The adenosine A2A receptor agonist CGS 21680decreases ethanol self-administration in bothnon-dependent and dependent animals
Hakim Houchi1, Wolfgang Persyn1,2,3, Rémi Legastelois1 & Mickaël Naassila1
Groupe de Recherche sur l’Alcool & les Pharmacodépendances (GRAP), INSERM ERi 24, UFR de Pharmacie, Université de Picardie Jules Verne, France1, Serviced’Alcoologie, Centre SESAME, CH Philippe Pinel2 and Département Universitaire de Psychiatrie, Unité de Psychiatrie Adulte (UPA), CHU Amiens, France3
ABSTRACT
There is emerging evidence that the adenosinergic system might be involved in drug addiction and alcohol dependence.We have already demonstrated the involvement of A2A receptors (A2AR) in ethanol-related behaviours in mice. Here,we investigated whether the A2AR agonist CGS 21680 can reduce ethanol operant self-administration in both non-dependent and ethanol-dependent Wistar rats. To rule out a potential involvement of the A1R in the effects of CGS21680, we also tested its effectiveness to reduce ethanol operant self-administration in both heterozygous andhomozygous A1R knockout mice. Our results demonstrated that CGS 21680 (0.065, 0.095 and 0.125 mg/kg, i.p.) hada bimodal effect on 10% ethanol operant self-administration in non-dependent rats. The intermediate dose was alsoeffective in reducing 2% sucrose self-administration. Interestingly, the intermediate dose reduced 10% ethanol self-administration in dependent animals more effectively (75% decrease) when compared with non-dependent animals(57% decrease). These results suggest that the A2AR are involved in CGS 21680 effects since the reduction of ethanolself-administration was not dependent upon the presence of A1R in mice. In conclusion, our findings demonstrated theeffectiveness of the A2AR agonist CGS 21680 in a preclinical model of alcohol addiction and suggested that theadenosinergic pathway is a promising target to treat alcohol addiction.
Correspondence to: Hakim Houchi, UFR de Pharmacie, INSERM ERi 24 GRAP, 1 rue des Louvels, 80000 Amiens, France. E-mail:[email protected]
INTRODUCTION
Ethanol abuse and dependence are serious medical andsocial problems. Currently, available alcohol dependencepharmacotherapies are only moderately successful(Mann 2004; Heilig et al. 2011), and thus, there areimportant expectancies for finding novel treatments toimprove clinical outcomes and help to reduce the devas-tating consequences of the disease.
Among the numerous neurotransmission systemsthat have been targeted to reduce ethanol self-administration in preclinical studies, the adenosinergicpathway has been shown to be of interest. Molecularstudies have shown that acute or chronic ethanol expo-sure induces indirect A2A signalling activation, throughthe inhibition of equilibrative nucleoside transporter 1(ENT1) and increases in extracellular adenosine levels(Choi et al. 2004). The resulting signal cascade involvescyclic adenosine monophosphate (cAMP) signalling,
Protein kinase A (PKA) activation and translocation, andcAMP response element (CRE)-dependent gene regula-tions (Fredholm et al. 2007). Moreover, A2AR activationseems to be involved in the brain-derived neurotrophicfactor (BDNF) pathway (Tebano et al. 2010), which isknown to gate ethanol self-administration (Jeanblancet al. 2009).
Adenosine, via four G-protein-coupled receptors (A1,A2A, A2B and A3 receptors) is a signalling molecule thattriggers numerous physiological responses and has alsobeen involved in different pathologies such as drug addic-tion (Ferré et al. 2007; Brown & Short 2008). Regardingall adenosine receptors, the A1R and A2AR have beenthe most studied; the former are widely expressed in thebrain, whereas the latter are concentrated in the basalganglia nuclei.
Both the A1R and A2AR have been involved inethanol-related behaviours. The A1R pathway seems tobe involved in anxiety-like behaviour, whereas the A2AR
pathway is associated with rewarding effects (Prediger,Batista & Takahashi 2004; Prediger et al. 2006).
We have previously suggested that activation ofA2AR may play a role in suppressing ethanol drinkingbehaviour in ethanol-preferring mice (Naassila, Ledent& Daoust 2002) and shown that mice lacking A2ARdisplay reduced ethanol-induced rewarding effects inthe conditioned place preference paradigm (Houchiet al. 2008). One study in Long-Evans rats showedthat a high dose of the A2AR antagonist 3,7-dimethylpropargylxanthine (DMPX) had a bimodal effecton ethanol self-administration, with low and high dosesincreasing and decreasing self-administration, respec-tively. Conversely, administration of an A1R antagonist,DPCPX, had no effect (Arolfo et al. 2004). Interestingly, arecent study confirmed that the A2AR agonist CGS21680 (0.1 mg/kg), at a dose devoid of any effect on totalfluid self-administration and locomotion, reduced 10%ethanol-operant self-administration in Marchigian Sar-dinian ethanol-preferring rats (Micioni Di Bonaventuraet al. 2011). However, the A2AR antagonists DMPX andSCH 58261 have been shown to reduce ethanol operantself-administration in rats (Thorsell, Johnson & Heilig2007; Adams et al. 2008). Regarding other drugs ofabuse, several studies have shown mixed results with theuse of pharmacological agents and knockout (KO) mice.Motivational mechanisms involved in both maintenanceand extinction of cocaine self-administration has beensuggested to be mediated by adaptive changes of thepharmacological properties of A2AR (Frankowska et al.2012). Consistent with our hypothesis, of using an A2ARagonist to decrease drug reward, it has been shownthat A2AR agonists reduce cocaine self-administration(Knapp et al. 2001) and reinstatement of cocaine-seekingbehaviour in rats (Bachtell & Self 2009; O’Neill, Letendre& Bachtell 2012), while an A2AR antagonist reinstatedcocaine-seeking behaviour in baboons (Weerts & Griffiths2003). In contrast, another study on squirrel monkeysdemonstrated the A2AR antagonist’s ability to decreasethe reinforcing effects of D-9-TetraHydroCannabinol(THC) but not those of cocaine or food and thatthese effects may be mediated through presynapticA2AR located on glutamatergic projections to the Nac(Justinová et al. 2011).
