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Neuropharmacology 66 (2013) 179e186

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Neuropharmacology

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mGlu5R promotes glutamate AMPA receptor phosphorylation via activationof PKA/DARPP-32 signaling in striatopallidal medium spiny neurons

Maria Teresa Dell’Anno 1, Simone Pallottino, Gilberto Fisone*

Department of Neuroscience, Karolinska Institutet, Retzius väg 8, 171 77 Stockholm, Sweden

a r t i c l e i n f o

Article history:Received 13 December 2011Received in revised form23 March 2012Accepted 29 March 2012

Keywords:Adenosine A2A receptorBasal gangliaMetabotropic glutamate receptorMouseStriatum

Abbreviations: A2AR, adenosine A2A receptor; Amethylisoxazole-4-propionic acid; CGS21680, 4-[2-[[furanuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzeride; DARPP-32, dopamine- and cAMP-regulated phos(R,S)-3,5-dihydroxyphenylglycine; DMSO, dimethylD1 receptor; D2R, dopamine D2 receptor; LY367385, (methylbenzeneacetic acid; mGluR, metabotropic glmethyl-6-(phenylethynyl)pyridine hydrochloride; MSNMDA, N-methyl-D-aspartate; PKA, cAMP-dependprotein kinase C; SCH23390, (R)-(þ)-7-Chloro-8-hydroxtetrahydro-1H-3-benzazepine hydrochloride; SKF8tetrahydro-1-phenyl-1H-3-benzazepine hydrobromide2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino* Corresponding author. Tel.: þ46 8 52487375; fax:

E-mail address: gilberto.fisone@ki.se (G. Fisone).1 Present address: San Raffaele Scientific Institute, V

Italy.

0028-3908/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.neuropharm.2012.03.025

a b s t r a c t

Group Imetabotropic glutamate receptors (mGluRs), which comprisemGlu1Rs andmGlu5Rs, are enrichedin striatalmedium spiny neurons (MSNs), where theymodulate glutamatergic transmission. Here,we haveexamined the effect of group I mGluRs on the regulation of the state of phosphorylation of the GluA1subunit of the AMPA glutamate receptor.We found that incubation of mouse striatal slices with the group ImGluR agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) promotes GluA1 phosphorylation at the cAMP-dependent protein kinase (PKA) site, Ser845. This effect is prevented by 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), a selective mGlu5R antagonist. The increase in GluA1 phosphorylationproduced by DHPG is also prevented by blockade of adenosine A2A receptors (A2ARs), which are knownto promote cAMP signaling specifically in striatopallidal MSNs, as well as by enzymatic degradationof endogenous adenosine, achieved with adenosine deaminase. The ability of DHPG to increasePKA-dependent phosphorylation of GluA1 depends on concomitant activation of the dopamine- andcAMP-regulated phosphoprotein of 32 kDa (DARPP-32). Thus, inactivation of the PKA phosphorylation siteof DARPP-32 abolishes the effect of DHPG. Moreover, cell-specific knock out of DARPP-32 in striatopallidal,but not in striatonigral, MSNs prevents the increase in Ser845 phosphorylation induced by DHPG. Theseresults indicate that activation of mGlu5Rs promotes PKA/DARPP-32-dependent phosphorylation ofdownstream target proteins in striatopallidal MSNs and that this effect is exerted via potentiation of tonicA2AR transmission.

This article is part of a Special Issue entitled ‘Metabotropic Glutamate Receptors’.� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The basal ganglia are implicated in numerous neurodegenerativeand neuropsychiatric disorders, including Parkinson’s disease,Huntington’s disease, schizophrenia and drug addiction. One

MPA, a-amino-3-hydroxy-5-6-amino-9-(N-ethyl-b-D-ribo-nepropanoic acid hydrochlo-phoprotein of 32 kDa; DHPG,sulfoxide; D1R, dopamineS)-(þ)-a-amino-4-carboxy-2-utamate receptor; MPEP, 2-Ns, medium spiny neurons;ent protein kinase; PKC,y-3-methyl-1-phenyl-2,3,4,5-1297, (�)-6-chloro-2,3,4,5-; ZM241385, 4-(2-[7-amino-]ethyl)phenol.þ46 8 320988.

ia Olgettina 58, 20132 Milan,

All rights reserved.

