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The Journal of Neuroscience, August 15, 2000, 20(16):5949-5957

Functional Uncoupling of Adenosine A_2A Receptors and Reduced Response to Caffeine in Mice Lacking Dopamine D₂ Receptors

Nancy R. Zahniser^{1, 2}, Johanna K. Simosky¹, R. Dayne Mayfield¹, Cori A. Negri¹, Taleen Hanania¹, Gaynor A. Larson¹, Michele A. Kelly³, David K. Grandy⁴, Marcelo Rubinstein⁵, Malcolm J. Low³, and Bertil B. Fredholm⁶

¹ Department of Pharmacology and ² Neuroscience Program, University of Colorado Health Sciences Center, Denver, Colorado 80262, ³ Vollum Institute for Advanced Biomedical Research and ⁴ Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, Oregon 97201, ⁵ INGEBI (CONICET) and Department of Biology, University of Buenos Aires, Buenos Aires, Argentina, and ⁶ Department of Physiology and Pharmacology, Section of Molecular Neuropharmacology, Karolinska Institutet, Stockholm, Sweden

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Dopamine D₂ receptors (Rs) and adenosine A_2ARs are coexpressed on striatopallidal neurons, where they mediate opposing actions. In agreement with the idea that D₂Rs tonically inhibit GABA release from these neurons, stimulation-evoked GABA release was significantly greater from striatal/pallidal slices from D₂R null mutant (D₂R^/) than from wild-type (D₂R^+/+) mice. Release from heterozygous (D₂R^+/) slices was intermediate. However, contrary to predictions that A_2AR effects would be enhanced in D₂R-deficient mice, the A_2AR agonist CGS 21680 significantly increased GABA release only from D₂R^+/+ slices. CGS 21680 modulation was observed when D₂Rs were antagonized by raclopride, suggesting that an acute absence of D₂Rs cannot explain the results. The lack of CGS 21680 modulation in the D₂R-deficient mice was also not caused by a compensatory downregulation of A_2ARs in the striatum or globus pallidus. However, CGS 21680 significantly stimulated cAMP production only in D₂R^+/+ striatal/pallidal slices. This functional uncoupling of A_2ARs in the D₂R-deficient mice was not explained by reduced expression of G_s, G_olf, or type VI adenylyl cyclase. Locomotor activity induced by the adenosine receptor antagonist caffeine was significantly less pronounced in D₂R^/ mice than in D₂R^+/+ and D₂R^+/ mice, further supporting the idea that D₂Rs are required for caffeine activation. Caffeine increased c-fos only in D₂R^/ globus pallidus. The present results show that a targeted disruption of the D₂R reduces coupling of A_2ARs on striatopallidal neurons and thereby responses to drugs that act on adenosine receptors. They also reinforce the ideas that D₂Rs and A_2ARs are functionally opposed and that D₂R-mediated effects normally predominate.

Key words: adenosine A_2A receptor; dopamine D₂ receptor; D₂ receptor knock-out mouse; CGS 21680; mRNA; [³H]SCH 58261; [³H]CGS 21680; caffeine; striatopallidal pathway; GABA release; cAMP stimulation; G_s; G_olf; type VI adenylyl cyclase; locomotor activity; c-fos

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Efferent neurons from rodent striatum project either directly to the substantia nigra or indirectly via the globus pallidus (for review, see Gerfen, 1992). Both of these striatal projections are GABAergic. However, striatonigral and striatopallidal neurons express different combinations of peptides and receptors. Striatopallidal neurons are distinguished from striatonigral neurons by expression of the preproenkephalin gene and a high density of dopamine D₂ and adenosine A_2A receptors (Rs) (Schiffmann et al., 1991; Fink et al., 1992; Schiffmann and Vanderhaeghen, 1993; Svenningsson et al., 1997b).

The coexpression of A_2ARs and D₂Rs on striatopallidal neurons provides an anatomical basis for the opposing interaction that exists between these receptors. Opposing A_2AR/D₂R effects have been shown at several different levels, including behavior, neurotransmitter release, receptor binding, and gene expression (for review, see Ferré et al., 1992, 1997). For example, A_2AR stimulation reduces D₂R-mediated locomotor activity (Ferré et al., 1991). Likewise, A_2AR activation antagonizes the D₂R agonist-induced decrease in GABA release from globus pallidus (Ferré et al., 1993; Mayfield et al., 1996).

Two lines of D₂R null mutant (D₂R^/) mice have been generated (Baik et al., 1995; Kelly et al., 1997). The striatal D₂R density in heterozygous (D₂R^+/) mice is ~50% of that in wild-type (D₂R^+/+) mice, whereas no specific striatal D₂R binding is detectable in D₂R^/ mice. As anticipated, D₂R^/ mice show some impairments in spontaneous locomotor activity (Baik et al., 1995; Kelly et al., 1998). D₂Rs are inhibitory modulators of neurotransmitter release in the striatum. D₂ autoreceptor inhibition of dopamine release is abolished in striatal synaptosomes from D₂R^/ mice (L'hirondel et al., 1998). Interestingly, however, basal and potassium-evoked extracellular dopamine levels, measured with in vivo microdialysis, are similar in D₂R^+/+ and D₂R^/ mouse striata (Dickinson et al., 1999).