Because of these inconsistencies and the lack of evi-dence that the pharmacological activation of A2ARcould be effective in a preclinical model of ethanol addic-tion, the present work investigated the effect of A2ARactivation on excessive ethanol self-administration in ratsdisplaying both physical and behavioural ethanol addic-tion (Gilpin et al. 2008). Herein, Wistar rats underwentchronic and intermittent ethanol exposure using aninhalation procedure to induce dependent state, as wepreviously described (Simon O’Brien et al. 2011). They
were assessed for their motivation to self-administerethanol after acute treatment with the A2AR agonist CGS21680 during acute withdrawal (Gilpin et al. 2008;Simon O’Brien et al. 2011). We also tested whether theeffect of CGS 21680 on ethanol self-administration wasmediated specifically by the A2AR using mice lackingA1R.
MATERIALS AND METHODS
Animals
Male Wistar rats initially weighing 250 to 300 g wereobtained from Charles River (Les Oncins, France). Theywere group-housed (3/cage) during 1 week in ourclimate-controlled animal facility (21°C, 50% humidity,12-hour light/12-hour dark cycle (light between7:00 a.m. and 7:00 p.m.). Rats had ad libitum access towater and standard chow during this first week. After theacclimation period, all subjects were given a 4-day forcedexposure to 10% ethanol, followed by a 3-day free-choiceexposure to 10% ethanol and water (with continuous adlibitum access to chow).
Male wild-type (WT) and A1 -/- (KO) mice generatedon a C57BL background were also used in the presentstudy (Johansson et al. 2001). Briefly, the A1R gene wasinactivated in E14.1 embryonic stem cells and one of theclones was used to generate chimeric mice. Male chime-ras were mated to C57BL females, and A1R+/- mice fromthis 129/OlaHsd/C57BL hybrid offspring were inter-crossed to generate A1R+/+, A1R+/- and A1R-/- offspring.KO and WT control males (25–30 g) were housed 10 percage (each genotype in a separate cage) and were used inthe present study when they reached 35–40 g. There wasno significant genotypic difference in body weight. Foodand water were available ad libitum during all experi-ments. All animals used in a given experiment originatedfrom the same breeding series and were matched for ageand body weight.
The number of animals was kept to a minimum andall efforts were made to avoid animal suffering. Experi-ments were carried out in strict accordance with both theguide for care and Use of Laboratory Animals (NIH), theE.C. regulations for animal use in research (CEE no.86/609) and our local ethic committee (CREMEAP). Allexperiments were performed under blind conditions.
Drugs
Ethanol (96.3%, v/v) was obtained from VWR (Paris,France) and was diluted to 10% (v/v) in tap water.
Sucrose powder was obtained from Sigma Aldrich(Saint-Quentin Fallavier, France) and was diluted to 2%(w/v) in tap water. All ethanol and sucrose solutions wereprepared immediately before the operant session. The
A2AR selective agonist CGS 21680-HCl (Tocris Bio-sciences, Bristol, UK) was dissolved by warming in waterand aliquoted at 2 mg/ml. Each CGS 21680 dose wasprepared from an aliquot and concentrations wereadjusted with saline (0.9% NaCl). The dose range of CGS21680 was determined based on its effects on spontane-ous or on drug-induced motor activity (Rimondini et al.1997). Since CGS 21680 0.1 mg/kg has been shown tobe devoid of cataleptic effects, we used a dose rangeincluding 0.1 mg/kg (for rats and mice experimentationsrespectively Jones-Cage, Stratford & Wirtshafter 2012and Listos et al. 2011). CGS 21680 solutions were admin-istered 15 minutes before each operant task session, viathe intra-peritoneal (i.p.) route.
Operant self-administration
Apparatus
All operant sessions were conducted in 12 modularoperant chambers housed in sound attenuating and ven-tilated cubicles (Bioseb, Vitrolles, France). Chambers wereinterfaced to a PC computer equipped with PackWin soft-ware (Panlab S.L., Barcelona, Spain) that recorded leverpresses and reinforcer deliveries. The left side of eachchamber contained two manipulanda (levers for rats ornose-poke holes for mice) located on either side of acentral access receptacle where the reinforcer was deliv-ered. The response schedule was fixed ratio 1 (FR-1)during training and testing sessions (30 minutes for ratsand 2 hours for mice). The active trigger delivered a fixedvolume of solution (50 ml for rats and 20 ml for mice)with a 3 seconds cue light upon the lever. During thistime, lever presses were recorded but did not lead tofurther delivery. The inactive lever did not lead to lightcues or reinforcer deliveries, but a 3-second timeoutperiod was maintained.
