The role of the D2 dopamine receptor (D2R) in A2A adenosine receptor (A2AR)-mediated behavioral and cellular responses as revealed by A2A and D2 receptor knockout mice Jiang-Fan Chen*†, Rosario Moratalla‡, Francesco Impagnatiello§, David K. Grandy¶, Beatriz Cuellar‡, Marcelo Rubinsteinʈ, Mark A. Beilstein*, Elizabeth Hackett*, J. Stephen Fink*,**, Malcolm J. Low††, Ennio Ongini§, and Michael A. Schwarzschild* *Molecular Neurobiology Laboratory, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129; ‡Cajal Institute, Madrid, 28002 Spain; §Schering-Plough Research Institute, Milan 20132, Italy; ††Vollum Institute, ¶Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, OR 97201; ʈINGEBI, CONICET, and Department of Biological Science, University of Buenos Aires, Vuelta de Obligado 2490, Buenos Aires 1428, Argentina Edited by John W. Daly, National Institutes of Health, Bethesda, MD, and approved November 21, 2000 (received for review August 3, 2000) The A2AR is largely coexpressed with D2Rs and enkephalin mRNA in and APEC inhibit the psychomotor effects induced by dopamine the striatum where it modulates dopaminergic activity. Activation of agonists (9, 10). This antagonism between A2A and D2 receptors is the A2AR antagonizes D2R-mediated behavioral and neurochemical further supported by the neurochemical demonstration that acti- effects in the basal ganglia through a mechanism that may involve vation of the A2AR antagonizes the D2R agonist-mediated inhibi- direct A2AR–D2R interaction. However, whether the D2R is required tion of acetylcholine release in the striatum (11, 12) and ␥-ami- for the A2AR to exert its neural function is an open question. In this nobutyric acid (GABA) release in the striatum and globus pallidus study, we examined the role of D Rs in A R-induced behavioral and 2 2A (13), and potentiates D2R antagonist-induced expression of the cellular responses, by using genetic knockout (KO) models (mice immediate early gene c-fos in striatum (1, 3, 14). deficient in A Rs or D Rs or both). Behavioral analysis shows that the 2A 2 The antagonistic interaction between A2A and D2 receptors ؅ A2AR agonist 2–4-(2-carboxyethyl)phenethylamino-5 -N-ethylcarbox- has been explained by a model of receptor–receptor interaction, amidoadenosine reduced spontaneous as well as amphetamine- i.e., postsynaptic inhibition of D2Rs by A2ARs in striatum (15). induced locomotion in both D2 KO and wild-type mice. Conversely, This model is based not only on the colocalization of A2ARs and the nonselective adenosine antagonist caffeine and the A2AR antag- D2Rs in striatopallidal neurons, but also on pharmacological onist 8-(3-chlorostyryl)caffeine produced motor stimulation in mice findings that some psychomotor effects of adenosine agonists lacking the D2R, although the stimulation was significantly attentu- and antagonists depend on an intact nigrostriatal dopaminergic ated. At the cellular level, A2AR inactivation counteracted the increase system (1). In addition, neurochemical studies have shown that in enkephalin expression in striatopallidal neurons caused by D2R activation of A2ARs reduces the binding affinity of D2 agonists deficiency. Consistent with the D KO phenotype, A R inactivation 2 2A to their receptors. This A –D receptor–receptor interaction partially reversed both acute D R antagonist (haloperidol)-induced 2A 2 2 has been demonstrated in striatal membrane preparations of rats catalepsy and chronic haloperidol-induced enkephalin mRNA expres- (16) as well as in fibroblast cell lines after cotransfection with sion. Together, these results demonstrate that A Rs elicit behavioral 2A A R and D R cDNAs (17, 18). In agreement with an intramem- and cellular responses despite either the genetic deficiency or phar- 2A 2 brane interaction, A –D receptor interactions have been dem- macological blockade of D Rs. Thus, A R-mediated neural functions 2A 2 2 2A onstrated in membrane preparations without ATP addition and are partially independent of D Rs. Moreover, endogenous adenosine 2 in transfected cell lines without cotransfection of adenylyl cy- acting at striatal A2ARs may be most accurately viewed as a facilitative modulator of striatal neuronal activity rather than simply as an clase (1, 15). Furthermore, A2AR-mediated direct inhibition of D2Rs also has been suggested to contribute to A2AR modulation inhibitory modulator of D2R neurotransmission. of GABA release in the striatum and globus pallidus (6). However, the direct receptor–receptor antagonistic model does 2ARs are highly concentrated in the basal ganglia where they not adequately explain recent findings that activation of the A2AR Amodulate dopaminergic activity (1–3). Within the striatum, exerts a tonic