Absence of quasi-morphine withdrawal syndrome in adenosine A 2A receptor knockout mice

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Rationale Caffeine and other methylxanthines induce behavioral activation and anxiety responses in mice via antagonist action at A2A adenosine receptors. When combined with the opioid antagonist naloxone, methylxanthines produce a characteristic
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  Psychopharmacology (2006) 185: 160  –  168DOI 10.1007/s00213-005-0284-0 ORIGINAL INVESTIGATION Ainhoa Bilbao .Andrea Cippitelli .Ana B. Martín .Noelia Granado .Oscar Ortiz .Erwan Bezard .Jiang-Fan Chen .Miguel Navarro .Fernando Rodríguez de Fonseca .Rosario Moratalla  Absence of quasi-morphine withdrawal syndrome in adenosine A  2A   receptor knockout mice Received: 22 June 2005 / Accepted: 21 November 2005 / Published online: 10 February 2006 # Springer-Verlag 2006 Abstract  Rationale:  Caffeine and other methylxanthinesinduce behavioral activation and anxiety responses inmice via antagonist action at A 2A  adenosine receptors.When combined with the opioid antagonist naloxone,methylxanthines produce a characteristic quasi-morphinewithdrawal syndrome (QMWS) in opiate-naive animals. Objectives:  The aim of this study was to establish the roleof A 2A  receptors in the quasi-morphine withdrawalsyndrome induced by co-administration of caffeine andnaloxone and in the behavioral effects of caffeine.  Methods:  We have used A 2A  receptor knockout (A 2A R  − /  − )mice in comparison with their wild-type and heterozygouslittermates to measure locomotor activity in the open fieldand withdrawal symptoms induced by caffeine and nalox-one. Naïve wild-type and knockout mice were alsoexamined for enkephalin and dynorphin mRNA expression by in situ hybridization and for  μ  -opiate receptor by ligand binding autoradiography to check for possible opiatereceptor changes induced by A 2A  receptor inactivation.  Results:  Caffeine increases locomotion and anxiety inwild-type animals, but it has no psychomotor effects inA 2A R  − /  − mice. Co-administration of caffeine (20 mg/kg)and naloxone (2 mg/kg) resulted in a severe quasi-morphine withdrawal syndrome in wild-type mice that was almost completely abolished in A 2A R  − /  − mice.Heterozygous animals exhibited a 40% reduction inwithdrawal symptoms, suggesting that there is no genet-ic/developmental compensation for the inactivation of oneof the A 2A R alleles. A 2A R  − /  − and wild-type mice havesimilar levels of striatal  μ  -opioid receptors, thus the effect is not due to altered opioid receptor expression. Conclusions:  Our results demonstrate that A 2A  receptorsare required for the induction of quasi-morphine with-drawal syndrome by co-administration of caffeine andnaloxone and implicate striatal A 2A  receptors and  μ  -opiatereceptors in tonic inhibition of motor activity in thestriatum. Keywords  Caffeine .Abstinence .Abuse . Naloxone .Basal ganglia .Anxiety .Locomotor activity .Behavior  .Morphine Introduction Adenosine, a neuromodulator in the brain (Haas andSelbach 2000; Dunwiddie and Masino 2001), exerts its  physiological actions through activation of a family of seven transmembrane domain, G protein-coupled recep-tors, including the A 1 , A 2A , A 2B , and A 3  receptors(Fredholm et al. 2001; Ribeiro et al. 2003). In contrast to the widespread distribution of A 1 , A 2B , and A 3  receptorswithin the central nervous system, A 2A  receptors (A 2A R)are almost exclusively localized in the striatum andolfactory tubercle (Johansson et al. 1997; Moreau andHuber  1999; Rebola et al. 2005). A. Bilbao .A. Cippitelli .M. Navarro .F. Rodríguez de FonsecaDepartamento de Psicobiología,Instituto Universitario de Drogodependencias,Universidad Complutense,Madrid 28223, SpainA. B. Martín . N. Granado .O. Ortiz .R. Moratalla ( * )Consejo Superior de Investigaciones Científicas,Instituto Cajal,C/Doctor Arce, 37Madrid 28002, Spaine-mail: moratalla@cajal.csic.esTel.: +34-915854705 A. Bilbao .A. Cippitelli .F. Rodríguez de FonsecaFundación IMABIS,Hospital Regional Universitario Carlos Haya,Málaga 29010, SpainE. BezardPhysiologie et Physiopathologie de la Signalization celullaire,UMR-CNRS 5543, Universite Victor Segalen Bordeaux2,33076 Bordeaux, FranceJ.