Induction procedure for rats
We used a total number of 33 rats that were trained torespond for a liquid reinforcer in a two-lever free-choicesituation and were further used in within-subjectsexperiments (Richards et al. 2008; Walker et al. 2008).Regarding the experiments on the effects of CGS 21680,three groups of rats were used: basal conditions (n = 9),ethanol dependent (n = 8) and controls (n = 8, airexposed). An additional group of naive animals (n = 8)was used to test the effects of CGS on 2% sucrose self-administration. The learning procedure for ethanolself-administration was sucrose fading, as described bySamson (1986), with modifications as we previouslydescribed (Simon O’Brien et al. 2011). Rats were waterdeprived in their home cage for 22 hours only before thefirst two starting sessions.
Sucrose fading procedure
Self-administration training was initiated on day 1 witha free delivery per minute of 10% sucrose to shapeapproach to the reinforcer deliveries. During this firstsession, lever pressing also produced a reinforcer delivery.On day 2, a response on one of the two levers produced a10% sucrose delivery, while a response on the other oneresulted in no free deliveries. On days 3–5, presses on thetwo levers produced a mixed 10% ethanol-10% sucrosedelivery. On days 6–10, one lever was inactive and theother produced a mixed 10% ethanol/10% sucrose deliv-ery. The sucrose fading procedure started on day 11.Sucrose concentration was gradually decreased aftereach block of three sessions (10%, 8%, 6%, 4%, 2%, 0%),while the ethanol concentration was maintained at 10%.
After the learning procedure, rats stabilized their self-administration during 12–15 sessions with one activelever delivering 10% ethanol. Only animals with consist-ent responding (response variation during three sessions< 20%) were allowed to continue the study.
Ethanol vapour inhalation in rats
After a learning period, a group of rats (n = 24) was indi-vidually housed in standard cages. All cages were placedinto two clear plastic chambers, one for ethanol inhala-tion (n = 8) and the other for control air inhalation(n = 16), as we previously described (Naassila et al. 2000;Simon O’Brien et al. 2011). Ethanol vapour was gener-ated by introducing pressured air into 96% ethanolthrough a submerged hose. Another leaky hose wasimmerged in water to produce both sufficient hygromet-ric conditions in chambers and diluents for ethanol. Thisdesign permitted us to eliminate vapour condensationsand to reach range of blood ethanol levels (BELs) between150 and 200 mg/dl during vapour exposure by mixingethanol vapour and wet air. Blood samples were collectedfrom the tail vein root immediately after the end of dailyexposure to determine BELs and to adjust the ethanol/airmix. The ethanol vapour concentration was graduallyincreased to reach targeted BELs. Ethanol vapour inhala-tion was maintained during 10 weeks in daily intermit-tent cycles (ethanol vapour turned on at 7:00 p.m.during 14 hours).
Testing procedure
Once animals reached stable levels of ethanol operantself-administration (response variation < 20% duringthree sessions), the 10% ethanol responding was exam-ined. The testing procedure began after at least 3 days ofbaseline without injection followed by at least 3 days ofbaseline with vehicle (i.p. injection of NaCl 0.9%).Finally, as soon as rats (n = 9) showed at least threestable sessions of NaCl baseline, the dose effect of the
A2AR agonist was tested in a Latin square design anda within-subjects experiment (Richards et al. 2008;Walker et al. 2008). Vehicle or CGS 21680 was admin-istered by i.p. injection, 15 minutes before operant tasksessions.
For rats (n = 24), we chose the dose of CGS 21680(0.095 mg/kg), which is able to decrease operant behav-iour without inducing cataleptic effects. We thus testedthis dose on FR-1 operant 10% ethanol responding inboth non-dependent (n = 8 control air) and dependent(n = 8 ethanol inhalation) rats. We also tested this doseon FR-1 operant 2% sucrose responding in an additionalgroup of non-dependent rats (n = 8 control air). Depend-ent rats were tested 6 hours after the end of the vapourinhalation exposure, when BELs returned to zero (Gilpinet al. 2008; Simon O’Brien et al. 2011).
Induction procedure for mice
Mice (n = 25: n = 8 WT, n = 9 heterozygous (HET), n = 8KO) were trained to respond in two nose-poke holes (oneactive and one inactive) on a FR-1 schedule. The learningprocedure for ethanol self-administration was sucrosefading, as described by Samson (1986), with modifica-tions. During the learning stage, we used a cooperativeprocedure in which four mice were placed in the operantchambers to boost acquisition of the operant task and weused the same sucrose fading procedure as for rats. Onlyanimals with constant responding (responses variationduring three sessions < 20%) were allowed to continuethe study. Two HET mice were excluded because of theirinconsistent responding.
Testing procedure
All mice (n = 23) received increasing doses of CGS 21680(0.065, 0.095, 0.125 mg/kg) before operant tasksessions, and washout periods (three baseline sessionswithout variation) were carried out between the differentCGS 21680 doses. Experimental procedures were per-formed was done by an experimenter blind to the geno-type of the mice.
Statistical analysis
Both active and inactive responses were recorded. Sincethe level of responding on the inactive lever was low(Richards et al. 2008), only active triggers data wereanalyzed in the operant self-administration experimen-tations. The sucrose operant self-administration data inrats were analyzed using a one-way repeated measuresanalysis of variance (ANOVA) and data on ethanoloperant self-administration in rats were analyzed usinga two-way ANOVA. The latency to press the lever wasanalyzed using a Student’s t-test. Data on alcoholoperant self-administration in mice were analyzed with a
two-way repeated measures ANOVA. All statisticalanalyses were performed using SigmaStat software and,in all cases, post hoc analyses were performed using aTukey’s test. The criterion for statistical significance wasfixed at 0.05.
