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Neuropharmacology 79 (2014) 559e566

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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

Haloperidol-induced Nur77 expression in striatopallidal neurons isunder the control of protein phosphatase 1 regulation by DARPP-32

Natalia Sánchez a,b,c,d,1, Renata Coura b,c,d,1, Olivia Engmann b,c,d,Lucile Marion-Poll b,c,d, Sophie Longueville b,c,d, Denis Hervé b,c,d,María E. Andrés a,**,1, Jean-Antoine Girault b,c,d,*,1

aMillennium Nucleus in Stress and Addiction (NEDA), Department of Cellular and Molecular Biology, Faculty of Biological Sciences,Pontificia Universidad Católica de Chile, Chileb Inserm, UMR-S 839, Paris, FrancecUniversité Pierre et Marie Curie (UPMC), Paris, Franced Institut du Fer à Moulin, Paris, France

a r t i c l e i n f o

Article history:Received 27 September 2013Received in revised form7 December 2013Accepted 7 January 2014

Keywords:StriatumDopamineOrphan nuclear receptorsAntipsychotics

Abbreviations: A2AR, adenosine 2A receptor; CDK5DAPI, 40 ,60-Diamidino-2-phenylindole; DARPP-32, dopphosphoprotein of 32 kDa; D1R, D2R, and D3R, dopaenhanced green fluorescent protein; ERK, extracelluGFP, green fluorescent protein; IEG, immediate-earlyglutamate receptor 5; MSN, medium-sized spiny nefactor-inducible gene B; NR4A1, nuclear receptor groclear hormone receptor 77; PBS, phosphate-buffered sprotein kinase; PKC, protein kinase C; PP1, protein perror of the mean; TBS, tris-buffered saline; TR3, test* Corresponding author. Inserm, UPMC UMR-S 839

rue du fer à moulin, 75005 Paris, France. Tel.: þ33 1** Corresponding author. NEDA, Department of CelFaculty of Biological Sciences, Pontificia Universidad CChile, Chile.

E-mail addresses: mandres@bio.puc.cl (M.E. Aninserm.fr (J.-A. Girault).

1 Equal contribution.

0028-3908/$ e see front matter � 2014 Elsevier Ltd.http://dx.doi.org/10.1016/j.neuropharm.2014.01.008

a b s t r a c t

Impaired dopaminergic signaling in the striatum is involved in diseases as diverse as Parkinson’s disease,addiction, and schizophrenia. An important pathophysiological aspect is the loss of balance betweenstriatopallidal and striatonigral pathways. Nur77 is an orphan nuclear receptor and dopamine-regulatedimmediate-early gene. Classical antipsychotic drugs widely used in the treatment of schizophrenia, suchas haloperidol, increase Nur77 mRNA expression in the striatum. However, little is known about theintracellular signaling pathways involved in Nur77 induction. Here, using pharmacological approachesand transgenic mutant mice, we investigated the mechanisms underlying the up-regulation of Nur77protein expression in the dorsal striatum after haloperidol injection. In drd1a::EGFP transgenic mice thatexpress GFP in D1 neurons, Nur77 up-regulation induced by haloperidol occurred predominantly in GFP-negative neurons. In Gaolf heterozygous mutant mice, in which cAMP production in response to A2A

stimulation is impaired in the striatum, haloperidol effect was not altered. In contrast, in DARPP-32knock-in mutant mice bearing a T34A point mutation of the site responsible for cAMP-dependentphosphatase 1 inhibition, Nur77 up-regulation by haloperidol was prevented. Haloperidol alsoinduced Nur77 protein in D2 neurons of the nucleus accumbens core of wild type but not T34A knock-inmice. Thus, our results show that expression of Nur77 is induced by haloperidol in D2 receptors-expressing medium-sized spiny neurons, through cAMP-dependent regulation of protein phosphatase1, which is likely to modulate the effects of other protein kinases. Our results clarify the mechanisms ofNur77 induction by antipsychotic and its possible contribution to extrapyramidal effects.