D₂R deficiency may alter release of other striatal neurotransmitters, e.g., GABA. Indeed, Baik et al. (1995) observed an increase in mRNA expression of the GABA synthetic enzyme glutamic acid decarboxylase (GAD) in the striatum of D₂R^/ mice, suggesting that GABA release from striatal projection neurons is increased. Enhanced GABA release from striatopallidal neurons in D₂R^/ mice would be predicted whether GABA release is tonically inhibited by D₂Rs and/or the action of A_2ARs is unopposed by D₂Rs. The adenosine receptor antagonist caffeine induces locomotor activation, and this behavioral hyperactivity has been shown to involve A_2ARs, D₂Rs, and GABA (Mukhopadhyay and Poddar, 1995; Svenningsson et al., 1997a; Khisti et al., 2000). A better understanding of the interactions between A_2ARs and D₂Rs may lead to novel therapies to treat basal ganglia movement disorders, such as Parkinson's disease and Huntington's disease (for review, see Ongini and Fredholm, 1996; Sebastião and Ribeiro, 1996; Ferré et al., 1997; Moreau and Huber, 1999). Therefore, we have further examined the significance of A_2AR/D₂R interactions using D₂R-deficient animals. We determined whether the ability of the selective A_2AR agonist CGS 21680 to modulate GABA release and induce cAMP production, as well as the ability of the adenosine receptor antagonist caffeine to induce locomotor activity and c-fos expression, differed among D₂R^+/+, D₂R^+/, and D₂R^/ mice.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Subjects. Male and female N₅ congenic mice (20-35 gm; Vollum Institute, Portland, OR) were used in most of the experiments. Detailed methods by which these mice were produced have been reported (Kelly et al., 1997, 1998). Briefly, a D₂R-genomic clone from a 129/SvEv library was isolated and used for the construction of a replacement-type targeting vector. Homologous recombination in D3 embryonic stem cells produced a mutant D₂R allele, which has a deletion of exon 8 sequences encoding the sixth and seventh putative transmembrane domains, the third extracellular loop, and the cytoplasmic C-terminal tail. Germ-line transmitting chimeras derived from targeted embryonic stem cells were mated to wild-type females to generate F1 heterozygous mice on the mixed 129S2/SvPas × C57BL/6J background. The mutated D₂R allele was then backcrossed an additional five generations by successive matings of heterozygous mice to wild-type C57BL/6J mice. The congenic N₅ mice used here were derived from intercrosses of heterozygous mice that yielded all three possible genotypes in normal Mendelian proportions: D₂R^+/+, D₂R^+/, and D₂R^/. The genotypes of all mice were confirmed by Southern blotting. Male C57BL/6J mice (20-30 gm) obtained from The Jackson Laboratory (Bar Harbor, ME) were used only in the experiments testing the effects of raclopride alone and in combination with CGS 21680 on GABA release. Mice were housed in groups of two to five under a 12 hr light/dark cycle with food and water available ad libitum. All animal-use procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center.

In vitro GABA release. Mice were killed by cervical dislocation, and 400 µm coronal brain slices were cut before dissecting out the region containing the striatum/globus pallidus (Franklin and Paxinos, 1997). During preparation the tissue was maintained in ice-cold modified Krebs' buffer (118 mM NaCl, 4.7 mM KCl, 11.1 mM D-glucose, 25 mM NaHCO₃, 1.2 mM MgCl₂, 1.0 mM NaH₂PO₄, 2.6 mM CaCl₂, and 4.0 µM Na₂-EDTA, saturated with 95% O₂/5% CO₂, pH 7.4, at 34°C). The slices were equilibrated in a metabolic shaker at 34°C for 30 min before being transferred to the superfusion chambers.

The GABA release method has been described previously (Mayfield and Zahniser, 1993). Briefly, striatal/pallidal slices were superfused at a rate of 0.25 ml/min with modified Krebs' buffer (34°C) containing 1-(2- (((diphenylmethylene)imino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NO-711) (10 µM) to inhibit GABA uptake. Three trains of monophasic rectangular pulses (12 Hz, 2 msec; 30 mA, 1 min) were applied at t = 72, 104, and 136 min after the start of superfusion, with the collection of 24 consecutive 1 ml superfusate fractions beginning at t = 60 min (Fig. 1). CGS 21680 (100 nM; stock solution made up in DMSO), raclopride (1 µM), or vehicle was included in the buffer at t = 120 min, 16 min before the third stimulation.

View larger version (15K): [in this window] [in a new window]   Figure 1.   Time course of a representative endogenous GABA release experiment showing the adenosine A2AR agonist CGS 21680-induced augmentation of evoked release from a D2R+/+ mouse striatal/pallidal slice. The GABA uptake blocker NO-711 (10 µM) was present throughout the experiment. Three periods of electrical field stimulation (S; 12 Hz, 2 msec; 30 mA, 1 min) were applied as indicated by the arrows. CGS 21680 (100 nM) was included in the superfusion buffer from 120 min through the end of the experiment (horizontal bar). Drug effects were determined from the S3/S2 ratio, the ratio of GABA release evoked above baseline in response to the third stimulation compared with that in response to the second stimulation.

GABA was quantified by HPLC with electrochemical detection after precolumn derivatization with o-pthaldialdehyde (Mayfield and Zahniser, 1993). The mobile phase was 1 M disodium phosphate, pH 6.4 (0.5 M final concentration), 0.86 mM NaCl, and 37.5% acetonitrile. The lower sensitivity limit was < 25 pg of GABA per 50 µl injection. Spontaneous GABA outflow, designated PS1, PS2, or PS3, was defined as the mean concentration of GABA in the three fractions immediately preceding each of the three periods of stimulation, respectively. Stimulation-evoked GABA overflow, designated S1, S2, or S3, was determined from the summed amount of GABA release that exceeded PS1, PS2, or PS3, respectively.

cAMP levels. Striatal/pallidal slices were prepared as outlined above and incubated at 34°C in multiwell plates in a metabolic shaker, with a change in modified Krebs' buffer at 30 and 45 min during a 1 hr equilibration period. Slices were then incubated in 0.5 ml of fresh buffer containing either no addition, 1 µM CGS 21680, 10 µM CGS 21680, or 1 µM CGS 21680 + 150 µM 8-(p-sulfophenyl)theophylline (8-p-SPT) at 34°C for 15 min. The incubation was terminated by the addition of 0.25 ml of 2.4% perchloric acid, sonication, and centrifugation at 30,000 × g for 15 min (Lu and Ordway, 1997). Pellets were dissolved in 0.2 ml of 0.1N NaOH for protein determination (Bradford, 1976) using bovine serum albumin as the standard. The supernatant was neutralized with excess (~20 mg) CaCO₃ (Thion et al., 1977). After centrifugation, the supernatant was evaporated to dryness and reconstituted in 0.25 ml of 50 mM sodium acetate buffer, pH 6.2, and cAMP was measured in duplicate 0.1 ml samples by radioimmunoassay (NEN Life Science Products, Boston, MA).