RESULTS
Dose effect of CGS 21680 on 10% ethanol operantself-administration within non-dependent rats (Fig. 1)
On Fig. 1, since the baseline operant responding for 10%ethanol was not significantly different among all groupsbefore any drug treatments, the mean of data wereanalyzed. During the three successive days of the NaClbaseline treatment, the mean of lever presses were41.5 � 2.6, 41.8 � 1.9, 45.5 � 2.5 and were notsignificantly different [F(2,27) = 0.9 P = 0.42]. CGS0.065 mg/kg significantly increased FR-1 operantethanol-reinforced responding 68.5 � 82 [F(3,36) = 4.8P < 0.002] (Fig. 1). Higher doses of CGS 21680 (0.095and 0.125 mg/kg) significantly decreased FR-1 operantethanol-reinforced responding [respectively, 28.7 � 5.8;F(3,36) = 2.9 P < 0.05 and 25.9 � 6.2; F(3,36) = 3.5P < 0.05] (Fig. 1). The ethanol consumption is also givenas grams of pure ethanol consumed by kilogram of bodyweight in Table 1.
Because high doses of CGS 21680 are known forinducing locomotor impairments (Rimondini et al. 1997;Wardas, Konieczny & Pietraszek 2003; Micioni DiBonaventura et al. 2011), the latency to lever pressingwas evaluated using the first and fifth lever press afterNaCl treatment (first lever press 31.7 seconds � 12.9seconds and fifth lever press 93.9 seconds � 23.8seconds). Since the latency to the first lever press dis-played high interindividual variability and thus was notsufficiently informative, we also measured the latency tothe fifth lever press. Compared with the NaCl treatment innon-dependent rats, a significant increase in the latenciesto the first and the fifth lever presses was observed only atthe highest dose (0.125 mg/kg first press 876.6 sec-onds � 280.8 seconds and fifth press 970.1 sec-onds � 202.1 seconds: P < 0.01). In addition, we did notfind any significant differences between any doses on thetime elapsed between the first and the fifth lever presses(0.065 mg/kg: first press 27.6 seconds � 7.0 secondsand fifth press 80.8 seconds � 14.0 seconds: P > 0.05and 0.095 mg/kg: first press 142.3 seconds � 85.7seconds and fifth press 228.3 seconds � 127.0 seconds:P > 0.05). These results indicate that the intermediateeffective dose did not display cataleptic effects. Moreover,a representative schematic pattern of operant behaviourextracted from PackWin software (Fig. 2a) provided abetter demonstration of this result.
Effects of CGS 21680 0.095 mg/kg on 2%sucroseoperant self-administration within non-dependent rats(Fig. 3)
The 0.095 mg/kg dose of CGS 21680 was also tested onsucrose operant responding (Fig. 3). The 2% sucrose con-centration was chosen to reach a comparable level ofresponding within non-dependent rats as the oneachieved with alcohol (Czachowski & Samson 2002). Aone-way ANOVA with repeated measures revealed a sig-nificant decrease in 2% sucrose operant responding afterCGS 21680 treatment [F(1,21) = 18.5 P < 0.001]. (Thevolume of 2% sucrose consumed is presented in Table 2).
Effects of CGS 21680 0.095 mg/kg on 10% ethanoloperant self-administration within dependentrats (Fig. 4)
The lowest dose of CGS 21680 previously found todecrease FR-1 operant ethanol-reinforced responding innon-dependent rats was chosen to test the effects of CGS
21680 on 10% ethanol operant responding withindependent rats. Here, we used an additional non-dependent group (n = 8), which was different from theone described above. As expected, dependent rats dis-played a significant increase in baseline level of FR-1operant ethanol-reinforced responding when theywere compared to non-dependent rats, [F(1,48) = 9.3P < 0.001, Fig. 4], thus revealing an excessive ethanolintake in dependent animals (Rimondini et al. 2002;Gilpin et al. 2008; Simon O’Brien et al. 2011).
Regarding NaCl or CGS 21680 (0.095 mg/kg) treat-ment within the dependent and non-dependent ratgroups, the two-way ANOVA revealed main effects ofboth drug factor [F(1,64) = 37.9 P < 0.001] and ratgroup factor [F(1,64) = 13.3 P < 0.001] and a significantinteraction between drug and rat group factors[F(1,64) = 14.5 P < 0.001]. Both rat groups showeddecrease of the FR-1 operant alcohol responding afterCGS 21680 (0.095 mg/kg) treatment compared withNaCl baseline [F(1,48) = 9.9 P < 0.001].
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Figure 1 Dose effect of CGS 21680 on 10% ethanol responding, after a 15 minutes systemic administration within non-dependent rats.Three doses of CGS 21680 were tested in a group of nine non-dependent rats in a Latin-square design: 0.065 mg/kg (n = 11), 0.095 mg/kg(n = 8), 0.125 mg/kg (n = 8). Data show the mean number of active lever presses (+/- SEM) during 30-minute operant sessions. White barrepresents means of 3 days of basal responses after NaCl i.p administration and black bars represent mean of treatment day responses afterCGS 21680 i.p administrations. @ denote significant differences between NaCl and CGS 21680 treatment on operant self-administration;@@ P < 0.01; and @ P < 0.05
Table 1 Diagram showing experimental design within non-dependent and dependent rats.
N-Dep n = 9 21 days 15 days 10% ethanol X 3 days w/o IP 3 days IP 0.065a 0.095a 0.125a
Dep n = 8 10 weeks—EtOH 0.095N-Dep n = 8 10 weeks—AirN-Dep n = 8 2% sucrose
aCGS 21680 doses (0.065, 0.095 and 0.125 mg/kg) have been administrated via i.p. root, using a Latin square design. All doses have been preceded by3 days of NaCl i.p. injections. Dep = dependent rats; N-Dep = non-dependent rats; IP = intra-peritoneal injection; w/o IP = without intra-peritonealinjection.
