Psychopharmacology (2006) 185: 160–168 DOI 10.1007/s00213-005-0284-0
ORIGINA L IN VESTI GATION
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 A2A receptor knockout mice Received: 22 June 2005 / Accepted: 21 November 2005 / Published online: 10 February 2006 # Springer-Verlag 2006
Abstract 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 quasi-morphine withdrawal syndrome (QMWS) in opiate-naive animals. Objectives: The aim of this study was to establish the role of A2A receptors in the quasi-morphine withdrawal syndrome induced by co-administration of caffeine and naloxone and in the behavioral effects of caffeine. Methods: We have used A2A receptor knockout (A2AR−/−) mice in comparison with their wild-type and heterozygous littermates to measure locomotor activity in the open field and withdrawal symptoms induced by caffeine and naloxone. Naïve wild-type and knockout mice were also
A. Bilbao . A. Cippitelli . M. Navarro . F. Rodríguez de Fonseca Departamento de Psicobiología, Instituto Universitario de Drogodependencias, Universidad Complutense, Madrid 28223, Spain A. B. Martín . N. Granado . O. Ortiz . R. Moratalla (*) Consejo Superior de Investigaciones Científicas, Instituto Cajal, C/Doctor Arce, 37 Madrid 28002, Spain e-mail:
[email protected] Tel.: +34-915854705 A. Bilbao . A. Cippitelli . F. Rodríguez de Fonseca Fundación IMABIS, Hospital Regional Universitario Carlos Haya, Málaga 29010, Spain E. Bezard Physiologie et Physiopathologie de la Signalization celullaire, UMR-CNRS 5543, Universite Victor Segalen Bordeaux2, 33076 Bordeaux, France J.-F. Chen Department of Neurology, Boston University School of Medicine, Boston, MA 02218, USA
examined for enkephalin and dynorphin mRNA expression by in situ hybridization and for μ-opiate receptor by ligand binding autoradiography to check for possible opiate receptor changes induced by A2A receptor inactivation. Results: Caffeine increases locomotion and anxiety in wild-type animals, but it has no psychomotor effects in A2AR−/− mice. Co-administration of caffeine (20 mg/kg) and naloxone (2 mg/kg) resulted in a severe quasimorphine withdrawal syndrome in wild-type mice that was almost completely abolished in A2AR−/− mice. Heterozygous animals exhibited a 40% reduction in withdrawal symptoms, suggesting that there is no genetic/developmental compensation for the inactivation of one of the A2AR alleles. A2AR−/− and wild-type mice have similar levels of striatal μ-opioid receptors, thus the effect is not due to altered opioid receptor expression. Conclusions: Our results demonstrate that A2A receptors are required for the induction of quasi-morphine withdrawal syndrome by co-administration of caffeine and naloxone and implicate striatal A2A receptors and μ-opiate receptors in tonic inhibition of motor activity in the striatum. Keywords Caffeine . Abstinence . Abuse . Naloxone . Basal ganglia . Anxiety . Locomotor activity . Behavior . Morphine
Introduction Adenosine, a neuromodulator in the brain (Haas and Selbach 2000; Dunwiddie and Masino 2001), exerts its physiological actions through activation of a family of seven transmembrane domain, G protein-coupled receptors, including the A1, A2A, A2B, and A3 receptors (Fredholm et al. 2001; Ribeiro et al. 2003). In contrast to the widespread distribution of A1, A2B, and A3 receptors within the central nervous system, A2A receptors (A2AR) are almost exclusively localized in the striatum and olfactory tubercle (Johansson et al. 1997; Moreau and Huber 1999; Rebola et al. 2005).