Locomotor activity. Mice were allowed to acclimate in individual transfer cages to the behavioral testing room for 30 min. Saline or caffeine (15 mg/kg) was then injected intraperitoneally in a volume of 1 ml/100 gm. Each mouse received only a single injection. Immediately after injection, mice were placed into individual activity chambers (San Diego Instruments, San Diego, CA). The room lights were turned off, and locomotor activity was recorded as the total distance traveled during 5 min periods for 120 min. Caffeine-induced locomotor activity was determined at each time point as a "difference score" by subtracting the mean activity of each genotype after saline injection from the activity of each mouse of that same genotype after caffeine injection.

Quantitative receptor autoradiography. Mice were killed 4 hr after saline or caffeine injection. Brains were frozen in powdered dry ice and stored at 70°C. Coronal sections (10 µm) were cut at 20°C at the levels of the rostral striatum/nucleus accumbens and the caudal striatum/globus pallidus. Binding of [³H]SCH 58261 and [³H]CGS 21680 was assayed using the published methods of Fredholm et al. (1998). Briefly, specific binding of 0.3 nM [³H]SCH 58261, defined with 50 µM 5'-N-ethylcarboxamidoadenosine (NECA), was measured in 170 mM Tris-HCl buffer, pH 7.4, containing 2 U/ml adenosine deaminase. For [³H]CGS 21680, the assay buffer also contained 10 mM MgCl₂. Indirect saturation curves were generated with [³H]CGS 21680 (2.5 nM) by incubating slide-mounted brain sections with either no drug (total binding), one of nine concentrations of unlabeled CGS 21680 (1 nM-10 µM), or 2-chloroadenosine (20 µM; nonspecific binding). [³H]raclopride binding was measured as described by Johansson et al. (1997). Specific binding of 2 nM [³H]raclopride, defined with 1 µM (+)-butaclamol, was measured in 170 mM Tris-HCl buffer, pH 7.6, containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, and 0.001% ascorbic acid. Slides and tritium standards were apposed to tritium-sensitive film for either 3 weeks ([³H]SCH 58261), 6 weeks ([³H]CGS 21680), or 8 weeks ([³H]raclopride).

Films were analyzed using computer-based imaging systems (Imaging Research, St. Catherines, Ontario, Canada). Inplot software (Graph Pad, San Diego, CA) was used to fit the indirect saturation curves. The affinity (K_d) and number of receptors (B_max) were determined from the equations published by DeBlasi et al. (1989). For statistical analyses, data from saline- and caffeine-treated mice of the same genotype were pooled on the basis of the observations that (1) caffeine does not interfere in binding assays because it is easily dissociated from A_2ARs by washing (Johansson et al., 1996) and (2) no differences between the saline- and caffeine-treated groups were detected when these data sets were analyzed separately.

In situ hybridization. Mice were killed 4 hr after saline or caffeine injection. Previously published methods were used to measure mRNA for A_2ARs and c-fos (Svenningsson et al., 1997a,b, 1999); these papers also describe the specificity of the probes. Briefly, consecutive coronal brain sections (14 µm) were cut with a cryostat and thaw-mounted onto poly-L-lysine-coated slides. The A_2AR probe was a riboprobe, 208 bases long, encoding amino acids 1196-1405 of the rat A_2AR protein (Svenningsson et al., 1997b) and was ³⁵S labeled by in vitro transcription using ³⁵S-labeled UTP. The c-fos probe was an oligodeoxynucleotide probe, 48 bases long, encoding amino acids 137-152 of the rat c-Fos protein. The oligodeoxynucleotide probe (Scandinavian Gene Synthesis AB, Köping, Sweden) was labeled using terminal deoxynucleotidyl transferase (Pharmacia, Uppsala, Sweden) and ³⁵S-labeled -dATP (NEN Life Science Products, Stockholm, Sweden) to a specific activity of ~10⁹ cpm/µg. Sections were hybridized in 50% deionized formamide (Fluka, Buchs, Switzerland), 4× standard sodium citrate, 1× Denhardt's solution, 1% sarcosyl, 0.02 M NaPO₄, pH 7.0, 10% dextran sulfate, 0.5 mg/ml yeast tRNA (Sigma Labkemi, Stockholm, Sweden), 0.06 M dithiothreitol, 0.1 mg/ml sheared salmon sperm DNA, and 10⁷ cpm/ml probe. After hybridization for 15 hr at 42°C, the sections were washed four times, for 15 min each, in 1× standard sodium citrate at 55°C; dipped briefly in water and 70, 95, and 99.5% ethanol; and air-dried. The sections were exposed to tritium-sensitive film for 2-5 weeks. The films were analyzed as in the autoradiographic studies above.