Comparing dependent and non-dependent rats, nodifference on lever presses was observed after CGS21680 (0.095 mg/kg) treatment. However, significantinteraction [treatment ¥ rat groups (F(1,64) = 14.5P < 0.001)] revealed that there is a difference in CGS21680-induced decrease in FR-1 operant ethanol-reinforced responding. More precisely, CGS 21680 treat-ment was more effective in dependent rats (75.5% versus51.7% decrease). The ethanol consumption is also givenas grams of pure ethanol consumed by kilogram of bodyweight in Table 1.
Dose effect of CGS 21680 (0.065, 0.095 and0.125 mg/kg) on 10% ethanol operantself-administration within A1R KO mice (Fig. 5)
Figure 5 shows that before any drug treatment, the base-line operant responding for 10% ethanol after NaCltreatment was not significantly different among geno-types (WT, HET and KO). More precisely, respectively, foreach CGS 21680 dose (NaCl, 0.065, 0.095, 0.125 mg/kg) and expressed in gram of pure ethanol per kilogramof body weight, WT mice consumed 2.0 � 0.33 g/kg,
(a) 10% Ethanol / non-dependent rat
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Figure 2 Dose effect of CGS 21680 on 10% ethanol responding, after a 15-minute systemic administration within non-dependent anddependent rats. Schematic patterns of 30-min sessions of operant behaviour.A representative pattern was extracted from data recorded withPackWin software. Each vertical tick on the x-axis indicates the time within the 30-minute testing session at which an event was recorded.For each pattern, the two first lines display active and inactive lever presses (respectively lever 1 and lever 2).The last line, entitled ‘detectorinside’, gives indication to beam breaks triggered by rats licking 10% ethanol solution. (a) Individual ethanol self-administration event recordsof representative subjects from each experimental group, NaCl, CGS 0.065 mg/kg, 0.095 mg/kg and 0.125 mg/kg. (b) 2% sucrose self-administration event records of a representative non-dependent subject treated with NaCl and 0.095 mg/kg. (c) 10%-ethanol self-administration event records of a representative dependent subject treated with NaCl and 0.095 mg/kg
0.13 g/kg, 0.9 � 0.23 g/kg. The two-way repeatedmeasures ANOVA did not reveal a main effect of geno-type factor [F(2,89) = 0.01, P = 0.984] but revealed asignificant main effect of drug factor [F(3,88) = 18.8,P < 0.001], revealing that the A2AR agonist is effectivein reducing FR-1 operant alcohol-reinforced responding.The two-way repeated measures ANOVA did not show asignificant interaction between genotype and drugfactors [F(6, 85) = 0.33 P = 0.916].
DISCUSSION
Our data demonstrated that the A2AR agonist CGS21680 had a bimodal effect on FR-1 operant ethanol-reinforced responding that was not mediated by A1R.
Interestingly, we showed that its effectiveness at the inter-mediate dose (0.095 mg/kg) was devoid of catalepticeffects and produced greater decreases in ethanol rein-forcer responding in ethanol-dependent animals com-pared to non-dependent animals. As expected, activationof A2AR may have a general impact on the brain rewardsystems since CGS 1680 also reduced 2% sucrose self-administration. Altogether, our results strengthen thehypothesis that activation of the A2AR pathway is effec-tive in reducing excessive drinking during acute with-drawal in ethanol-dependent animals.
The present findings confirmed and extended our pre-vious data obtained in C57BL/6J mice showing that CGS21680 decreased both 10% ethanol intake and prefer-ence in the two-bottle choice paradigm (Houchi et al.2008). Those data suggested that adenosine actingthrough A2AR pathway is implicated in the rewardingeffects of ethanol (Naassila et al. 2002; Houchi et al.2008). The bimodal outcome on 10% ethanol operant
self-administration could be explained by high- and low-affinity subtypes of A2AR (Cunha et al. 1996; El Yacoubiet al. 2000). It is noteworthy that the high concentrationof CGS 21680 could also activate A1R (Lopes et al. 2004;Cristalli, Müller & Volpini 2009), and that adenosinergiceffects on locomotor behaviour are principally mediatedby the A1R pathway (Dar 2001). Moreover, using mice
lacking A2AR, we have already shown that A2AR arenot involved in 2 g/kg ethanol-induced behavioural loco-motor sensitization (Houchi et al. 2008). Consequently,we used the intermediate dose of CGS 21680 inducing adecrease of ethanol operant self-administration withoutany sedative or cataleptic effect within non-dependentrats to assess the outcome in dependent subjects.