� 2014 Elsevier Ltd. All rights reserved.

, cyclin-dependent kinase 5;amine- and cAMP-regulatedmine receptors 1, 2, 3; EGFP,lar signal-regulated kinase;gene; mGluR5, metabotropicuron; NGFI-B, nerve growthup 4A number 1; Nur77, nu-aline; PKA, cAMP-dependenthosphatase 1; SEM, standardicular receptor 3., Institut du Fer à Moulin, 17,45 87 61 52.lular and Molecular Biology,atólica de Chile, Santiago de

drés), jean-antoine.girault@

All rights reserved.

1. Introduction

Nur77 (also known as NGFI-B, TR3, and NR4A1) is animmediate-early gene (IEG) of the nuclear receptor superfamily anda ligand-independent transcription factor (Lim et al., 1987;Milbrandt, 1988). As the other two receptors of the NR4A sub-group (Nurr1 and Nor-1), Nur77 rapidly responds to changes incellular environment. A wide range of stimuli, including growthfactors, cytokines, peptide hormones, stress, and neurotransmitterscan induce Nur77 expression. In the brain, Nur77 expression hasbeen closely associated to dopamine neurotransmission (Beaudryet al., 2000; Gervais et al., 1999; Langlois et al., 2001; Maheuxet al., 2005; Werme et al., 2000). Nur77 is detected in targetareas of dopamine neurons including the dorsal striatum, the

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nucleus accumbens, and the prefrontal cortex (Gervais et al., 1999;Xiao et al., 1996; Zetterstrom et al., 1996). Work from many labo-ratories indicates that Nurr77 mediates some long-term effects ofdopamine receptors blockade or stimulation (reviewed in Levesqueand Rouillard, 2007).

Typical and atypical antipsychotic drugs, currently used in thetreatment of schizophrenia, induce distinct patterns of Nur77expression in the brain (Beaudry et al., 2000; Werme et al., 2000).Up-regulation of Nur77 mRNA expression in the rodent striatum isinduced by blockade of D2/D3 dopamine receptors (D2R/D3R)(Ethier et al., 2004a; Gilbert et al., 2006; Maheux et al., 2005).Nur77 induction correlates with the affinity of antipsychotic agentsfor D2R and D3R (Maheux et al., 2005) and persists during long-term administration (Beaudry et al., 2000; Langlois et al., 2001).These observations led to the hypothesis that Nur77, together withother nuclear receptors, might mediate some of the effects ofantipsychotic agents (see Levesque and Rouillard, 2007). Moreover,typical antipsychotics can produce extrapyramidal motor symp-toms that are thought to primarily involve the dorsal striatum, aregion in which these drugs have a strong effect on Nur77expression (Beaudry et al., 2000; Werme et al., 2000). Nur77knockout mice have an augmented basal locomotor activity(Bourhis et al., 2009; Gilbert et al., 2006) and are resistant to cat-alepsy induced by D2R antagonists (Ethier et al., 2004a). Chronichaloperidol treatment of mice induces the appearance of vacuouschewing movements reminiscent of tardive dyskinesias in humansand this response is more pronounced in Nur77 KO mice (Ethieret al., 2004b). These results suggest that Nur77 could modulategenes mediating antipsychotic motor lateral effects.