common neuronal substrate for these pathological conditions arethe GABAergic medium spiny neurons (MSNs) of the striatum.MSNsare innervated by glutamategic inputs from cortex, thalamus andlimbic areas, which play a critical role in basal ganglia transmission(Alexander et al., 1986). In the striatum, metabotropic glutamatereceptors (mGluRs) are implicated in the control of ionotropic [e.g.a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)and N-methyl-D-aspartate (NMDA)] glutamate receptors andsynaptic plasticity (Gubellini et al., 2004). Group I mGluRs, whichcomprise mGlu1Rs and mGlu5Rs, are highly expressed in striatalMSNs (Kerner et al., 1997; Tallaksen-Greene et al., 1998; Testa et al.,1994). mGlu5Rs have been shown to potentiate NMDA-mediatedresponses (Domenici et al., 2003; Pisani et al., 2001) and stimulateglutamate release (Pintor et al., 2000). In addition, injection of groupI mGluR agonists in the dorsal striatum increases the phosphoryla-tion of the GluA1 subunit of the AMPA receptors (Ahn and Choe,2009), an effect known to facilitate glutamate transmission (Bankeet al., 2000; Derkach et al., 1999; Roche et al., 1996).

Striatal MSNs comprise two large populations, with distinctfunctional properties. One group of MSNs projects directly to theoutput nuclei of the basal ganglia and promotes motor activity

M.T. Dell’Anno et al. / Neuropharmacology 66 (2013) 179e186180

(Albin et al., 1989; Alexander and Crutcher, 1990; DeLong, 1990).These neurons, which form the so-called direct, or striatonigralpathway, are selectively enriched in dopamine D1 receptors (D1Rs)(Gerfen et al., 1990). The other group of MSNs is connected to theouput stations of the basal ganglia indirectly and, when activated,depresses motor function (Albin et al., 1989; Alexander andCrutcher, 1990; DeLong, 1990). These neurons, which form theindirect or striatopalidal pathway, express high levels of dopamineD2 receptors (D2Rs) and adenosine A2A receptors (A2ARs)(Fink et al., 1992; Gerfen et al., 1990; Schiffmann et al., 1991). Thedistinct functional properties of the MSNs of the direct and indirectpathway indicate the importance of identifying changes producedby activation of mGluRs in one or the other population.

Group I mGluRs are expressed in both striatonigral and striato-pallidal MSNs (Kerner et al., 1997; Tallaksen-Greene et al., 1998;Testa et al., 1994). In the striatum, mGlu5 receptors have beenproposed to form heteromeric complexes with adenosine A2ARs(Ferre et al., 2002). In support of this idea, it has been shownthat combined activation of A2ARs and mGlu5Rs promotes theexpression of the immediate early gene, c-fos (Ferre et al., 2002) andreduces D2R binding (Ferre et al., 1999). Moreover, A2ARs playa permissive role on the potentiation produced by mGlu5Rs onNMDA receptor-mediated transmission (Domenici et al., 2004). Thislatter effect is abolished by inhibition of cAMP-dependent proteinkinase (Domenici et al., 2004). Notably, A2ARs are required formGlu5R-mediated activation of the dopamine and cAMP-regulatedphosphoprotein of 32 kDa (DARPP-32), a critical mediator of cAMPsignaling (Nishi et al., 2003).

In this study, we examined the role of A2AR and cAMP/DARPP-32 signaling in mGlu5R-mediated regulation of GluA1 phosphory-lation. In addition, we provide evidence indicating that suchregulation occurs specifically in striatopallidal MSNs.

2. Materials and methods

2.1. Animals

Male C57BL/6 mice (25e30 g) were purchased from Taconic (Tornbjerg,Denmark). Mice in which DARPP-32 was conditionally deleted in D1R-, or D2R-expressing MSNs (D32f/fD1RCreþand D32f/fD2RCreþ conditional knock out mice)were generated by breeding floxed DARPP-32mice to either D1R-Cre (EY262 line) orD2R-Cre (ER44 line) BAC transgenic mice (Gong et al., 2007), as described in Bateupet al. (2010). Knock-in mice expressing a mutated form of DARPP-32, in which thephosphorylation site for PKA (Thr34) was substituted with an Ala (T34A DARPP-32mutant mice), were generated as described in Svenningsson et al. (2003). Allexperiments were carried out in accordance with the guidelines of the SwedishAnimal Welfare Agency.