Based on the analysis of event records extracted fromthe individual patterns of ethanol self-administration(Fig. 2a), the highest dose (0.125 mg/kg) dramaticallyincreased the latency to the first lever press, as expected.Other studies have revealed a motor effect at the equiva-lent dose of CGS 21680. (Rimondini et al. 1997; Wardaset al. 2003; Micioni Di Bonaventura et al. 2011). Inaccordance with other investigators, we did not observesigns of sedation when using higher doses of A2ARagonist (such as 0.250 mg/kg, data not shown). However,below the dose of 0.1 mg/kg, our data showed no altera-tion of the latency to either the first or the fifth lever press(see patterns in Fig. 2a,b,c). Moreover, it has been alsodescribed that doses under 0.1 mg/kg could be consideredas weaker doses since they did not significantly affect base-line locomotion or drug-induced hyper-locomotion, likethat produced by cocaine or amphetamine (Rimondiniet al. 1997; Baldo, Koob & Markou 1999; Knapp et al.2001; Filip et al. 2006; Micioni Di Bonaventura et al.2011; Jones-Cage et al. 2012). Interestingly, the inter-mediate dose of CGS 21680 (0.095 mg/kg) decreasedthe FR-1 operant ethanol-reinforced responding andincreased the inter-reinforcement intervals. Although
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Figure 3 Effect of CGS 21680 0.095 mg/kg on 2% sucrose respond-ing, after a 15 minute systemic administration within non-dependentrats (n = 8). Data show the mean number of active lever presses (+/-SEM) during 30-minute operant sessions. Black bars represent meansof 3 days of basal responses after NaCl i.p administration and whitebars represent mean of treatment day responses after CGS 216800.095 mg/kg i.p administrations. @ denote significant differencesbetween basal and treatment responses; @@@ P < 0.001
Table 2 Amount of alcohol and sucrose consumed corresponding to lever presses or nose-pokes.
CGS 21680
Baseline 0.065 mg/kg 0.095 mg/kg 0.125 mg/kg
Non-dependent ratsPure ethanol (g/kg) n = 9 0.41 0.02 0.66@@@ 0.05 0.28@ 0.06 0.25@ 0.06Mean � SEMPure ethanol (g/kg) n = 8 0.55 0.07 0.25@ 0.10Mean � SEM2% sucrose (ml) n = 8 4.9 0.6 1.5@@@ 0.7Mean � SEM
Dependent ratsPure ethanol (g/kg) n = 8 0.98### 0.06 0.27@@@ 0.08Mean � SEM
Non-dependent micePure ethanol (g/kg) n = 23 1.96 0.12 1.33@@ 0.16 0.76@@@ 0.11 1.04@@@ 0.19Mean � SEM
Means (bold values) � SEM (italicized values) of pure alcohol expressed in grams per kilograms and means � SEM of 2% sucrose volume expressed inmillilitres. Baselines show means of a block of 3 days and each CGS 21680 doses show mean of the test day. For non-dependent rats, data correspondto 10% ethanol and 2% sucrose self-administration, respectively, after 0.065, 0.095 and 0.125 mg/kg and 0.095 mg/kg treatments. For dependent rats,data show 10% ethanol self-administration after 0.095 mg/kg treatment. For non-dependent mice, data correspond to 10% ethanol self-administrationafter 0.065, 0.095 and 0.125 mg/kg treatments. @ denote significant differences between basal and treatment responses: @P < 0.05, @@P < 0.01,@@@P < 0.001. # denotes significant differences between non-dependent and dependent rats in their basal responses: ###P < 0.001.
CGS 21680 (0.095 mg/kg)-treated rats clearly showeda slower rate of ethanol self-administration, animalsmaintained an active level of responding, especiallyduring the first 15–20 minutes (Fig. 2a–c). Regardingnon-dependent animals, the fact that there was nodifference in the operant behaviour between CGS 21680-treated and NaCl-treated rats, ruled out the possibilityof a motor impairment produced by CGS 21680(0.095 mg/kg).
On the other hand, the lowest dose (0.065 mg/kg)induced an increase in both the FR-1 operant ethanol-reinforced responding and the inter-reinforcement inter-vals associated with maintenance of responding until theend of the 30-minute session. This finding may thus indi-cate that there not strong correlation between the motoreffects of CGS 21680 and its effects on operant behaviour.Hence, it is unlikely that the decrease in FR-1 operant
ethanol-reinforced responding at the 0.095 mg/kg dosecould be due to motor effects of the A2AR agonist.
We also observed a significant reduction in 2% sucroseself-administration within non-dependent animals dis-playing the same level of responses compared to ethanol-dependent animals. As observed with ethanol, our datashowed a decrease in the rate of self-administration butrats maintained a significant level of response during thefirst half of the session indicating only a modest influenceof the potential motor effects of CGS 21680 (Fig. 2a–c).These findings suggest a more general impact of activat-ing A2AR on the brain reward systems, and are inaccordance with a previous study that showed a decreasein lever pressing for food or sucrose after systemic andhigher doses of intra-nucleus accumbens (NAc) adminis-tration of a higher doses of A2AR agonists (Font et al.2008). In contrast, O’Neill et al. (2012) did not observe
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# # #Figure 4 Effect of CGS 21680 0.095 mg/kg on 10% ethanol responding, after a 15-minute systemic administration within non-dependent and dependent rats. Data showthe mean number of active lever presses(+/- SEM) during 30-minute operant ses-sions within non-dependent (n = 8) anddependent rats (n = 8). Black bars representmeans of 3 days of basal responses afterNaCl i.p. administration and white bars rep-resent mean of treatment day responsesafter CGS 21680 0.095 mg/kg i.p adminis-trations. @ denote significant differencesbetween basal and treatment responses:@@@ P < 0.001 and @ P < 0.05, # denotessignificant differences between non-dependent and dependent rats in theirbasal responses: ### P < 0.001
Figure 5 Dose effect of CGS 21680 on 10% ethanol responding, after a 15-minute systemic administration within Adora1 deleted mice on129 ¥ B6 genetic background.Three doses of CGS 21680 were tested: 0.065 mg/kg (n = 23), 0.095 mg/kg (n = 23), 0.125 mg/kg (n = 23). Datashow the mean number of active lever presses (+/- SEM) during 2 hours operant sessions. NaCl treatment represents means of three daysof basal responses after NaCl i.p administration and CGS 21680 treatment represent mean of treatment day responses after CGS 21680 i.padministrations. # denotes significant differences between basal and treatment responses; ### P < 0.001; and ## P < 0.01
any effect of intra-NAc CGS 21680 injections on sucroseseeking in Sprague-Dawley rats trained to self-administersucrose pellets after a period of extinction. Othermolecules that are effective in reducing ethanol self-administration have also been shown to have an effect onself-administration of natural reinforcers such as theserotonergic and noradrenergic transporter inhibitormilnacipran (Simon O’Brien et al. 2011), the CB1Rantagonist SR-141716A (Economidou et al. 2006) andthe g-aminobutyric (GABA) B receptors agonist Baclofen(Anstrom et al. 2003). It is noteworthy that CGS 21680,SR-141716A and Baclofen target presynaptic and post-synaptic receptors that are widely distributed in the brainreward systems, and thus, may explain, at least in part,their effects on the mesolimbic dopaminergic pathwayand on the rewarding properties of both drugs of abuseand sucrose.