The expression of mRNA for the  subunits of G_s and G_olf, as well as for type VI adenylyl cyclase (AC VI), was examined using cRNA ³⁵S-riboprobes essentially as described previously (Le Moine et al., 1997; Svenningsson et al., 1997b) using sense and antisense probes for the corresponding rat proteins (Jones and Reed, 1987, 1989; Glatt and Snyder, 1993). The transcription was performed using MAXI-script in vitro transcription kits according to the manufacturer's protocol (Ambion, Austin, TX). The probes were separated from unincorporated ribonucleotides using Sephadex G-50 chromatography. No signals were detected with the sense probes.

Drugs. [³H]SCH 58261 was a gift from Dr. Ennio Ongini (Schering-Plough, Milan, Italy); [³H]raclopride and [³H]CGS 21680 were obtained from NEN Life Science Products (Boston, MA, or Stockholm, Sweden); and NO-711, CGS 21680, raclopride, 8-p-SPT, caffeine, NECA, 2-chloroadenosine, and (+)-butaclamol were obtained from Sigma/RBI (St. Louis, MO).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

GABA release

Endogenous GABA release was evoked from striatal/pallidal slices by three periods of electrical stimulation (Fig. 1). The GABA uptake inhibitor NO-711 (10 µM) was present throughout the entire experiment. Control release was assessed in response to the first two stimuli. Spontaneous GABA outflow was measured before each of these stimuli and equaled ~0.18 ng of GABA per mg wet weight of tissue per ml in the D₂R^+/+ and D₂R^+/ mice (Fig. 2A). In comparison with both the D₂R^+/+ and D₂R^+/ mice, spontaneous GABA release was significantly elevated by ~40% in the D₂R^/ mice (Fig. 2A). In response to each of the two stimuli, GABA overflow was significantly increased in all three genotypes. However, as seen in rat striatal/pallidal slices (Mayfield and Zahniser, 1993; Mayfield et al., 1996), the second stimulation evoked consistently lower GABA release than did the first stimulation in all of the mice (Fig. 2B). This is not caused by depletion of releasable pools of GABA but rather involves activation of GABA_BRs (Mayfield and Zahniser, 1993). Nonetheless, both the first and second stimuli increased GABA overflow to a significantly greater extent, by 71 and 63%, respectively, in the D₂R^/ versus the D₂R^+/+ mice (Fig. 2B). Stimulated GABA overflow in the D₂R^+/ mice was intermediate and did not differ statistically from that of either of the other two genotypes (Fig. 2B). Because the S2/S1 ratios were similar in all three genotypes, the data indicate that GABA_BR-mediated autoinhibition is unaltered by elimination of D₂Rs.

View larger version (37K): [in this window] [in a new window]   Figure 2.   GABA release was higher from D2R/ than from D2R+/+ mouse striatal/pallidal slices. A, Spontaneous GABA release before the first (PS1) and second (PS2) periods of stimulation. See Figure 1 for experimental details. Mean values ± SEM are shown for n = 15 mice (D2R+/+), n = 22 mice (D2R+/), or n = 18 mice (D2R/). Two-factor ANOVA followed by Newman-Keuls post hoc comparisons: *p < 0.05, D2R/ versus D2R+/+ or D2R+/. B, Stimulated GABA release evoked by the first (S1) and second (S2) periods of stimulation. Mean values ± SEM are shown for n = 15 mice (D2R+/+), n = 18 mice (D2R+/), or n = 20 mice (D2R/). Two-factor ANOVA with repeated measures (stimulation period) followed by Newman-Keuls post hoc comparisons: #p < 0.05, within genotype S2 versus S1; *p < 0.05, D2R/ versus D2R+/+.

The A_2AR agonist CGS 21680 was introduced before the third period of stimulation (Fig. 1) because preliminary experiments showed a more consistent A_2AR-mediated modulation of GABA release when the effects of CGS 21680 were tested during the third, rather than the second, stimulation. The more consistent results could reflect the fact that the second and third stimuli released similar amounts of GABA (Fig. 3; control S3/S2 ratios = ~1) whereas the second stimulus evoked less release than did the first stimulus (Fig. 2B; S2/S1 ratios < 1). The inclusion of 100 nM CGS 21680 in the superfusion buffer 16 min before the third stimulation did not alter spontaneous GABA outflow from striatal/pallidal slices from any of the three genotypes (data not shown). However, in the D₂R^+/+ mice, exposure to 100 nM CGS 21680 significantly increased stimulation-evoked GABA overflow by 90% (Fig. 3; S3/S2 ratio). In contrast, stimulated release was not significantly altered in the presence of CGS 21680 in either the D₂R^+/ or D₂R^/ mice (Fig. 3).

View larger version (36K): [in this window] [in a new window]   Figure 3.   CGS 21680 (100 nM) significantly enhanced stimulation-evoked GABA release from striatal/pallidal slices from D2R+/+ mice but not from D2R+/ or D2R/ mice. See Figure 1 for experimental details. Mean values ± SEM are shown for n = 6 mice (D2R+/+, D2R/) or n = 8 mice (D2R+/). Two-factor ANOVA with repeated measures (drug) followed by Newman-Keuls post hoc comparisons: *p < 0.05, D2R+/+ control versus CGS 21680.

To test the effect of acute blockade of D₂Rs on CGS 21680 modulation during the GABA release assay, we measured release in C57 mouse striatal/pallidal slices in the presence of maximally effective concentrations of CGS 21680 and/or the D₂R antagonist raclopride. Neither drug altered spontaneous GABA outflow (data not shown). In agreement with the results of the D₂R^+/+ mice, CGS 21680 (100 nM) significantly increased stimulation-evoked GABA overflow from the C57 mouse slices (Fig. 4; 154% above control). Raclopride (1 µM) also significantly increased evoked release by 119% (Fig. 4). When CGS 21680 and raclopride were combined, overflow was increased to a significantly greater extent (256%) than when raclopride was present alone (Fig. 4). These results demonstrate that in the presence of D₂R blockade, CGS 21680 increased GABA release from mouse striatal/pallidal slices and suggest that an acute absence of D₂Rs cannot explain the lack of A_2AR modulation in the D₂R-deficient mice.