2.2. Drugs

(R,S)-3,5-Dihydroxyphenylglycine (DHPG) was purchased from Ascent Scientific(Bristol, UK). 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), (S)-(þ)-a-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385)and (�)-6-chloro-2,3,4,5-tetrahydro-1-phenyl-1H-3-benzazepine hydrobromide(SKF81297) were purchased from Tocris Bioscience (Bristol, UK). (R)-(þ)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride(SCH23390), 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680) and quinpirolewere purchased from SigmaeAldrich (Stockholm, Sweden). Adenosine deaminasewas purchased from Roche (Mannheim, Germany). DHPG, MPEP, SCH23390, quin-pirole and ADA were dissolved in water. LY367385, ZM241385 and CGS21680 weredissolved in dimethyl sulfoxide (DMSO).

2.3. Preparation and incubation of striatal slices

Mice were killed by decapitation and the brains were rapidly removed. Coronalslices (250 mm) were prepared using a vibratome (Leica, Nussloch, Germany). Dorsalstriatawere dissected out fromeach slice under amicroscope. Two sliceswere placedin individual 5 ml polypropylene tubes containing 2 ml of Krebs-Ringer’s bicar-bonate buffer [118 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl2, 1.5 mM MgSO4, 1.2 mMKH2PO4, 25 mM NaHCO3 and 11.7 mM glucose, equilibrated with 95% O2/5% CO2

(vol/vol), pH 7]. The samples were equilibrated at 30 �C for two 30-min intervals,each followed by replacement of the medium with 2 ml fresh Krebs-Ringer’sbicarbonate buffer. Test substances were then added for various durations as spec-ified. After incubation, the solutions were rapidly removed, the slices were sonicatedin 1% SDS and the samples analyzed by Western blotting as described below.

2.4. Western blot assay of phosphorylated GluA1 and DARPP-32

Aliquots (5 ml) of the homogenatewere used for protein determinationusing a BCA(bicinchoninic acid) assay kit (Pierce Europe, Oud Beijerland, the Netherlands). Equalamounts of protein (30 mg) for each samplewere loaded onto 10% polyacrylamide gels.Proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresisand transferred overnight to polyvinylidene fluoride membranes (AmershamPharmacia Biotech, Uppsala, Sweden) (Towbin et al., 1979). The membranes wereimmunoblotted using antibodies against phospho-Ser845-GluA1 (1:1000), phospho-Thr34-DARPP-32 (PhosphoSolutions, Aurora, CO, USA), or phospho-Ser831-GluA1(1:400) (Upstate Ltd., Milton Keynes, UK). Antibodies against GluA1 (1:1000) (UpstateLtd., Milton Keynes, UK) and DARPP-32 (1:10000) (Hemmings and Greengard, 1986)that are not phosphorylation state specific were used to estimate the total amount ofproteins. Detection was based on fluorescent secondary antibody binding andquantified using a Li-Cor Odyssey infrared fluorescent detection system (Li-Cor,Lincoln, NE, USA). The amount of each phosphoproteinwas normalized for the amountof the corresponding total protein detected in the sample.

3. Results

3.1. DHPG increases the phosphoryation of GluA1 at Ser845via activation of mGlu5Rs

We started by examining the effect of the group I mGluR agonist,DHPG, on the phosphorylation of GluA1 at Ser845, which isregulated by PKA, and Ser831, which is instead regulated by proteinkinase C (PKC) and Ca2þ/calmodulin protein kinase II (Barria et al.,1997; Roche et al., 1996). Incubation of striatal slices with DHPGpromoted the phosphorylation of GluA1 at Ser845. This effectwas concentration-dependent, with a maximal, two-fold increasereached with 25 mM DHPG (Fig. 1A). The increase in Ser845 phos-phorylation produced by DHPG was reached within 10 min ofincubation and declined at 20 min (Fig. 1B). In contrast, incubationof striatal slices for 10 min with DHPG (10e50 mM) did not produceany effect on GluA1 phoshorylation at Ser831 (Fig. 1C).

We next examined the contribution of mGlu1Rs and mGlu5Rsto the effect of DHPG on Ser845 phosphorylation. We found thatpreincubation of striatal slices with the mGluR5 antagonist, MPEP(10 mM), abolished the increase in Ser845 phosphorylation producedby DHPG (Fig. 2A). In contrast, the effect of DHPG was not affectd bypreincubation with the mGlu1R antagonist, LY367385 (100 mM;Fig. 2B). These results indicated that mGlu5Rs are specificallyinvolved in PKA-dependent regulation of GluA1 phosphorylation.

