract APE09002ESA), Institut de France/Fondation NRJ ‘Biology of addiction’ and ERDF Grant/INTERREG IVA program N°4096 ‘AlcoBinge’. R.L. issupported by a doctoral Fellowship from the Ministère del’Enseignement supérieur et de la Recherche. B.B. issupported by a post-doctoral fellowship from the CRP.The funders had no role in study design, data collectionand analysis, decision to publish or preparation of themanuscript.
Conflict of Interest
The authors have declared that no competing interestsexist.
Authors Contribution
RL, BB and MN designed the research; RL, BB, JJ and FCperformed the research; RL, BB and MN analyzed thedata; and RL, BB and MN wrote the paper.
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SUPPORTING INFORMATION
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Figure S1 Concentration-dependent increase in EtOHdrinking. Four groups of mice were submitted to a two-bottle free choice between tap water and EtOH at concen-trations ranging from 3 to 20% (n = 16–18). Results fromEtOH intake (a) and preference (b) are presented asmean ± SEM. ***P < 0.001 indicates a difference between3 or 6% and 10 or 20% EtOH concentrationsFigure S2 Individual sensitization levels are stable allalong the experiments. From day 9 to day 18, micereceived 10 consecutive once-daily i.p. injections of2.5 g/kg EtOH (EtOH/Rep-E and water/Rep-E groups,n = 40 and 30, respectively) immediately followed by a 5minute-long locomotor activity recording session. At day54, challenge day, mice received a single i.p. injection of
2.5 g/kg EtOH. The correlation between locomotoractivity of mice from the EtOH/Rep-E group at day 18 andday 54 was investigatedFigure S3 Individual differences in blood EtOHconcentrations (BECs). Mice from the EtOH/Rep-S, EtOH/Rep-E and water/Rep-E groups were euthanized 30minutes after a 2.5 g/kg EtOH injection at day 54(n = 80). Blood was immediately collected and BECsmeasured. (a) Results from BECs are presented asmean ± SEM. (b) The correlation between sensitizationscores and BECs was investigated in the EtOH/Rep-Egroup (n = 40). (c) BECs were measured in another batchof mice at the challenge day 5 minutes after a 2.5 g/kgethanol injection in acute, resistant and sensitizedmice (n = 13–15). Each histogram represents the meanvalue (± SEM)Figure S4 Daily EtOH intake before and after EIBS.Mice from the EtOH/Rep-E group were exposed to a two-bottle choice paradigm (10% EtOH and tap water) before(day 1–day 6) and after (day 19–day 24) EIBS (n = 40).From day 25 to day 30, mice were exposed to a two-bottle choice paradigm with free access to 20% EtOHand tap water. Results from EtOH intakes are presentedas mean ± SEM. EIBS: EtOH-induced behavioralsensitizationFigure S5 Repeated EtOH injections increase both 10and 20% EtOH drinking. Mice from the EtOH/Rep-Egroup were exposed to a two-bottle choice paradigm(10% EtOH and tap water) before (day 1–day 6) and after(day 19–day 24) EIBS (n = 40). Results from 10% EtOHintake (a) and preference (b) and from 20% EtOH intake(c) are presented as mean ± SEM. *P < 0.05, **P < 0.01versus 10% EtOH drinking before EIBS. EIBS: EtOH-induced behavioral sensitizationFigure S6 Repeated EtOH injections do not changewater intake. Mice from the water/Rep-E group hadaccess to tap water before (day 1–day 6) and after (day19–day 24) EIBS (n = 30). The change in water intakewas calculated as (water intake after EIBS)—(waterintake before EIBS). The correlation between thesensitization score and the change in water intake wasinvestigatedFigure S7 Repeated saline injections do not change EtOHconsumption. Mice from the EtOH/Rep-S group hadaccess to 10% EtOH before (day 1–day 6) and after (day19–day 24) repeated saline injections (n = 10). Resultsfrom 10% EtOH intake (a) and preference (b) are pre-sented as mean ± SEMFigure S8 Correlations between the sensitization scoreand 10% EtOH drinking (before and after EIBS) in theresistant and sensitized subpopulations of mice. Micefrom the EtOH/Rep-E (n = 40) group were submitted to atwo-bottle choice paradigm (10% EtOH and tap water)before (day 1–day 6) and after (day 19–day 24) EIBS.
Changes in EtOH drinking (EtOH intake and preference)were calculated as (10% EtOH drinking after EIBS)—(10% EtOH drinking before EIBS). Correlations wereinvestigated in resistant (n = 22) and sensitized (n = 18)groups between the sensitization score and changes in(1) 10% EtOH intake (a) and preference (d) before EIBS,(2) 10% EtOH intake (b) and preference (e) after EIBS and(3) 10% EtOH intake (c) and preference (f). EIBS: EtOH-induced behavioral sensitizationFigure S9 Individual differences in sensitivity to quinineaversive effect. Mice from the EtOH/Rep-E group weresubmitted to a two-bottle free choice between tap waterand 0.15 g/l quinine in tap water (day 31–day 36)(n = 40). (a) The correlation between quinine solutionpreference and the sensitization score was investigated.Mice were split into resistant and sensitized groups basedon their sensitization score (n = 18–22). (b) Results fromquinine preference are presented as mean ± SEM
Figure S10 Correlations between the sensitization scoreand 20% EtOH drinking (in the absence or presence ofquinine) in the resistant and sensitized subpopulations ofmice. Mice from the EtOH/Rep-E group were submitted toa two-bottle choice (20% EtOH and tap water) betweenday 25 and day 30 (n = 40). They were then submitted toanother two-bottle choice paradigm with one bottle con-taining tap water and the other one 0.15 g/l quinine in20% EtOH between day 37 and day 42. Changes in EtOHdrinking (EtOH intake and preference) were calculated as(quinine + 20% EtOH drinking)—(20% EtOH drinking).Correlations were investigated in resistant (n = 22) andsensitized (n = 18) groups between the sensitization scoreand changes in (1) 20% EtOH intake (a) and preference(d) in absence of quinine, (2) 20% EtOH intake (b) andpreference (e) in presence of 0.15 g/l quinine and (3)20% EtOH intake (c) and preference (f). EIBS: EtOH-induced behavioral sensitization