View larger version (54K): [in this window] [in a new window]   Figure 4.   CGS 21680 (100 nM) increased evoked GABA release from C57 mouse striatal/pallidal slices whether or not D2Rs were blocked by raclopride (1 µM). See Figure 1 for experimental details. Mean values ± SEM are shown for n = 6-8 mice/condition. One-factor ANOVA followed by Newman-Keuls post hoc comparisons: *p < 0.05, CGS 21680 or raclopride versus control; **p < 0.05, CGS 21680 + raclopride versus control or raclopride.

A_2AR mRNA, A_2ARs, and D₂Rs

Downregulation of A_2ARs would be one explanation for the lack of CGS 21680-modulated GABA release in the D₂R-deficient mice. A_2AR mRNA could be quantitated in the striata and nucleus accumbens, areas containing cell bodies of neurons expressing A_2ARs, but was not detectable in the globus pallidus, the area containing terminals of neurons expressing A_2ARs (Fig. 5). In all three genotypes, lower levels of A_2AR mRNA were expressed in the nucleus accumbens than in the striatum (Table 1). However, within each brain region, no significant differences among the genotypes were observed in A_2AR mRNA expression.

View larger version (93K): [in this window] [in a new window]   Figure 5.   mRNA for A2ARs was highly expressed in the caudal striatum, whereas specific A2AR binding was detected in both the caudal striatum (Str) and globus pallidus (GP). Autoradiograms from coronal hemibrain sections of a D2R/ mouse are shown; these are representative of all three genotypes (see Table 1). Left, In situ hybridization with a 35S-riboprobe designed against the rat A2AR protein. Right, Specific binding of the selective A2AR antagonist [3H]SCH 58261 (0.3 nM).

View this table: [in this window] [in a new window]   Table 1.   Levels of A2AR mRNA, specific binding of the A2AR antagonist [3H]SCH 58261, and specific binding of the D2R antagonist [3H]raclopride in D2R+/+, D2R+/, and D2R/ mouse brain regions

[³H]SCH 58261 is a relatively new antagonist that is highly selective for A_2ARs versus A₁Rs (~800-fold) (Fredholm et al., 1998). Its binding was quantitated by autoradiographic analysis in the striatum, nucleus accumbens, and globus pallidus of the D₂R^+/+, D₂R^+/, and D₂R^/ mice. An approximate K_d concentration of [³H]SCH 58261 was used (0.3 nM) (Fredholm et al., 1998). In contrast with A_2AR mRNA, specific A_2AR antagonist-binding sites were observed in both the striatum and globus pallidus (Fig. 5). Levels of specific binding were approximately threefold higher in the striatum than in the globus pallidus, with that in the nucleus accumbens being intermediate (Table 1). However, in agreement with the A_2AR mRNA determinations, similar levels of binding were observed within each brain region of the three genotypes (Table 1). Binding of the D₂R antagonist [³H]raclopride was also measured in the striatum and nucleus accumbens of these mice. In comparison with the D₂R^+/+ mice, levels of D₂R binding were 50% lower in both brain regions of the D₂R^+/ mice and were not detectable in the D₂R^/ mice (Table 1). These results agree with those reported for the F₂ generation of these mice (Kelly et al., 1997).

Indirect saturation curves were generated using the A_2AR agonist [³H]CGS 21680 and quantitative autoradiographic analysis in the striatum, nucleus accumbens, and globus pallidus in the D₂R^+/+, D₂R^+/, and D₂R^/ mice. Assays contained 10 mM Mg²⁺ to induce the high-affinity agonist-binding state, and the curves were best fit by a single-site model. The affinities, ranging from 23 to 29 nM, were similar in all brain regions of the three genotypes (Table 2). In agreement with the [³H]SCH 58261 results, the receptor densities measured with [³H]CGS 21680 were approximately threefold higher in the striatum than in the globus pallidus, with that in the nucleus accumbens being intermediate (Table 2). Likewise, there were no differences among genotypes in the densities of A_2AR agonist-binding sites within a single brain region.

View this table: [in this window] [in a new window]   Table 2.   Similar affinities and densities for the A2AR agonist [3H]CGS 21680 in D2R+/+, D2R+/, and D2R/ mouse brain regions

A_2AR-stimulated cAMP

An alternative explanation to receptor downregulation that could underlie the lack of CGS 21680 modulation of GABA release in the D₂R^+/ and D₂R^/ mice would be an uncoupling of A_2ARs from adenylyl cyclase. To test this hypothesis, basal and CGS 21680-stimulated cAMP levels were measured in striatal/pallidal slices from the three genotypes. Preliminary experiments confirmed that, similar to previous results in rats (Lupica et al., 1990), maximal stimulation was produced by 1 and 10 µM CGS 21680. Furthermore, preliminary experiments showed that the adenosine receptor antagonist 8-p-SPT (150 µM) blocked the increases in cAMP induced by 1 µM CGS 21680, confirming that this is an A_2AR-mediated response. Basal levels of cAMP were similar in the three genotypes (Fig. 6; legend). CGS 21680 induced a significant 64% increase in cAMP formation in the D₂R^+/+ mice, whereas cAMP levels in the D₂R^/ mice were not altered by CGS 21680 (Fig. 6). Although there was a trend for CGS 21680 to increase cAMP (34% above basal) in the D₂R^+/ mice, this change was not statistically significant (Fig. 6). These results suggest that normal A_2AR signaling via increased cAMP production is disrupted in the striatopallidal neurons in the D₂R-deficient mice.