excitatory effect on c-fos expression in dopamine- A2AR mRNA is largely coexpressed with D2R as well as enkephalin depleted animals and on D R antagonist-(haloperidol)-induced mRNA in striatopallidal neurons (4, 5) (although the expression of 2 phosphorylation of dopamine- and cAMP-regulated phosphopro- A R mRNA also has been detected in striatal cholinergic inter- 2A tein of 32 kDa (DARPP-32) in striatum (19). For example, the neurons; ref. 6). For example, in situ hybridization studies reveal A2AR agonist CGS21680 induced c-fos expression in the 6-hydroxy- that 93% of D2R mRNA-bearing cells contain A2AR mRNA, and 95% of A2AR mRNA-bearing cells have D2R mRNA in striatum (4, 5). This colocalization of A2AR and D2R mRNAs suggests that the This paper was submitted directly (Track II) to the PNAS office. striatal efferent system is an important site for the integration of Abbreviations: A2AR, A2A adenosine receptor; CGS21680, 2–4-(2-carboxyethyl)phenethyl- adenosine and dopamine signaling in brain. Indeed, behavioral amino-5Ј-N-ethylcarboxamidoadenosine; CSC, 8-(3-chlorostyryl)caffeine; D2R, D2 dopa- analyses show that the nonselective adenosine antagonists caffeine mine receptor; DARPP-32, dopamine- and cAMP-regulated phosphoprotein of 32 kDa; DPCPX, 8-cyclopentyl-1,3-dipropylxanthyne; KO, knockout; PD, Parkinson’s disease; WT, and theophylline as well as the more selective A2AR antagonists wild type; GABA, ␥-aminobutyric acid. SCH58261 {7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3- †To whom reprint requests should be addressed. E-mail: [email protected]. c]-1,2,4-triazolo-[1,5,-c]-pyrimidine} and KW6002 [(E)-1,3-diethyl- **Present address: Department of Neurology, Boston University School of Medicine, 8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6- Boston, MA 02118. dione] potentiate dopamine-mediated psychomotor stimulant The publication costs of this article were defrayed in part by page charge payment. This effects (2, 7, 8) whereas the A2AR agonists 2–4-(2-carboxyethyl)- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. phenethylamino-5Ј-N-ethylcarboxamidoadenosine (CGS21680) §1734 solely to indicate this fact. 1970–1975 ͉ PNAS ͉ February 13, 2001 ͉ vol. 98 ͉ no. 4 dopamine (6-OHDA)-lesioned striatum, but failed (at doses up to were habituated to the testing environment and basal spontaneous 50-fold higher) to stimulate c-fos expression in normal striatum locomotion was recorded for 120 min. Mice were monitored during ͞ (20). Also, the A2AR antagonist 8-(3-chlorostyryl)caffeine (CSC) the light phase of the light dark cycle to obtain low baseline has been shown to inhibit D2R antagonist-induced c-Fos immuno- locomotor activity in the studies with A2AR antagonists and dopa- reactivity in reserpinized rats (21). Furthermore, the D2R antago- minergic agents, or conversely, in the dark phase to obtain high nist eticlopride induces DARPP-32 phosphorylation in the striatum baseline locomotor activity in the studies with A2AR agonists. All ͞ of wild-type (WT) mice but not A2A knockout (KO) mice (22), drugs were administered i.p. in a volume of 0.1 ml 10 g of body suggesting that DARPP-32 phosphorylation requires tonic stimu- weight, and locomotor behavior was monitored for 120–480 min. lation of A2ARs, independent of D2R blockade. These results may WT, A2A KO, D2 KO, and A2A-D2 double KO mice (male and be best explained by a proposed model of opposing, independent female littermates from 3–8 months old) were used for this study. A2A and D2 receptor modulation of cellular responses, i.e., A2AR Locomotor activity. Horizontal locomotor activity was assessed in activation by endogenous adenosine may exert an excitatory influ- standard polypropylene cages (15 ϫ 25 cm) that were placed into ence on striatopallidal neurons by a D2R-independent mechanism adjustable frames equipped with seven infrared photocell beams (19, 23). (San Diego Instruments, San Diego). Ambulation (sequential Thus, whether or not striatal A2AR functions depend, in part or breaks in two adjacent beams) were recorded and analyzed on a entirely, on D2Rs is a central but open question. This is critical to computer as described (24, 27). our understanding not only of the cellular mechanisms underlying Catalepsy score. Catalepsy behavior was induced by the D2R adenosine–dopamine interaction, but also of the physiology of antagonist haloperidol (1.5 mg͞kg s.c.). Thirty minutes after hal- endogenous adenosine at A2ARs: Endogenous adenosine may act operidol treatment, mice underwent a habituation session (pretest) at A2ARs not only as an inhibitory modulator of dopaminergic and then 150 min later, the extent of catalepsy was evaluated by the neurotransmission (as proposed by A2AR–D2R