In this way, available data on the rewarding effects ofelectrical brain stimulation indicate that adenosine, viaA2AR, may inhibit central reward process (Baldo et al.1999). In this study, it has been suggested that the A2ARagonist elevates reward thresholds possibly by opposingthe effects of dopamine and that this effect may explainthe impact on rewarding effects of electrical brain stimu-lation and more ‘natural’ stimuli such as Baldo et al.(1999). Adenosine A2AR activation negatively modu-lates postsynaptic effects of dopamine by a potentialantagonistic interaction with DRD2 which is able tomodulate the striatal GABAergic-enkephalinergic(GABA-ENK) neurons (Fink et al. 1992; Ferré et al. 1997;Hillion et al. 2002). In a homeostatic state, the dopamin-ergic tonus, from the ventral tegmental area (VTA) toventral striatum, leads to enkephalin release from GABA-ENK neurons, known to be involved in positive reinforce-ment (Belluzzi & Stein 1977; Skoubis et al. 2005). It isnoteworthy that DRD2 are localized on these GABA-ENKneurons and contribute to the decrease in enkephalinrelease (Shirayama & Chaki 2006). Considering thishealthy state, this pathway could participate in naturalseeking of hedonic values. Taking into account theantagonistic interaction between DRD2 and A2AR,A2AR agonists are able to block DRD2 outcomes (Fontet al. 2008) and thus contribute to an increase inenkephalin release leading to a pharmacologicallyinduced sated state. Moreover, A2AR agonist generatesdopamine antagonist-like outcomes without extrapy-ramidal side effects reported with neuroleptics andantipsychotic treatments (Ferré et al. 1997). These phe-nomena occur principally in the dorsal striatum andNAc, both of which display a high density of both DRD2and A2AR. This antagonistic interaction leads to thesame behavioural effects observed during an increase inelectrical brain stimulation reward threshold (Baldo et al.1999). It is noteworthy that an interaction between
A2AR and CB1R also has been demonstrated (Ferréet al. 2010). Interestingly, this interaction has beendescribed both at the presynaptic or postsynaptic leveldepending on the type of GABAergic neuron, i.e.GABA-dynorphinergic (GABA-DYN) and GABA-ENK,respectively. To our knowledge, no data are availableregarding the potential role of this interaction in ethanolself-administration.
In addition, ethanol exposure has been shown tochange adenosine synaptic levels through two pathways:Acute ethanol treatment inhibits ENT1 while chronicethanol exposure decreases expression of ENT1 (Gordonet al. 1990; Nagy et al. 1990). With respect to both theA2AR/DRD2 antagonist interaction and the neuro-modulator role in adenosine, it has been suggested thatthe increase of adenosine synaptic levels could reinforcethe A2AR-signalling pathway, whereas the decrease ofadenosine synaptic levels may facilitate the DRD2 signal-ling pathway. Thus, local injection of A2AR agonistinhibits DRD2-like effects (Ferré et al. 2007) and thatinhibition could be linked to the decrease in motivation toexert efforts (Font et al. 2008). Finally, these A2AR-DRD2heterodimers, which are carried by GABA-ENK neurons(Svenningsson et al. 1999; Torvinen et al. 2004; Fuxeet al. 2005, 2007; Brown & Short 2008), may alterenkephalin levels and thus have an impact on rewardmechanisms (Gianoulakis 2009).
As suggested by the ‘reward deficiency syndrome’ (LeFoll et al. 2009), dependent rats seem to ascribe a higherhedonic value to ethanol and the dependent state couldbe associated with a disrupted mesolimbic dopaminergictonus. Moreover, DRD2 activation, which is associated torewarding properties, can be reinforced by the lack ofA2AR activation. It is noteworthy that an A2AR agonistcould elevate enkephalin levels and subsequently ele-vate hedonic threshold because of the antagonisticinteraction between A2AR and DRD2. The A2ARpathway could be targeted to attenuate motivation to self-administer ethanol since it has been demonstrated thatthe decrease of motivation during the exertion of effortscould be mediated, in part, through the A2AR pathwayin the NAc (Font et al. 2008). In addition, it has beensuggested that within dependent rats, chronic ethanolexposure induces ENT1 desensitization to the inhibitoryeffect of ethanol, and as we could expect, leads to adecrease of extracellular adenosine. Thus, low synapticadenosine levels could lead to a decrease in sated thresh-old. In the other hand, regarding the DRD2 antagonistpathway, it has been shown that antipsychotic effectscould decrease motivation to consume ethanol, butextrapyramidal and anhedonia side effects dramaticallylimit this use against ethanol craving. Consequently,during a dependent state, it could be interesting to use anA2AR agonist that is able to pharmacologically elevate
hedonic threshold to counteract excessive ethanol con-sumption without side effects.