Nur77 mRNA is induced by haloperidol in neurons that expressenkephalin (Beaudry et al., 2000), presumably striatopallidalGABAergic medium-size spiny neurons (MSNs) expressing D2R(Gerfen et al., 1990). Haloperidol selectively activates severalsignaling pathways in striatopallidal MSNs (Bateup et al., 2008;Bertran-Gonzalez et al., 2008, 2009), which co-express D2R andadenosine A2A receptors (A2AR) (Svenningsson et al., 1999b). Inthese neurons, D2R is coupled to Gi/o protein that inhibits adenylylcyclase in response to dopamine, whereas A2AR is coupled to Golfprotein that activates adenylyl cyclase (Corvol et al., 2001; Hervéet al., 2001; Kull et al., 2000). The blockade of D2R by haloperidolactivates cAMP-dependent protein kinase (PKA) signaling byremoving the inhibition exerted by D2R on adenylyl cyclase, whichis stimulated by A2AR under basal conditions (Fink et al., 1992;Svenningsson et al., 1999b). This antagonistic interaction betweenreceptors results in fine modulation of gene expression(Svenningsson et al., 1999a). cAMP effects in MSNs are amplifiedand diversified through the phosphorylation by PKA of dopamine-and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) onthreonine 34 (T34), which results in a potent inhibition of proteinphosphatase 1 (PP1; for recent reviews see Le Novere et al., 2008;Yger and Girault, 2011). Haloperidol selectively increases T34phosphorylation in D2R-expressing MSNs (Bateup et al., 2008) andselective deletion of DARPP-32 in these neurons abolisheshaloperidol-induced catalepsy (Bateup et al., 2010).

In cells in culture, transcription of Nur77 is regulated by PKA,extracellular signal-regulated kinase (ERK) and its effectors,mitogen and stress activated kinases (MSKs; Darragh et al., 2005;Fass et al., 2003; Kovalovsky et al., 2002). Although little isknown about the signaling pathways mediating the induction ofNur77 by antipsychotic drugs, a recent work using high doses ofkinase inhibitors provided evidence that both PKC and ERK areinvolved in the effects of eticlopride, a selective D2R/D3R antago-nist (Bourhis et al., 2008). Here, we usedmutant mice to investigatethe signaling pathways regulating Nur77 protein expression afterhaloperidol injection. Our results show that Nur77 expression

occurs predominantly in striatopallidal neurons and involves in-hibition of protein phosphatase 1 through DARPP-32.

2. Material and methods

2.1. Animals

Male C57Bl/6J wild type mice (Charles River, France) aged 9e12 weeks wereused in this study. We also used three lines of transgenic mice. C57Bl/6JeSwissWebster hybrid mice that carry a bacterial artificial chromosome encoding theenhanced green fluorescent protein under the control of D1R promoter(drd1a::EGFP; Gong et al., 2003) were generated by GENSAT (Rockefeller University,New York, NY). The Gnalþ/� mice, which carry a heterozygous disruption of the Gnalgene that encodes the Gaolf protein (Belluscio et al., 1998), were kindly provided by L.Belluscio (NINDS, NIH, Bethesda, MD, USA), and backcrossed for eight generationswith C57BL/6J wild-type mice. They were mated with C57BL/6J mice for producingheterozygous Gnal mutant mice and wild-type littermates. The DARPP-32 T34Aknock-in mice, in which T34 was replaced by alanine (Svenningsson et al., 2003),was kindly provided by Paul Greengard (the Rockefeller university, New York, NY,USA). Mice were group-housed (four mice/cage) for one week to acclimatize to theanimal facility under a 12/12 h light/dark cycle (light on at 8.00 am) and stableconditions of temperature (22 �C), with food and water ad libitum. All experimentalprocedures were carried out in accordance with the ethical standard defined byInserm and European Community guidelines for care of laboratory animals (n�86/609). All procedures were conducted in order to reduce the number of mice usedwhen possible and to reduce their level of pain and discomfort as much as possible.

2.2. Drug treatments

Haloperidol (0.5 mg/kg, SigmaeAldrich) was dissolved in saline containing 5%(vol/vol) acetic acid and the pH was adjusted to 6.0 with 1 M NaOH. Quinpirole(1 mg/kg, SigmaeAldrich) was dissolved in saline. Mice were injected intraperito-neally (i.p) with the drug or the corresponding vehicle in their home cage and killed2 h later as described below.