View larger version (62K): [in this window] [in a new window]   Figure 6.   CGS 21680 significantly enhanced cAMP formation in striatal/pallidal slices from D2R+/+ mice but not from D2R+/ or D2R/ mice. Basal levels of cAMP were similar in the three genotypes (D2R+/+, 325 ± 92 pmol of cAMP/mg of protein per 15 min; n = 6; D2R+/, 400 ± 134; n = 6; D2R/, 351 ± 168; n = 5). Mean values ± SEM are shown as a percentage of the basal cAMP value for each mouse in the presence of a maximally effective concentration of the agonist CGS 21680 (1-10 µM). One-factor ANOVA followed by Tukey post hoc comparisons: *p < 0.05, D2R+/+ versus D2R/.

Levels of mRNA for the stimulatory G-proteins G_olf and G_s and for AC VI were measured by in situ hybridization in the D₂R^+/+, D₂R^+/, and D₂R^/ mice. These experiments were conducted as an initial investigation into putative downstream-signaling mechanisms that might explain the compromised ability of A_2ARs to stimulate cAMP accumulation in the striatum/globus pallidus of D₂R-deficient mice. The isoform(s) of adenylyl cyclase to which striatopallidal A_2ARs couple is unknown. However, we focused on AC VI because its mRNA is present in both the striatum and globus pallidus (Liu et al., 1998) and A_2ARs are known to activate AC VI in pheochromocytoma 12 cells (Chern et al., 1995). Interestingly, G_olf and AC VI mRNAs, but not G_s mRNA, were readily detected in the striatum (Fig. 7). Thus, in this respect the mouse appears similar to the rat (Hervé et al., 1993). However, among the three genotypes, no differences were observed in the levels of expression of any of these mRNAs (Fig. 7).

View larger version (117K): [in this window] [in a new window]   Figure 7.   Expression of mRNAs for the  subunits of stimulatory G-proteins (Golf and Gs) and the catalytic subunit of AC VI was not different in the caudal striata of the D2R+/+, D2R+/, and D2R/ mice. Left, Representative autoradiograms from the in situ hybridization assays in coronal brain sections from a D2R/ mouse are shown for Golf (A), Gs (B), and AC VI (C). 35S-riboprobes designed against the respective rat proteins were used. Right, Mean values ± SEM from the quantitative analysis of these assays are shown for n = 9 (D2R+/+), n = 5-6 (D2R+/), and n = 8 (D2R/).

Caffeine-induced locomotor activity

Caffeine is known to stimulate locomotion secondarily to blockade of A_2ARs (Ledent et al., 1997; Svenningsson et al., 1997a; Hauber et al., 1998). Caffeine-induced locomotor activity was therefore measured as an additional test of A_2AR function in the D₂R^+/+, D₂R^+/, and D₂R^/ mice. After acclimatization to the behavioral testing room, the mice were injected with either saline or caffeine (15 mg/kg, i.p.), and locomotor activity was measured for 2 hr (Fig. 8A,B). During the first 45 min after saline injection, the activity of the D₂R^/ mice was significantly lower than that of the D₂R^+/+ and/or D₂R^+/ mice (Fig. 8A). To factor out baseline activity differences, caffeine-induced difference scores were generated (Fig. 8C). Mice of all three genotypes injected with caffeine were more active than were the respective controls injected with saline (Fig. 8C). Over the 2 hr period after caffeine injection, the total caffeine-induced distance traveled by the D₂R^+/+ mice was 42100 ± 9750 cm (n = 5), that traveled by the D₂R^+/ mice was 47200 ± 6970 cm (n = 5), and that traveled by the D₂R^/ mice was 24200 ± 3100 cm (n = 9). Statistical analysis revealed that the less-pronounced caffeine-induced activation of the D₂R^/ mice reflected the fact that their activity was significantly lower than that of the D₂R^+/+ and/or D₂R^+/ mice from 45 to 100 min after injection (Fig. 8C). Direct observation of D₂R^/ mice injected with caffeine revealed that the lower caffeine-induced locomotor activation was not caused by an increase in stereotypic behaviors (data not shown).

View larger version (26K): [in this window] [in a new window]   Figure 8.   Caffeine-induced locomotor activity was reduced in D2R/ mice relative to that in D2R+/+ and D2R+/ mice. Saline or caffeine was administered at time = 0. A, Distance traveled after saline injection (1 ml/100 gm, i.p.). Mean values ± SEM are shown for n = 4 mice (D2R/) or n = 5 mice (D2R+/+, D2R+/). Two-factor ANOVA with repeated measures (time) followed by Tukey post hoc comparisons: *p < 0.05, D2R/ versus D2R+/+ and D2R+/; xp < 0.05, D2R/ versus D2R+/+. B, Distance traveled after caffeine injection (15 mg/kg, i.p.). Mean values ± SEM are shown for n = 5 mice (D2R+/+, D2R+/) or n = 9 mice (D2R/). C, Caffeine-induced locomotor activity determined at each time point as a difference score by subtracting the mean activity of each genotype after saline injection from the activity of each mouse of that same genotype after caffeine injection. Mean values ± SEM are shown. Two-factor ANOVA with repeated measures (time) followed by Tukey post hoc comparisons: *p < 0.05, D2R/ versus D2R+/+ and D2R+/; xp < 0.05, D2R/ versus D2R+/+; +p < 0.05, D2R/ versus D2R+/.