-F. ChenDepartment of Neurology,Boston University School of Medicine,Boston, MA 02218, USA  The striatum is the main component of the motor circuit of the basal ganglia, processing information received fromthe primary somatosensory and motor cortices to allowsequencing of motor behaviors (Hauber  1998) and changesin gene expression (Yano and Steiner  2005). The selectivedistribution of A 2A Rs in the region strongly suggests a rolefor these receptors in the control of motor behaviors (Kase2001). This is supported by the finding that the A 2A R agonist CGS 21680 induces catalepsy in rats and A 2A R antagonists ameliorate motor deficits in several rodent  behavioralmodels(Ferréetal.1997;Fredduzzietal.2002). A 2A Rs are co-localized with dopamine D 2  receptors onstriatopallidal neurons (Johansson et al. 1997), and aden-osine and dopamine can influence each other  ’ s function(Ferré et al. 1997; Chen et al. 2001). Loss of dopaminergic regulation of striatal neuronal activity results in alteredmotor functions, thus adenosine could play a key role in thecontrol of movements through its effects on striataldopamine transmission. As an example, inactivation of adenosine A 2A Rs results in hypo-dopaminergic activity instriatum (Dassesse et al. 2001) and attenuates behavioralresponses induced by psychostimulants (Chen et al. 2000,2003). It is thought that these antagonistic interactions between adenosine and dopamine receptors in the striatumare at least in part responsible for the motor-stimulant effects of adenosine receptor antagonists (Moreau andHuber  1999).Caffeine antagonizes A 1  and A 2A  adenosine receptor subtypes, facilitating dopaminergic neurotransmission andthereby inducing hyper-locomotion (Ferré et al. 1997;Chen et al. 2001). However, this effect is biphasic: caffeineis stimulatory at low to moderate concentrations but  becomes a motor depressant at higher concentrations(Daly and Fredholm 1998; Hauber  1998; Kase 2001; Halldner et al. 2004). Caffeine also exhibits anxiogenic properties in humans and anxiogenic-like effects in rodent models of phobic anxiety (Moreau and Huber  1999).Adenosine and its receptors seem to play a pivotal role inthe modulation of behaviors associated with drug depen-dence. Adenosine, via A 2A  receptors, modulates behaviorsassociated with acute and chronic exposure to opiates(Kaplan and Sears 1996; Capasso 2000; Berrendero et al. 2003; Bailey et al. 2004), cannabinoids (Soria et al. 2004), and psychostimulants (Chen et al. 2000, 2003). In opiate- naïve rats, administration of a methylxanthine followed bya small dose of naloxone induces a state of behavioralexcitation closely resembling the characteristic withdrawalsyndrome precipitated by naloxone in morphine-dependent rats (Collier et al. 1974; Butt et al. 1979; Cowan 1981). The  behavioral, pharmacological, and neurochemical responsesto methylxanthines followed by naloxone are so similar tomorphine withdrawal syndrome that this phenomenon has beentermedquasi-morphinewithdrawalsyndrome(QMWS),and it has been suggested that these syndromes sharecommon neural substrates. However, this hypothesis hasnot been validated experimentally. In the present study, wecharacterized the effects of caffeine and caffeine-inducedQMWS in A 2A  receptor knockout (A 2A R  − /  − ) and hetero-zygous (A 2A R  +/  − ) mice to determine the role of A 2A receptors in QMWS. Materials and methods AnimalsAdult male wild-type, heterozygous A 2A R  − /  − , and A 2A R  +/  − mice weighting 25  –  30 g were used throughout. A 2A R  − /  − mice were generated as described (Chen et al. 1999). Thegenotype of each mouse was determined by genomicSouthern blot analysis as described (Chen et al. 1999). Allanimals used in a given experiment srcinated from thesame breeding series and were matched for age and weight.Mice were housed in groups of four to five per cage in clear  plastic cages and maintained in a temperature (22°C)- andhumidity-controlled room on a 12-h light   –  dark schedulewith food and water provided ad libitum. The maintenanceof the animals, as well as the experimental procedures,followed guidelines from European Union Council Direc-tive 86/609/EEC. All efforts were made to minimize thenumber of animals used and their suffering. The experi-mental protocols involving animals were approved by theCSIC ethic committee.Drugs Naloxone hydrochloride and caffeine were obtained fromSigma Chemicals (St. Louis, MO, USA). Both drugs weredissolved in sterile 0.9% sodium chloride solution andinjected in a volume of 0.1 ml/10 g body weight.Behavioral studiesAs psychostimulants, such as caffeine, generally increase psychomotor activity, mice were studied after