Ethanol addiction is a complex behavioural disorder,which is difficult to characterize using animal models.However, some traits have been clearly identified in pre-clinical studies. For example, following chronic exposureto high doses of ethanol vapours, rats display increasedanxiety-like behaviour, ethanol drinking and motivationto work for ethanol during acute withdrawal (Gilpin et al.2008; Simon O’Brien et al. 2011). Excessive consump-tion is one of traits largely explored using animal modelsof the dependent state during acute withdrawal. Thisincrease in ethanol self-administration is likely to be theconsequence of both positive and negative reinforcement,elevated reward threshold and ethanol withdrawal-induced anxiety and stress (Gilpin et al. 2008). Subse-quently, we proposed the hypothesis that dependent ratscould give a higher hedonic value to ethanol during acutewithdrawal and that, consequently, CGS 21680 could bemore effective on motivation to self-administer 10%ethanol.
Interestingly, CGS 21680 (0.095 mg/kg) wasmore effective in dependent animals compared withnon-dependent ones. The fact that CGS 21680 had agreater effect on ethanol self-administration in ethanol-dependent rats during acute withdrawal suggests thatchronic intermittent ethanol exposure may inducechanges in the adenosinergic system (Gordon et al. 1990),rendering rats more sensitive to CGS 21680. The mecha-nism by which CGS 21680 modulates ethanol self-administration remains to be elucidated but it is unlikelythat it could be related to anxiety. Anxiety-like behaviourand aversive effects of ethanol have been shown to involveA1R signalling pathway in GABA-DYN (Florio et al. 1998;Steiner & Gerfen 1998; Johansson et al. 2001; Lang et al.2003; Prediger et al. 2004; Gieryk et al. 2010; Asatryanet al. 2011; Maximino et al. 2011). In addition, A1R couldbe activated under higher doses of the A2AR agonist CGS21680 (Cristalli et al. 2009). Considering the potentialcrosstalk between A1R and A2AR on pharmacologicaloutcomes, we ruled out the involvement of the A1Rpathway on the CGS 21680-induced decrease in ethanolself-administration.
To our knowledge, the current study is the first to usethe A1-KO mouse strain (Johansson et al. 2001) to assessboth genotypic differences and CGS 21680 effects on 10%ethanol operant responding. Using A1-KO, A1-HET andA1-WT mice under basal conditions, our results demon-strated that there was no genotypic difference in 10%ethanol operant self-administration after NaCl treatment.We are aware of the spontaneous ethanol preference dis-played by mice with a C57BL genetic background(Yoneyama et al. 2008). However, in our operant para-digm conditions, the Adora1 genetic difference did not
significantly affect ethanol operant self-administration.Based on these results and as expected, CGS 21680treatment equally decreased ethanol operant self-administration within all three genotypes. As alreadyreported in rats, A1R agonists and antagonists did notaffect 10% ethanol operant self-administration (Arolfoet al. 2004; Thorsell et al. 2007). However, A2AR ligandsare known for altering ethanol self-administration.Adenosine A2AR agonists and antagonists, respectively,decrease and increase ethanol self-administration(Houchi et al. 2008; Micioni Di Bonaventura et al. 2011).Moreover, it is unlikely that these observations are theconsequence of Adora2a neuroadaptation because thereare no compensatory adaptive changes in adenosinereceptors in KO mice (-/- and -/+) (Lopes et al. 2004;Yang et al. 2009). Altogether, these results indicatethat the reduction of ethanol self-administration byCGS 21680 may not involve A1R. Interestingly, non-dependent mice did not show the biphasic response withincreasing doses of CGS 21680 that we observed in non-dependent rats. One possible explanation could be thatthese mice were mated using a C57BL genetic backgrounddisplaying spontaneous ethanol preference and probablyassociated with a hypodopaminergic state (George et al.1995). Thus, we suggest that A2AR agonists could bemore effective in ethanol-preferring animals.
Taken together, our results showed that the A2ARagonist CGS 21680 decreases both 10% ethanol operantself-administration and 2% sucrose solution self-administration. Interestingly, the decreasing effect on10% ethanol operant self-administration was more effec-tive within dependent than non-dependent rats. Usingmice lacking the Adora1 gene, we clearly ruled out therole of A1R pathway on the effects CGS 21680 onethanol self-administration. Finally, with respect to theeffect size on operant task, our results confirmed ourhypothesis and showed that pharmacological activationof A2A receptors decreases motivation to consume bothalcohol and a natural reinforcer.
Acknowledgements
This study was supported by the Conseil Re?gional dePicardie (CRP), the Inter-ministerial Mission for the Fightagainst Drugs and Drug Addiction (MiLDT)-NationalInstitute of Health and Medical Research (INSERM)-Institute of Cancer (InCa) (Contracts A08095ES andA09119ES) Institut de France/Fondation NRJ ‘Bio-logy of addiction’ and IREB. RL is supported by a docto-ral fellowship from the French Ministry of Researchand Technology. We thank Ludovic Didier for his technicalassistance, Dr. Bertil Fredholm for providing A1R mutantmice and Professor Margaret Martinetti for helpful discus-sions and careful reading of the manuscript.
HH conducted behavioural tests, analyzed data andwrote the draft of the manuscript. WP participated inbehavioural tests. RL genotyped mice. MN supervisedbehavioural tests and revised the manuscript.
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