3.2. mGlu5R-dependent phosphorylation of GluA1 requires intactA2AR, but not D1R, transmission

In the striatum, activation of adenosine A2ARs promoteGolf-dependent cAMP signaling (Fredholm, 1977; Herve et al.,2001; Svenningsson et al., 1998). Moreover, mGlu5Rs and A2ARshave been shown to interact synergistically (Domenici et al., 2004;Ferre et al., 2002, 1999; Nishi et al., 2003). Based on these obser-vations, we examined the involvement of A2ARs in mGlu5R-dependent increase of GluA1 phosphorylation. Striatal slices wereincubated with DHPG (25 mM) alone, or in combination with theA2AR antagonist, ZM 241385 (1 mM). We found that ZM 241385abolished the increase in Ser845 phosphorylation produced byDHPG (Fig. 3A). In parallel experiments, we examined whether theeffect of DHPG was dependent on D1Rs, whose activation is alsocoupled to stimulation of cAMP signaling (Nishi et al., 1997; Stoofand Kebabian, 1981; Svenningsson et al., 1998). We found thatthe D1R antagonist SCH23390 (1 mM) did not affect the ability ofDHPG to increase Ser845 phosphorylation (Fig. 3B).

Fig. 2. DHPG-induced phosphorylation of GluA1 at Ser845 depends on activation ofmGlu5Rs. Striatal slices were preincubated for 10 min in the presence of MPEP (10 mM)(A), or LY367395 (100 mM) (B) and then for a further 5 min in the presence of DHPG(25 mM). The levels of GluA1 phosphorylated at Ser845 were determined by Westernblotting as described in Methods. Upper panels show representative immunoblots oftotal GluA1 (GluA1) or phosphorylated GluA1 (P-GluA1). Lower panels showsummaries of data expressed as means� SEM (n¼ 9). **p< 0.01 and ***p< 0.001 vs.Control, xx p< 0.01 vs. DHPG; one-way ANOVA followed by Bonferroni-Dunn post-hoctest.

Fig. 1. DHPG increases the phosphorylation of GluA1 at Ser845, but not at Ser831.Striatal slices were incubated for 5 min in the presence of 10, 25, or 50 mM DHPG(A and C), or for 5, 10, or 20 min in the presence of 25 mM DHPG (B). The levels ofGluA1 phosphorylated at Ser845 (A and B), or Ser831 (C) were determined byWestern blotting as described in Methods. Upper panels show representativeimmunoblots of total GluA1 (GluA1) or phosphorylated GluA1 (P-GluA1). Lowerpanels show summaries of data expressed as means� SEM (n¼ 9). **p< 0.01 and***p< 0.001 vs. control; one-way ANOVA followed by Bonferroni-Dunn post-hoc test.

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3.3. Tonic activation of A2ARs is required for mGlu5R-dependentSer845 phosphorylation

The ability of ZM241385 to prevent DHPG-induced phosphory-lation of GluA1 suggested that tonic activation of A2ARs playsa critical role in striatal mGlu5R-mediated transmission. We furtherexamined this possibility by measuring the response to DHPGfollowing removal of endogenous adenosine. Striatal slices werefirst incubated for 60 min with adenosine deaminase, whichconverts adenosine into inosine, and then stimulated with DHPG.

Fig. 3. DHPG-induced GluA1 phosphorylation at Ser845 depends on tonic A2AR activation. Striatal slices were preincubated for 10 min in the presence of ZM241385 (1 mM) (A), orSCH23390 (1 mM) (B) and then for a further 5 min in the presence of DHPG (25 mM). (C and D), Adenosine deaminase (ADA; 10 mg/ml) was added 60 min prior incubation for 5 minwith DHPG (25 mM) (C and D), or for 10 min with CGS21680 (5 mM) (D). When combined with DHPG (D), CGS21680 was added 5 min earlier. The levels of GluA1 phosphorylated atSer845 were determined by Western blotting as described in Methods. Upper panels show representative immunoblots showing immunoreactivity corresponding to total GluA1(GluA1) or phosphorylated GluA1 (P-GluA1). Lower panels show summaries of data expressed as means� SEM (n¼ 9). ***p< 0.001 vs. Control, xx p< 0.01 and xxx p< 0.001 vs.DHPG, and ## p< 0.01 vs. CGS21680; one-way ANOVA followed by Bonferroni-Dunn post-hoc test.