habituationto the handling procedures, to the observational arenas, andthe open field during the light phase of the light   –  dark cycleto obtain low baseline activity (Fernández-González et al.2004). Studies performed included open field test andquasi-morphine withdrawal syndrome. Open field test   This test was used to assess the animal ’ sexploratory and locomotor activity and drug-inducedarousal/anxiety as previously described (Fernández-Gonzálezetal.2004).Experimentswereconductedbetween9:00and12:00 hours. Mice were moved into the behavioral testingroomatleast1hpriortotesting. Theopenfield consistedof a40×40-cmarenadividedinto25squaresbylinesdrawnonthe floor of the apparatus. The nine squares not bounded bythe walls of the test were referred to as center squares. Micewere habituated for 20 min to the open field, 24 h before behavioral observation. Each mouse was placed into thecentral square of the arena and allowed to freely explore for 20 min. Thefollowing parameters were recorded during the 161  20-min session: number of squares crossed, number of center squares entered, time in the center of the field, andimmobility time. The apparatus was cleaned between testswith a weak acetic acid solution. Illumination of the test room was the same as the mouse colony room (100 lux).Mice were monitored by a video tracking system equippedwith a camera (Smart, Panlab S.A., Barcelona, Spain).To evaluate the dose response to caffeine in the threegenotypes of mice, three different caffeine doses wereselected (vehicle and 20, 50, and 100 mg/kg of caffeine).The purpose of this experiment was to compare the behavioral effects of caffeine after deletion of A 2A  geneand to select a dose to be used in the quasi-morphinewithdrawal experiment. Caffeine was administered sub-cutaneously (s.c.) and its effects were evaluated for a 20-min observation period in the open field. Quasi-morphine withdrawal syndrome (QMWS)  On theday of the experiment, mice were injected with 20 mg/kgof caffeine followed 10 min later by injection of naloxone(2 mg/kg, i.p.), as previously described (Navarro et al.1991). Mice were placed into clear observation boxes andwithdrawal signs were assessed for 30 min immediatelyfollowing naloxone administration. Behavioral assessment of withdrawal syndrome was based on the procedures of Collier et al. (1974). The global withdrawal score wasexpressed as the percentage of the incidence of each signfollowing naloxone challenge (Collier et al. 1981). Thesigns were divided into two categories: quantitative(expressed as the frequency) and qualitative (expressedas the presence or absence). The frequency of jumps,abdominal contractions, facial rubbing, paw fluttering, andwet dog shakes were measured. The presence or absenceof other behavioral signs was noted, such as teethchattering, swallowing, ptosis, genital grooming, bodytremors, and abnormal posture (Berrendero et al. 2003;Bailey et al. 2004).Biochemical studies Tissue preparation for receptor autoradiographyand for in situ hybridization Adult male wild-type and A 2A R  − /  − mice were decapitatedand their brains were rapidly removed and placed in powdered dry ice until frozen. Transverse sections were cut at 12 μ  m on a cryostat, thaw mounted onto gelatine-coatedslides, air dried, and stored at   −  80°C until use. Opiate receptor autoradiography Opiate receptor binding was performed as indicated byMoratalla et al. (1992). Briefly, sections were incubated for 60 min at 4°C in 50 mM Tris  –  HCl buffer containing2.5 nM [ 3 H]naloxone (Du Pont/NEN, 30.5 Ci/mmol).Sections were washed three times in 50 mM PBS for 20 s at 4°C, briefly rinsed in distilled water, and dried. Nonspe-cific binding was determined by the addition of unlabellednaloxone, 1  μ  M, to the incubation solution. Slices were placed in the gas chamber of the  β -imager (BioSpace,France), which provides an accurate linear detection of counts per minute (cpm). The signal was quantified by adirect measurement of numeric images obtained fromactual counting of emitted  β -particles, therefore obtainingreal numbers. Data are expressed as counts per minute per square micrometer (cpm/  μ  m 2 ). The radioactivity wasassessed for striatal areas at the mid rostrocaudal levels(AP 0.7 from Paxinos and Franklin 2004), dividing thestriatum in four quadrants: dorsomedial (Dm), dorsolateral(Dl), ventromedial (Vm), and ventrolateral (Vl) (Rivera et al. 2002; Grande et al. 2004). Three animals per group, with four sections per animal were analyzed by anexaminer blinded with regard to the experimental condi-tion. Details for these methods have been previously published (Bailey et al. 2002; Pioli et al. 2004).  