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The striatum is the main component of the motor circuit of the basal ganglia, processing information received from the primary somatosensory and motor cortices to allow sequencing of motor behaviors (Hauber 1998) and changes in gene expression (Yano and Steiner 2005). The selective distribution of A2ARs in the region strongly suggests a role for these receptors in the control of motor behaviors (Kase 2001). This is supported by the finding that the A2AR agonist CGS 21680 induces catalepsy in rats and A2AR antagonists ameliorate motor deficits in several rodent behavioral models (Ferré et al. 1997; Fredduzzi et al. 2002). A2ARs are co-localized with dopamine D2 receptors on striatopallidal neurons (Johansson et al. 1997), and adenosine 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 altered motor functions, thus adenosine could play a key role in the control of movements through its effects on striatal dopamine transmission. As an example, inactivation of adenosine A2ARs results in hypo-dopaminergic activity in striatum (Dassesse et al. 2001) and attenuates behavioral responses induced by psychostimulants (Chen et al. 2000, 2003). It is thought that these antagonistic interactions between adenosine and dopamine receptors in the striatum are at least in part responsible for the motor-stimulant effects of adenosine receptor antagonists (Moreau and Huber 1999). Caffeine antagonizes A1 and A2A adenosine receptor subtypes, facilitating dopaminergic neurotransmission and thereby inducing hyper-locomotion (Ferré et al. 1997; Chen et al. 2001). However, this effect is biphasic: caffeine is 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 in the modulation of behaviors associated with drug dependence. Adenosine, via A2A receptors, modulates behaviors associated 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 opiatenaïve rats, administration of a methylxanthine followed by a small dose of naloxone induces a state of behavioral excitation closely resembling the characteristic withdrawal syndrome precipitated by naloxone in morphine-dependent rats (Collier et al. 1974; Butt et al. 1979; Cowan 1981). The behavioral, pharmacological, and neurochemical responses to methylxanthines followed by naloxone are so similar to morphine withdrawal syndrome that this phenomenon has been termed quasi-morphine withdrawal syndrome (QMWS), and it has been suggested that these syndromes share common neural substrates. However, this hypothesis has not been validated experimentally. In the present study, we characterized the effects of caffeine and caffeine-induced QMWS in A2A receptor knockout (A2AR−/−) and hetero-
zygous (A2AR+/−) mice to determine the role of A2A receptors in QMWS.
Materials and methods Animals Adult male wild-type, heterozygous A2AR−/−, and A2AR+/− mice weighting 25–30 g were used throughout. A2AR−/− mice were generated as described (Chen et al. 1999). The genotype of each mouse was determined by genomic Southern blot analysis as described (Chen et al. 1999). All animals used in a given experiment originated from the same 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)- and humidity-controlled room on a 12-h light–dark schedule with food and water provided ad libitum. The maintenance of the animals, as well as the experimental procedures, followed guidelines from European Union Council Directive 86/609/EEC. All efforts were made to minimize the number of animals used and their suffering. The experimental protocols involving animals were approved by the CSIC ethic committee. Drugs Naloxone hydrochloride and caffeine were obtained from Sigma Chemicals (St. Louis, MO, USA). Both drugs were dissolved in sterile 0.9% sodium chloride solution and injected in a volume of 0.1 ml/10 g body weight. Behavioral studies As psychostimulants, such as caffeine, generally increase psychomotor activity, mice were studied after habituation to the handling procedures, to the observational arenas, and the open field during the light phase of the light–dark cycle to obtain low baseline activity (Fernández-González et al. 2004). Studies performed included open field test and quasi-morphine withdrawal syndrome. Open field test This test was used to assess the animal’s exploratory and locomotor activity and drug-induced arousal/anxiety as previously described (Fernández-González et al. 2004). Experiments were conducted between 9:00 and 12:00 hours. Mice were moved into the behavioral testing room at least 1 h prior to testing. The open field consisted of a 40×40-cm arena divided into 25 squares by lines drawn on the floor of the apparatus. The nine squares not bounded by the walls of the test were referred to as center squares. Mice were habituated for 20 min to the open field, 24 h before behavioral observation. Each mouse was placed into the central square of the arena and allowed to freely explore for 20 min. The following parameters were recorded during the
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20-min session: number of squares crossed, number of center squares entered, time in the center of the field, and immobility time. The apparatus was cleaned between tests with 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 equipped with a camera (Smart, Panlab S.A., Barcelona, Spain). To evaluate the dose response to caffeine in the three genotypes of mice, three different caffeine doses were selected (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 A2A gene and to select a dose to be used in the quasi-morphine withdrawal experiment. Caffeine was administered subcutaneously (s.c.) and its effects were evaluated for a 20min observation period in the open field. Quasi-morphine withdrawal syndrome (QMWS) On the day of the experiment, mice were injected with 20 mg/kg of 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 and withdrawal signs were assessed for 30 min immediately following naloxone administration. Behavioral assessment of withdrawal syndrome was based on the procedures of Collier et al. (1974). The global withdrawal score was expressed as the percentage of the incidence of each sign following naloxone challenge (Collier et al. 1981). The signs were divided into two categories: quantitative (expressed as the frequency) and qualitative (expressed as the presence or absence). The frequency of jumps, abdominal contractions, facial rubbing, paw fluttering, and wet dog shakes were measured. The presence or absence of other behavioral signs was noted, such as teeth chattering, swallowing, ptosis, genital grooming, body tremors, and abnormal posture (Berrendero et al. 2003; Bailey et al. 2004).