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Preincubation with adenosine deaminase did not affect the basallevels of phospho-Ser845 GluA1. However, this treatment was ableto abolish the increase in Ser845 phosphorylation induced by DHPG(Fig. 3C). We also determined the effect produced by exposingadenosine deaminase-treated slices to the A2AR agonist CGS21680.We found that CGS21680 did not modify Ser845 phosphorylationwhen added alone, but it was able to rescue the effect of DHPGwhengiven in combination with the group I mGluR agonist (Fig. 3D).

3.4. mGlu5R-dependent phosphorylation of GluA1 requires PKA-mediated phosphorylation of DARPP-32 in striatopallidal MSNs

Previous work showed that, in striatal slices, activation ofmGlu5Rs increases PKA-dependent phosphorylation of DARPP-32

(Nishi et al., 2003). Therefore, we examined the possible involve-ment DARPP-32 in the regulation of GluA1 phosphorylation exertedby DHPG. In preliminary experiments, we confirmed the ability ofthis drug to promote DARPP-32 phosphorylation at the PKA site,Thr34. Incubation of striatal slices for 5 min with 25 mM DHPGresulted in a 1.5-fold increase in the levels of phospho-Th34 DARPP-32 (Fig. 4A).We then compared the effect produced byDHPG in slicesfromwild type mice with that produced in slices from T34A DARPP-32 mutant mice. The results showed that inactivation ofPKA-mediated regulation of DARPP-32 abolished the increase inGluA1 phosphorylation produced by DHPG at Ser845 (Fig. 4B).

We proceeded by determining in which subpopulation ofstriatal MSNs this regulation was occurring, using D32f/fD1RCre-þand D32f/fD2RCreþ conditional knock out mice. In agreement

Fig. 4. DHPG-induced phosphorylation of GluA1 at Ser845 requires PKA-mediated activation of DARPP-32 in striatopallidal MSNs. Striatal slices fromwild type mice (A and B), T34ADARPP-32 mutant mice (B), DARPP32f/fD2RCreþmice (D), DARPP32f/fD1RCreþ mice (E) and DARPP32f/fCre� mice (D and E) were incubated for 5 min in the presence of DHPG (25 mM).(C), Reduced levels of DARPP-32 in striatal slices from DARPP32f/fD2RCreþ and DARPP32f/fD1RCreþ mice compared to DARPP32f/fCre� mice. Total and phosphorylated DARPP-32 (A andC), or GluA1 (B, D and E) were determined by Western blotting as described in Methods. Upper panels show representative immunoblots showing immunoreactivity correspondingto total DARPP-32 (DARPP-32) (A and C), total GluA1 (GluA1) (B, D and E), phosphorylated DARPP-32 (P-DARPP-32) (A), or phosphorylated GluA1 (P-GluA1) (B, D and E). Lowerpanels show summaries of data expressed as means� SEM (n¼ 6e10). *p < 0.05 vs. Control (Ctr), ***p< 0.001 vs. Wild type Ctr, xxx p< 0.01 vs. DARPP32f/fCre� Ctr and # p< 0.05 vs.DARPP32f/fD1RCreþ Ctr; One-way ANOVA followed by Bonferroni-Dunn post-hoc test.

M.T. Dell’Anno et al. / Neuropharmacology 66 (2013) 179e186 183

Fig. 5. Activation of D2Rs abolishes DHPG-induced phosphorylation of GluA1 atSer845. Striatal slices were preincubated for 10 min in the presence of quinpirole(Quinp; 1 mM) (A and B), and then for a further 5 min in the presence of vehicle (A andB), DHPG (25 mM) (A), or SKF81297 (1 mM) (B). The levels of GluA1 phosphorylated atSer845 were determined by Western blotting as described in Methods. Upper panelsshow representative immunoblots showing immunoreactivity corresponding to totalGluA1 (GluA1) or phosphorylated GluA1 (P-GluA1). Lower panels show summaries ofdata expressed as means� SEM (n¼ 4). ***p< 0.001 vs. Control; one-way ANOVAfollowed by Bonferroni-Dunn post-hoc test.

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with previous work (Bateup et al., 2010), we confirmed that DARPP-32 was reduced by 60% and 35% in D32f/fD1RCreþand D32f/fD2RCreþ,respectively (Fig. 4C). Notably, we found that the increase producedby DHPG on Ser845 phosphorylationwas prevented by inactivationof DARPP-32 in D2R-expressing striatopallidal MSNs (Fig. 4D).In contrast, inactivation of DARPP-32 in the D1R-expressingstriatonigral MSNs did not modify the effect of DHPG (Fig. 4E).