Caffeine-induced c-fos expression

The differences in caffeine-induced behavioral activation among the three genotypes might reflect differences in the D₂R-mediated activity of intrinsic pallidal neurons. To investigate this possibility, we quantitated the levels of c-fos mRNA by in situ hybridization 4 hr after injection of either saline or caffeine (15 mg/kg). Expression of c-fos in the cerebral cortex, measured as a control, was similar in saline- and caffeine-treated mice of all three genotypes (data not shown). Expression of c-fos also did not differ significantly in the globus pallidus of saline- or caffeine-treated D₂R^+/+ and D₂R^+/ mice (Fig. 9). In contrast, c-fos expression was significantly increased by 94% in the globus pallidus of the caffeine-treated, versus the saline-treated, D₂R^/ mice (Fig. 9). Caffeine-induced c-fos expression in the D₂R^/ mice was also significantly higher than caffeine-induced c-fos expression in either the D₂R^+/+ or D₂R^+/ mice (Fig. 9).

View larger version (33K): [in this window] [in a new window]   Figure 9.   Expression of c-fos mRNA in the globus pallidus of D2R/ mice was increased by caffeine administration. mRNA levels were measured 4 hr after caffeine injection (15 mg/kg, i.p.). Mean values ± SEM are shown for n = 3 mice (D2R+/ saline, D2R+/ caffeine, D2R/ saline), n = 4 mice (D2R+/+ caffeine), or n = 5 mice (D2R+/+ saline, D2R/ caffeine). One-factor ANOVA followed by Newman-Keuls post hoc comparisons: ***p < 0.001, D2R/ caffeine versus all other groups. O.D., Optical density.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Evidence of tonic D₂R inhibition of striatopallidal GABA release

Activation of D₂Rs inhibits stimulation-evoked GABA release from striatopallidal neurons. Conversely, in striatal/pallidal slices from control C57 mice, we observed that the D₂R antagonist raclopride increased GABA release. We also hypothesized that GABA release from these striatal projection neurons would be elevated in mice lacking D₂Rs. In agreement with this hypothesis, we observed significantly greater (40-70%) spontaneous and electrically evoked GABA release from D₂R^/ than from D₂R^+/+ mouse striatal/pallidal slices. GABA release from D₂R^+/ mice was intermediate. Our observations show the essential correctness of the surmise of Baik et al. (1995). They found higher levels of striatal GAD mRNA in a different line of D₂R^/ mice and, on the basis of this observation, suggested that GABA release might be increased. Together, these observations strengthen the idea that D₂Rs tonically inhibit GABA release from striatopallidal neurons.

The reduced baseline locomotor activity observed here in the N₅ congenic D₂R^/ mice is compatible with disinhibited pallidal GABA release. During the initial 40 min after saline injection, the D₂R^/ mice were significantly less active than were the other two genotypes. Subtle differences in the initiation of movement were also observed between D₂R^+/+ and D₂R^/ mice in the F₂ generation (Kelly et al., 1998). The lower level of locomotor activity in the D₂R^/ mice is consistent with pharmacological studies showing that increased GABA_A receptor activation, as well as decreased dopamine receptor activation, reduces locomotor activity (Mukhopadhyay and Poddar, 1995).

Lack of A_2AR agonist effects in D₂R-deficient mice

An antagonistic interaction between D₂Rs and A_2ARs in striatopallidal neurons is well established (for review, see Ferré et al., 1992, 1997). This antagonistic interaction impacts GABA release. The A_2AR agonist CGS 21680 not only increases stimulation-evoked GABA release from rat striatal/pallidal slices but also abolishes the D₂R agonist-mediated inhibition of this release (Mayfield et al., 1993, 1996). Likewise, Ferré et al. (1993) observed that CGS 21680 antagonizes the D₂R-mediated reduction in extracellular GABA in vivo in rat globus pallidus. Here we observed that CGS 21680 also markedly increased stimulation-evoked GABA release from D₂R^+/+ and C57 mouse striatal/pallidal slices. Thus, we hypothesized that without opposing D₂Rs, the stimulation of GABA release by A_2ARs would be potentiated. Unexpectedly, however, CGS 21680 did not affect GABA release in either D₂R^+/ or D₂R^/ mice. It is unlikely that the lack of CGS 21680 potentiation in the D₂R-deficient mice was caused by an acute absence of D₂Rs during the assay. Stimulation-evoked GABA release from control mouse striatal/pallidal slices was still augmented by CGS 21680 when the D₂R antagonist raclopride was included in the assay. It is also unlikely that a ceiling effect, i.e., that GABA release was already maximal, explains the lack of CGS 21680 potentiation because stimulated GABA release from D₂R^+/ slices was not statistically higher than that from D₂R^+/+ slices (Fig. 2B).

Uncoupled A_2ARs in D₂R-deficient mice

Loss of A_2AR effects in the D₂R^+/ and D₂R^/ mice likely reflects changes, which may have occurred during development, to compensate for the reduced number of D₂Rs. Previous studies (Baik et al., 1995; Kelly et al., 1998) suggest that striatonigral activity is not increased in D₂R-deficient animals. Downregulation of A_2AR expression was the first potential change that we investigated. Both A_2ARs and D₂Rs are expressed at very low levels during early striatal development and reach adult levels only at the end of the second postnatal week (Johansson et al., 1997). Nonetheless, levels of A_2AR mRNA in the striatum or globus pallidus were similar in the D₂R^+/+, D₂R^+/, and D₂R^/ mice. The antagonist [³H]SCH 58261 has the same high affinity for both G-protein-coupled and -uncoupled states of the A_2AR (Fredholm et al., 1998). Thus, its binding measures the total complement of A_2ARs. In contrast, in the presence of 10 mM Mg²⁺, [³H]CGS 21680 detects primarily the G-protein-coupled state with high affinity for agonists (Johansson et al., 1992). However, we observed similar numbers of A_2ARs among the three genotypes in the striatum or globus pallidus with both [³H]SCH 58261 and [³H]CGS 21680. These results indicated that changes in A_2AR expression are not the basis for differences in A_2AR modulation of GABA release.