In situ hybridization: riboprobe synthesis and labeling  We used a 492-bp  35 S-labeled cRNA probe complementaryto rat preproenkephalin (Enk) cDNA (plasmid provided byDr. Sabol, Laboratory of Biochemical Genetic, NIH,Bethesda, MD) and 600-bp  35 S-labeled cRNA probecomplementary to rat prodynorphin (Dyn) cDNA (plasmid provided by Dr. Douglass, Vollum Institute, Portland, OR,USA). The radioactive cRNA probe for Enk was synthe-sized as in Moratalla et al. (1993) with a Promega labellingreaction kit (Madison, WI, USA). As in (Julián et al. 2003),1  μ  g of the appropriate template was reacted with 350  μ  Ciof a  35 S-CTP (1,000 Ci/mmol, DuPont, NEN, Boston, MA,USA),togetherwith50 μ  MofamixofunlabeledCTP,ATP,GTP, and UTP. Riboprobes were purified by ethanol precipitation, resuspended in TE buffer (10 mM Tris  –  HCl,1 mM EDTA, pH 7.6) with 40 units of RNasin (an RNaseinhibitor provided with the kit), and stored at   − 80°C untiluse.  In situ hybridization For in situ hybridization, radioactively labelled probes,diluted in the hybridization solution, were applied toselected sections and hybridized for 12 h at 50°C in ahumid chamber (Pavón et al. 2005). After hybridization,slides were rinsed in 2×SSC (saline sodium citrate)solution and then in 0.1×SSC at 60°C for 30 min andthen treated with RNase A, for 30 min at 37°C, rinsed for 15 min at room temperature, and washed twice for 30 minwith 0.1×SSC at 60°C. Slides were then dehydrated in 70,80, 95, and 100% ethanol for 2 min each. After dryingovernight, slides were exposed to Hyperfilm  β Max(Amersham Pharmacia Biotech, Barcelona, Spain). Filmswere subsequently developed and analyzed with the NIHImage Analyzer program (available on the Internet at  http:// rsb.info.nih.gov/nih-image). Optical density measurementswere made in the caudoputamen, which was divided for  162  this purpose into four quadrants, Dm, Dl, Vm, and Vl.Measurements from 12 sections per animal, taken at middlerostrocaudal levels of the striatum, were pooled. Valuesfrom autoradiographic standards on the films were used toconstruct a correlation curve to allow conversion of opticalmeasurement to activity values in microcurie per gram( μ  Ci/g) of tissue±SEM. Values from right and left hemi-spheres were also pooled. Statistical comparison betweenvalues obtained from WT and A 2A R  − /  − mice were carriedout by one-way ANOVA and by Student   t   test.StatisticsBehavioural data were analyzed using two-way analysis of variance (ANOVA) followed by a Student   –   Newman  –  Keuls post hoc test. Data for in situ hybridization studieswere quantified with a computer-assisted program (ScionImage, NIH, USA). Binding and in situ hybridization datawere analyzed using the Student   t   test. All data werenormally distributed, and significance levels of   t  -test comparisons were adjusted for inequality of varianceswhen appropriate. These analyses were completed usingthe STATA program (Intercooled Stata 6.0, Stata Corpora-tion, College Station, TX, USA). A probability level of 5%(  p <0.05) was considered significant. Results A 2A R depletion decreases basal locomotor activityin open field test Twenty-four hours after habituation, A 2A  adenosine recep-tor knockout mice (A 2A R  − /  − ) showed decreased locomotor activity and reduced exploration of the center of the fieldcompared with their wild-type littermates (Fig. 1a,b).Heterozygous mice (A 2A R  − /+ ) exhibited a significant decrease in locomotor activity similar to that seen in their A 2A R  − /  − littermates, while their exploration of the openfield was intermediate between that of A 2A R  − /  − and wild-type littermates and significantly different from both.These results are similar to those described previously in adifferent strain of A 2A R  − /  − animals (Ledent et al. 1997).Dose-dependent effects of caffeine on motor and exploratory activitiesAs expected, caffeine induced an increase in locomotion inwild-type animals. The maximal stimulatory effect wasseen at doses of 20 and 50 mg/kg. The highest dose,100 mg/kg, produced increased locomotion compared withuntreated animals, but this dose of caffeine was signifi-cantly less stimulatory than the lower doses (Fig. 2a).Caffeine also increased locomotion in A 2A R  +/  − littermates, but its stimulatory effects