3.5. mGlu5R-dependent phosphorylation of GluA1 isprevented by activation of D2Rs

The above results indicate that activation of mGlu5Rs promotesDARPP-32-dependent phosphorylation of GluA1 specifically instriatopallidal MSNs, which are selectively enriched in D2Rs(Gerfen et al., 1990). Since D2Rs are negatively coupled to adenylylcyclase activity (Kebabian and Calne, 1979), we examined the effectproduced by their activation on DHPG-induced GluA1 phosphory-lation. We found that incubation of striatal slices with quinpirole,a D2R agonist, prevented the increase in Ser845 phosphorylationproduced by DHPG (Fig. 5A). In contrast, quinpirole did not affectGluA1 phosphorylation induced by the D1R agonist SKF81297(Fig. 5B).

4. Discussion

The main conclusion of this study is that mGlu5Rs play animportant role in the regulation of cAMP/PKA signaling, specificallyat the level of the striatopallidal MSNs of the indirect pathway.Previous work in transfected cells indicates that mGlu5Rs areable to stimulate cAMP production (Joly et al., 1995). Moreover,incubation with a non-specific mGluR agonist has been shown toincrease the accumulation of cAMP in striatal and hippocampalslices (Wang and Johnson, 1995; Winder and Conn, 1993). Thepresent results are in line with these initial findings and showthat at least part of the increase in cAMP signaling produced byactivation of mGluRs is achieved through mGlu5Rs.

mGlu5Rs are conventionally coupled to Gq-dependent activa-tion of phospholipase C, which leads to increased release of Ca2þ

from intracellular stores and stimulation of PKC (Conn and Pin,1997). However, in this study, incubation of striatal slices withDHPG did not produce any increase in GluA1 phosphorylation atSer831, which is specifically regulated by PKC and Ca2þ/calmodulinprotein kinase II (Barria et al., 1997; Roche et al., 1996). Thiscontrasts with results showing that infusion of DHPG in the ratdorsal striatum increases GluA1 phosphorylation both at Ser845and Ser831 (Ahn and Choe, 2009). Such a discrepancy may be dueto the different type of experimental conditions (i.e. local injectionin intact animals vs. incubation of striatal slices), or to the differentspecies (rat vs. mouse) utilized in the studies.

We found that the ability of DHPG to promote PKA-dependentphosphorylation of GluA1 requires tonic activation of A2ARs. Asimilar dependence on A2ARs has been documented with respectto the ability of a non-selective mGluR agonist to stimulate cAMPproduction in brain slice preparations (Wang and Johnson, 1995;Winder and Conn, 1993). In addition, a number of functionalresponses, including increased c-fos expression (Ferre et al.,2002), potentiation of NMDA receptor-mediated transmission(Domenici et al., 2004) and phosphorylation of DARPP-32 (Nishiet al., 2003) have been shown to occur as a result of a synergisticinteraction between A2ARs and mGlu5Rs. In agreement withthese observations, we demonstrate that blockade of A2ARs, orenzymatic inactivation of endogenous adenosine achieved withadenosine deaminase, abolish the PKA-dependent phosphoryla-tion of GluA1 produced by DHPG. This supports the idea that,in slice preparations, the levels of extracellular adenosine are

comparable to those found in the intact brain and are sufficientto occupy a significant proportion of A2ARs (Fredholm et al.,1984). Interestingly, addition of the A2AR agonist, CGS21680, toadenosine-depleted (i.e. adenosine deaminase treated) striatalslices does not produce any significant increase in Ser845phosphorylation, whereas incubation with CGS21680 plus DHPGresults in a large increase in GluA1 phosphorylation. Theseobservations indicate that in the striatum activation of A2ARs isnot sufficient per se to induce PKA-dependent phosphorylation ofGluA1 and that concomitant activation of mGlu5Rs is required toachieve this effect.