2.3. Tissue preparation

Mice were deeply anaesthetized with an i.p. injection of pentobarbital (500 mg/kg, Sanofi-Aventis, France) and transcardially perfused with 4% (weight/vol) para-formaldehyde in 0.1 M sodium phosphate buffered saline (PBS), pH 7.4. Brains wererapidly removed and post-fixed overnight in the same solution at 4 �C. Thirty-mmthick sections were cut on a vibratome (Leica, France). Sections were collected in0.1 M PBS and stored at�20 �C in a solution containing 30% (vol/vol) ethylene glycol,30% (vol/vol) glycerol, and 0.1 M PBS, until they were processed for immunofluo-rescence. Brain regions were identified using a mouse brain atlas (Paxinos andFranklin, 2001), and sections including the medial nucleus accumbens and thedorsal striatum (antero-posterior distance from bregma, about 0.98 mm) wereselected for analysis.

2.4. Nur77 immunofluorescence

Immunofluorescent detection of Nur77 was performed as previously described(Campos-Melo et al., 2011). Briefly, free-floating brain sections were rinsed in Tris-buffered saline (TBS; 25 mM Tris and 150 mM NaCl, pH 7.5), incubated for 25 minat 80 �C in sodium citrate-buffer (10 mM sodium citrate and 0.05% (vol/vol) Tween20, pH 6) for antigen retrieval, and then rinsed three times in TBS for 10 min. Sec-tions were incubated in a 4% BSA-0.5% Triton X-100-TBS solution for 1 h. Sectionswere then incubated with the mouse anti-Nur77monoclonal antibody (1:100, SantaCruz Biotechnology) in the same solution for 36 h at 4 �C under agitation. Sectionswere rinsed three times in TBS for 10min and incubated in donkey anti-mouse Alexa488- or 568- (for colabeling with GFP) conjugated (1:500, Invitrogen) antibody in 4%BSA-0.5% Triton X-100-TBS for 90 min at room temperature. Finally, sections wererinsed twice during 10 min in TB (25 mM Tris) and mounted on slides with coverslipon Vectashield Mounting Mediumwith DAPI (Vector Laboratories INC., Burlingame,CA, USA).

2.5. Image analysis

Confocal microscopy and image analysis were carried out at the Institut du Fer àMoulin Imaging Facility. Double-labeled images were obtained bilaterally usingsequential laser scanning confocal microscopy (SP5, Leica). EGFP-labeled neuronswere visualized by direct detection of GFP fluorescence. Neuronal quantificationwasperformed in 375 mm � 375 mm confocal images by counting Nur77-immunofluorescent nuclei in the dorsolateral striatum, and in some experimentsthe nucleus accumbens core, using Image J software (NIH). In experiments withdrd1a::EGFP mice images were then combined with EGFP fluorescence. Image an-alyses were performed by an observer blind to treatment. Nur77-positive cells werecounted bilaterally in a 350 � 350 mm area, in 3 sections (i.e. 0.735 mm2).

Fig. 1. D2R/D3R control Nur77 protein expression in the dorsolateral striatum. Mice were injected with saline, haloperidol (0.5 mg/kg) or quinpirole (1 mg/kg) and perfused 2 hlater. A) Confocal sections of the dorsal striatum showing immunofluorescence for Nur77 (green, left panels) in each condition. Cell nuclei were stained with DAPI (blue). The rightpanels show a zoomed view of the area indicated by a square in the merge panel Scale bar: 100 mm. B) Quantification of the number of Nur77-positive cells in the dorsolateralstriatum of saline- or haloperidol-injected as illustrated in A). Data are means þ SEM. One-way ANOVA: F(2,9) ¼ 4.85, p ¼ 0.038. Post hoc Bonferroni test, quinpirole vs haloperidol,*p < 0.05.

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2.6. Statistical analysis

Immunofluorescence data were analyzed by one-way ANOVA followed byBonferroni’s post-hoc test or two-way ANOVA. Results are expressed as number ofcells positives for Nur77 immunoreactivity and correspond to group means � SEM.Statistical analyses were performed with Graphpad Prism 5.01 software.