on locomotion were significantlysmaller than in wild-type animals. A 2A R  − /  − mice showedno increase in locomotion in response to caffeine (Fig. 2a).However, when exploratory behavior was examined fol-lowing administration of 20 mg/kg caffeine, wild-type,heterozygous, and homozygous knockout animals all ex-hibited a similar decrease in exploratory activity (Fig. 2 b).Quasi-morphine withdrawal syndrome (QMWS)is dramatically reduced in A 2A R  − /  − miceWild-type mice injected with caffeine (20 mg/kg) followed10 min later by an injection of naloxone (2 mg per kilogram, i.p.) exhibited quasi-morphine withdrawal syn-drome, characterized by escape attempts, wet dog shakes,facial rubbings, abnormal posture, ptosis and swallowingmovements, as well as by other less frequent behaviors(Table 1 and Fig. 3). A 2A R  +/  − mice exhibited a 40% Fig. 1  Motor activity and exploratory behavior in wild-type,heterozygous, and A 2A  receptor knockout mice. Locomotor activity( a ) and exploration of the center of the open field ( b ) for threedifferent A 2A  adenosine receptor genotypes: wild-type (+/+),heterozygous (+/  − ), and homozygous knockout ( − /  − ) mice.Twenty-four hours after habituation to the open field arena, behavior was scored for 20 min. Data are means±SEM of at least 11determinations per group.  Asterisk   (*) indicates  p <0.01 vs wild-typegroup, and # indicates  p <0.01 vs homozygous knockout mice group(by Newman  –  Keuls test)163  reduction in global withdrawal score, while withdrawalscores for A 2A R  − /  − mice were reduced by 80%. In thecomplete absence of A 2A  receptors, most abnormal motor responses associated with the administration of caffeineand naloxone were nearly or completely abolished,including abnormal posture, tremor, teeth chattering, wet dog shakes, facial rubbing, and paw fluttering. However,mice heterozygous for the A 2A  adenosine receptor displayed a mixed response when individual behaviorswere evaluated. Jumps were nearly completely abolished inthe heterozygous animals, while wet dog shakes were partially reduced, and facial rubbing and paw flutteringwere unaffected (Fig. 3 b). Neither saline nor naloxonealone resulted in any behavioral signs related to QMWS(data not shown). Fig. 2  Absence of caffeine-induced motor activity and exploratory behavior in A 2A  knockout mice.  a  Effects of caffeine (20, 50, and100 mg/kg) on locomotor activity in the open-field arena for threedifferent A 2A  adenosine receptor genotypes: wild type (+/+), hetero-zygous (+/  − ), and homozygous knockout ( − /  − ) mice. Animals weretested for20min after asingle subcutaneous injection of caffeine.Dataare means±SEM of at least ten determinations per group. A Newman  –  Keuls test was used to determine statistical significance.  Asterisk   (*)indicates  p <0.01 vs vehicle-treated group; # indicates  p <0.01 vscaffeine-treated wild-type mice for each dose.  b  Effects of caffeine onthe exploratory activity in the center of the open field.  Asterisk   (*)indicates  p <0.01 vs vehicle-treated group; # indicates  p <0.01 vsvehicle-treated wild-type (by Newman  –  Keuls test) Fig. 3  Absence of quasi-morphine withdrawal syndrome in aden-osine A 2A  knockout mice. Behavioral signs for quasi-morphinewithdrawal syndrome were evaluated in three different A 2A adenosine receptor genotypes: wild type (+/+), heterozygous (+/  − ),and homozygous knockout ( − /  − ) mice. Animals were treated with asingle dose of caffeine (20 mg/kg) and, 10 min later, QMWS wasinduced by naloxone. Behavioral withdrawal signs were evaluatedfor a period of 30 min after naloxone injection. Data are means±SEM of at least ten determinations per group.  a  Global QMWSscore.  b  Quantification of behavior signs for QMWS: jumps, wet dog shakes ( WDS  ), abdominal constrictions (  ABD CON  ), facialrubbings (  FAC RUB ), and paw fluttering (  PAW FLU  ). *  p <0.01 vswild-type mice for A and B (Newman  –  Keuls test) Table 1  Percentage of animals exhibiting selected quasi-morphinewithdrawal symptoms after a sequential injection of caffeine (20 mg/ kg) and naloxone (2 mg/kg)A 2A R  +/+ A 2A R  +/  − A 2A R  − /  − Abnormal posture 100 100 0Body tremor 33.3 0 0Genital grooming 26.6 9.1 14.3Ptosis 86.6 72.7 0Swallowing 60 54.5 14.4Teeth chattering 33.3 9.1 0Values indicate percentage over total number of animals tested(A 2A R  +/+ ,  n =15; A 2A R  +/  − ,  n =11; and A 2A R  − /  − ,  n =14)164
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