A second potential mechanism for reduced A_2AR function is the uncoupling of A_2ARs from their effector molecule adenylyl cyclase. Whereas CGS 21680 significantly increased cAMP production in striatal/pallidal slices from D₂R^+/+ mice, it was ineffective in D₂R^+/ and D₂R^/ mice. Thus, despite unchanged agonist binding, the cAMP experiments suggested that A_2ARs are functionally uncoupled in the D₂R-deficient mice. Furthermore, the ability of CGS 21680 to increase GABA release likely depends on increases in cAMP (Wang and Johnson, 1995). The uncoupling does not appear to be caused by lower expression of the  subunits of stimulatory G-proteins (G_olf or G_s) or the catalytic subunit of AC VI. Alternatively, the A_2AR and G-protein could be kinetically uncoupled, the levels of G-protein and/or AC VI protein could be reduced, and/or the subcellular localization of the A_2AR relative to the G-protein and/or AC could be altered in the D₂R-deficient mice.

Altered effects of caffeine in D₂R^/ mice

Dopamine receptors are involved in mediating caffeine-induced locomotion, and the differential results in the D₂R^+/+ and D₂R^/ mice further support a role for D₂Rs. Characteristic of the D₁R/D₂R synergy required for many dopamine-mediated behaviors, locomotor stimulation induced by caffeine requires activation of both receptor subtypes and is blocked by either selective D₁R or D₂R antagonists (Garrett and Holtzman, 1994). Moreover, Fenu and Morelli (1998) have demonstrated that caffeine produces motor stimulation in 6-hydroxydopamine-lesioned rats, which have had prolonged compromised D₁R/D₂R stimulation, only when the rats have been primed with a dopamine receptor agonist before caffeine testing. Therefore, differential behavioral effects of caffeine were expected in the D₂R^+/+ and D₂R^/ mice. We observed an inability of the D₂R^/ mice to sustain caffeine-induced activation whereas the D₂R^+/+ and D₂R^+/ mice were activated to similar extents.

Caffeine has similar affinities for A₁Rs and A_2ARs (Daly, 1993; Fredholm, 1995; Fredholm et al., 1999). However, it is A_2AR antagonism that is required for locomotor activation (Ledent et al., 1997; Svenningsson et al., 1997a; Hauber et al., 1998). Our observation of uncoupled A_2ARs in the D₂R-deficient mice predicts that caffeine activation should have been precluded. This clearly was not the case with the D₂R^+/ mice and suggests that A_2ARs in these mice must be at least partially functional. We did observe a trend for CGS 21680 to increase cAMP in the D₂R^+/ striatum/pallidum (Fig. 6). In any case, it is clear that only half the normal complement of D₂Rs is sufficient to allow normal caffeine activation.

Although locomotor stimulation by caffeine was markedly reduced in the D₂R^/ mice, it was not totally absent. It is possible that blockade of A₁Rs could have also contributed. Indeed, from several rodent studies, combined blockade of A₁Rs and A_2ARs is known to produce larger locomotor responses than is blockade of only one receptor subtype (see Daly, 1993; Fredholm et al., 1999). Because the D₂R^/ mice are functionally devoid of A_2AR signaling, blockade of A₁Rs may produce greater effects than in intact animals. A₁Rs can negatively influence D₁R signaling by blocking dopamine release in the striatum (Jin et al., 1993; Harvey and Lacey, 1997), by increasing the firing of dopaminergic neurons (Stoner et al., 1988), and by direct interactions at striatonigral neurons (Ferré et al., 1998). Thus, blockade of A₁Rs, by antagonizing any or all of these mechanisms, can increase activity in striatonigral neurons (Ferré et al., 1996). Furthermore, activation of D₁Rs leads to a larger locomotor response in D₂R^/ mice than in D₂R^+/+ or D₂R^+/ mice (Kelly et al., 1998).

In rodents, c-fos expression in the globus pallidus is increased when higher doses of caffeine, which induce locomotor depression, are administered (Svenningsson et al., 1995; Svenningsson and Fredholm, 1997; Bennett and Semba, 1998). The induction of c-fos in the globus pallidus appears to require simultaneous inhibition of striatopallidal neurons and activation of striatonigral neurons (Le Moine et al., 1997). Indeed, inhibition of A_2ARs, combined with activation of D₁Rs, is very effective. Hence, our data showing pallidal c-fos induction after caffeine in the D₂R^/ animals might be explained if in these animals striatonigral activity is increased by caffeine, even though basal activity is not elevated.

Summary

The present results show that a targeted disruption of the D₂R influences responses to drugs that act on adenosine receptors. Thus, enhancement of GABA release induced by an A_2AR agonist was completely eliminated when D₂R expression was reduced. This likely involves some adaptive change(s), because acute treatment with a D₂R antagonist does not produce the same result. The adaptation is not simply a change in A_2AR expression or a loss of any key signaling component. However, the exact mechanism remains unclear. The motor stimulatory effect of caffeine, which depends on A_2AR blockade, was substantially reduced in mice lacking D₂Rs. These findings support the idea that some level of D₂R activity is required for the action of caffeine, as has been postulated previously on the basis of results with antagonists. The results also show that animals with a targeted disruption of the D₂R have a functional uncoupling of the A_2AR. This underscores that not all of the phenotypic changes reported for such mice can be necessarily attributed to the D₂R loss.

	FOOTNOTES

Received Feb. 10, 2000; revised May 23, 2000; accepted May 26, 2000.

This work was supported by National Institutes of Health Grants NS 26851 and DA 12062 and by the Swedish Medical Research Council Project No. 2553. We thank Drs. Tom Dunwiddie and Per Svenningsson for their helpful discussions.

Correspondence should be addressed to Dr. Nancy R. Zahniser, Department of Pharmacology C-236, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. E-mail: nancy.zahniser{at}uchsc.edu.

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