3. Results

3.1. Effects of acute haloperidol or quinpirole injection on Nur77protein levels

Although Nur77 mRNA expression has been shown to be up-regulated in rodent striatum after D2R/D3R blockade (Ethieret al., 2004a; Gilbert et al., 2006; Maheux et al., 2005), the

consequences of this up-regulation at the protein level are notknown. To address this question, C57Bl/6J wild type mice wereacutely injected with a D2R antagonist (haloperidol 0.5 mg/kg) oragonist (quinpirole 1 mg/kg) and Nur77 protein expression wasevaluated by counting Nur77-like immunofluorescence-positivecells. Haloperidol increased the number of cells expressing Nur77in the dorsal striatum (Fig. 1A, B), whereas quinpirole administra-tion had an opposite effect (Fig. 1A, B). These data show that Nur77protein levels are regulated by D2/D3 dopamine receptors.

3.2. Haloperidol selectively induces Nur77 expression in D2R MSNs

D1R and D2R are by far the most abundant dopamine receptorsexpressed in the striatum. These receptors are highly segregated in

Fig. 3. Heterozygous loss of Gaolf does not prevent haloperidol-induced Nur77expression in the dorsolateral striatum. WT and Gnal þ/� mice (Gnal is the genecoding for Gaolf) were injected with saline or haloperidol (0.5 mg/kg) and perfused 2 hlater. Nur77-positive cells were quantified as in Fig. 2. Data are means þ SEM. Two-wayANOVA: Interaction, F(1,37) ¼ 0.3, NS; genotype effect, F(1,37) ¼ 0.55, NS; treatment effectF(1,37) ¼ 47.6, p ¼ 0.0068.

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the two populations of MSNs, with the D1R mostly located inneurons of the striatonigral direct pathway, and D2R in striato-pallidal MSNs of the indirect pathway (Gerfen et al., 1990). Recentwork in EGFP BAC transgenic mice confirmed this segregation withan estimated colocalization of D1R and D2R in only 5% or less of thedorsal striatum MSNs (Ade et al., 2011; Bertran-Gonzalez et al.,2008; Matamales et al., 2009; Thibault et al., 2013). In order todetermine inwhich cell type Nur77 protein expression is regulated,we investigated its immunofluorescence after haloperidol admin-istration in Drd1a::EGFP mice. In these mice, 2 h after the admin-istration of haloperidol, a marked increase in the number of Nur77positive cells was observed in neurons that did not express GFP(Fig. 2A, B). These cells were medium-sized neurons and corre-sponded presumably mostly to D2R-expressing MSNs. A few EGFP-positive neurons expressing Nur77 were also observed in micetreated with haloperidol but not in mice treated with saline (Fig. 2).Thus, haloperidol induced Nur77 expression in the dorsolateralstriatum mostly in D2R-expressing neurons.

3.3. Haloperidol-induced Nur77 expression is not altered byheterozygous mutation of Gaolf, the G protein associated with A2AR

The D2R-positive MSN population expresses A2AR, which is Gprotein-coupled receptors activating adenylyl cyclase and the cAMPpathway (Fink et al., 1992; Schiffmann et al., 1991). A2AR opposesthe action of D2R (Svenningsson et al., 1999a). We investigatedwhether A2AR is implicated in the up-regulation of Nur77 expres-sion induced by haloperidol. We first found that SCH58261, an A2ARantagonist, had no effect on haloperidol-induced Nur77 expression(data not shown), in agreement with a previous report (Maheuxet al., 2012). We then tried a different A2AR antagonist, KW6002.However, this compound had an effect by itself suggesting a non-specific action (data not shown) and preventing evaluation of therole of A2AR in Nur77 regulation. We therefore turned to a differentapproach taking advantage of the fact that in the striatum A2AR iscoupled to adenylyl-cyclase through Gaolf-containing hetero-trimeric G protein (Corvol et al., 2001; Hervé et al., 2001). In Gnalþ/�

heterozygous mutant mice, which express about 50% of the wildtype levels of Gaolf, the activation of the cAMP pathway in vivo byA2AR is dramatically altered (Corvol et al., 2007). This impairmentblocks some signaling pathways activated in striato-nigral neurons