cific binding was determined by the addition of unlabelled naloxone, 1 μM, 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 a direct measurement of numeric images obtained from actual counting of emitted β-particles, therefore obtaining real numbers. Data are expressed as counts per minute per square micrometer (cpm/μm2). The radioactivity was assessed for striatal areas at the mid rostrocaudal levels (AP 0.7 from Paxinos and Franklin 2004), dividing the striatum 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 an examiner blinded with regard to the experimental condition. 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 35S-labeled cRNA probe complementary to rat preproenkephalin (Enk) cDNA (plasmid provided by Dr. Sabol, Laboratory of Biochemical Genetic, NIH, Bethesda, MD) and 600-bp 35S-labeled cRNA probe complementary to rat prodynorphin (Dyn) cDNA (plasmid provided by Dr. Douglass, Vollum Institute, Portland, OR, USA). The radioactive cRNA probe for Enk was synthesized as in Moratalla et al. (1993) with a Promega labelling reaction kit (Madison, WI, USA). As in (Julián et al. 2003), 1 μg of the appropriate template was reacted with 350 μCi of a 35S-CTP (1,000 Ci/mmol, DuPont, NEN, Boston, MA, USA), together with 50 μM of a mix of unlabeled CTP, 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 RNase inhibitor provided with the kit), and stored at −80°C until use.
Biochemical studies Tissue preparation for receptor autoradiography and for in situ hybridization Adult male wild-type and A2AR−/− mice were decapitated and their brains were rapidly removed and placed in powdered dry ice until frozen. Transverse sections were cut at 12 μm on a cryostat, thaw mounted onto gelatine-coated slides, air dried, and stored at − 80°C until use. Opiate receptor autoradiography Opiate receptor binding was performed as indicated by Moratalla et al. (1992). Briefly, sections were incubated for 60 min at 4°C in 50 mM Tris–HCl buffer containing 2.5 nM [3H]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-
In situ hybridization For in situ hybridization, radioactively labelled probes, diluted in the hybridization solution, were applied to selected sections and hybridized for 12 h at 50°C in a humid 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 and then treated with RNase A, for 30 min at 37°C, rinsed for 15 min at room temperature, and washed twice for 30 min with 0.1×SSC at 60°C. Slides were then dehydrated in 70, 80, 95, and 100% ethanol for 2 min each. After drying overnight, slides were exposed to Hyperfilm βMax (Amersham Pharmacia Biotech, Barcelona, Spain). Films were subsequently developed and analyzed with the NIH Image Analyzer program (available on the Internet at http:// rsb.info.nih.gov/nih-image). Optical density measurements were made in the caudoputamen, which was divided for
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this purpose into four quadrants, Dm, Dl, Vm, and Vl. Measurements from 12 sections per animal, taken at middle rostrocaudal levels of the striatum, were pooled. Values from autoradiographic standards on the films were used to construct a correlation curve to allow conversion of optical measurement to activity values in microcurie per gram (μCi/g) of tissue±SEM. Values from right and left hemispheres were also pooled. Statistical comparison between values obtained from WT and A2AR−/− mice were carried out by one-way ANOVA and by Student t test. Statistics Behavioural data were analyzed using two-way analysis of variance (ANOVA) followed by a Student–Newman– Keuls post hoc test. Data for in situ hybridization studies were quantified with a computer-assisted program (Scion Image, NIH, USA). Binding and in situ hybridization data were analyzed using the Student t test. All data were normally distributed, and significance levels of t-test comparisons were adjusted for inequality of variances when appropriate. These analyses were completed using the STATA program (Intercooled Stata 6.0, Stata Corporation, College Station, TX, USA). A probability level of 5% (p