M.T. Dell’Anno et al. / Neuropharmacology 66 (2013) 179e186 185

The mechanism conferring to mGluR5 the ability to promoteA2AR-dependent cAMP signaling remains to be characterized. Oneplausible hypothesis is that mGlu5Rs and A2ARs interact physicallyby forming functionally active heteromers. In support of this possi-bility, complexes containing these receptors have been immuno-precipitated from transfected cells and striatal tissue (Ferre et al.,2002). These complexes may preferentially couple to Golf, insteadthan to Gq, thereby conferring to DHPG the ability to promote cAMPsignaling (Fiorentini et al., 2010; Hasbi et al., 2010). Further studieswill be necessary to identify specific domains involved in thisinteraction and to determine the role playedby heteromer formationin mGlu5R-mediated phosphorylation of GluA1.

One important conclusion stemming from the present study isthat the increase produced by DHPG on PKA-dependent GluA1phosphorylation requires concomitant phosphorylation of DARPP-32 at Thr34. Phospho-Thr34-DARPP-32 is a potent inhibitor ofprotein phosphatase-1 (PP-1), which is involved in the dephos-phorylation of GluA1 (Snyder et al., 2000). Thus, activation ofmGlu5Rs promotes phosphorylation of GluA1 by increasingPKA-dependent phosphorylation and suppresing PP-1-dependentdephosphorylation at Ser845.

The requirement of A2ARs in DHPG-dependent activation ofcAMP signaling suggests that this regulation is exerted specificallyin the striatopallidal MSNs which form the indirect pathway. Thisidea is further supported by the observation that specific inacti-vation of DARPP-32 in striatopallidal, but not in striatonigral,MSNs abolishes the increase in PKA-dependent phosphorylationproduced by DHPG. DARPP-32 is also necessary for the increase inGluA1 phosphorylation at Ser845 produced in the striatum bypsychostimulants, such as cocaine and amphetamine. This effect ismimicked by stimulation of D1Rs and is most likely occurring in theMSNs of the direct pathway (Snyder et al., 2000). Therefore, in thestriatum, mGlu5Rs and D1Rs produce similar activations of thecAMP/PKA/DARPP-32 cascade acting on striatopallidal andstriatonigral MSNs, respectively.

In this study, we show that the ability of DHPG to promotecAMP/DARPP-32-mediated phosphorylation of GluA1 is abolished bythe D2R agonist quinpirole. This effect indicates the existence of anantagonistic relationship between mGlu5Rs and D2Rs in striato-pallidal MSNs. Previous work indicated that quinpirole counteractsthe increase in GABA release produced in striatopallidal MSNsby combined activation of mGlu5Rs and A2ARs (Diaz-Cabialeet al., 2002). Moreover, the ability of quinpirole to inducecontralateral turning behavior in rats lesioned unilaterally with6-hydroxydopamine (a toxin used to generate a model of parkin-sonism) is decreased by injecting an mGlu5R agonist in the striatum(Popoli et al., 2001). The present results suggest that these antago-nistic actions may occur, in striatopallidal MSNs, as a result of theopposite regulation exerted bymGlu5Rs andD2Rs on cAMP signaling.

In conclusion, this study shows that activation of mGlu5Rs in thestriatum leads to cAMP/PKA/DARPP-32-dependent phosphoryla-tion of GluA1 at Ser845. Importantly, this effect: 1) occursselectively in the striatopallidal MSNs of the indirect pathway, 2) itis exerted via potentiation of tonic A2AR transmission and 3) it isprevented by activation of D2Rs. The antagonistic actions exertedon cAMP signaling by mGlu5Rs and D2Rs may be at the basis of theantipsychotic properties displayed by mGlu5R positive allostericmodulators (Kinney et al., 2005; Liu et al., 2008). Indeed, admin-istration of conventional antipsychotic drugs is also known topromote cAMP/DARPP-32 signaling specifically in striatopallidalMSNs and increase GluA1 phosphorylation at Ser845 (Bateup et al.,2008; Bonito-Oliva et al., 2011; Håkansson et al., 2006). Furtherstudies will be necessary to determine the involvement of changesin cAMP-mediated transmission in the therapeutic properties ofdrugs acting at mGlu5Rs.

Acknowledgments

We thank Dr. Paul Greengard for kindly providing D32f/fD1RCreþand D32f/fD2RCreþ conditional knock out mice and T34ADARPP-32 mutant mice. This work was supported by SwedishResearch Council Grant 13482, the Swedish Brain Foundation andthe Agence Nationale de la Recherche (ANR) in the frame of Era-NetNEURON (project ANR-08-NEUR-006-01) (to GF). SP was a recipientof a postgraduate fellowship from “Sapienza” University of Rome.

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