Fig. 2. Haloperidol increases Nur77 protein expression predominantly in D2R-expressingtreated with saline or haloperidol (0.5 mg/kg) and perfused 2 h later. A) Confocal sections in(red, left panels) or in combination with GFP fluorescence (green, middle panels). A magnifipanel (zoom). Scale bar: 100 mm. B) Quantification of the number of Nur77-positive cells aminjected Drd1a::EGFP mice as illustrated in A. Data are means þ SEM. Two-way ANOVA: InF1,16 ¼ 6.24, P ¼ 0.027; effect of haloperidol F1,16 ¼ 11.11, P ¼ 0.005. Post hoc Bonferroni testnegative, *p < 0.05.

by psychostimulants or L-DOPA (Alcacer et al., 2012; Corvol et al.,2007) and in striato-pallidal neurons by antipsychotic drugs(Valjent et al., 2011).We therefore investigated the consequences ofGaolf heterozygous mutation on the effects of haloperidol on Nur77expression using Gnalþ/� mice (Gnal is the gene coding for Gaolf,Fig. 3). The haloperidol-induced increase in Nur77-positive neuronswas not altered in Gnalþ/� mice as compared to wild type mice(Fig. 3). These results revealed that the partial impairment of Golfsignaling, which dramatically alters some A2AR responses in striatalneurons (Corvol et al., 2001, 2007) was not sufficient to preventNur77 induction, suggesting the existence of potent amplificatorymechanisms for Nur77 induction.

3.4. DARPP-32 is involved in the control of Nur77 expression

DARPP-32 is a key protein integrating signaling in MSNs whichis involved in a wide variety of biochemical, physiological, and

MSNs. Mice expressing EGFP under the control of D1R promoter (Drd1a::EGFP) werethe dorsal striatum of Drd1a::EGFP mice showing immunofluorescence for Nur77 alone,cation of the area indicated by a white square in the merge panel is shown in the rightong GFP-positive or negative cells in the dorsolateral striatum of saline- or haloperidol-teraction between cell population and drug, F1,16 ¼ 5.33, P ¼ 0.038; effect of cell type, haloperidol in GFP-positive vs GFP-negative, ��p < 0.01, haloperidol vs saline in GFP-

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behavioral responses to pharmacological treatments (reviewed inLe Novere et al., 2008; Svenningsson et al., 2004; Walaas et al.,2011; Yger and Girault, 2011). DARPP-32 has diverse actionsdepending on the stimuli. When it is phosphorylated on T34 byPKA, DARPP-32 is a potent inhibitor of PP1 (Hemmings et al., 1984),whereas when it is phosphorylated on T75 by CDK5 it becomes aninhibitor of PKA (Bibb et al., 1999). To address specifically the role ofPP1 regulation, we used knock-in mice bearing a point mutation bywhich T34 is replaced by alanine (Svenningsson et al., 2003). Inthese mice the inhibition of PP1 by active DARPP-32 is selectivelyimpaired, whereas the other properties of the protein are un-changed. The results we obtained in the T34A mutant mice werevery clear (Fig. 4). The effects of haloperidol and of genotype weresignificant as well as their interaction (F and p values in Fig. 4legend). The number of Nur77-positive nuclei was lower insaline-injected mutant mice than in wild type littermates andhaloperidol injection had virtually no effect. These results clearlyshowed that DARPP-32 phosphorylation on T34 is critical forhaloperidol-induced Nur77 expression in the striatum. Therebythey demonstrate the importance of PP1 inhibition in Nur77regulation in striatal neurons in vivo.

Fig. 4. Thr-34 in DARPP-32 is essential for haloperidol-induced Nur77 induction in thedorsolateral striatum. WT or DARPP-32 T34A knock-in mutant mice were treated withsaline or haloperidol (0.5 mg/kg) and perfused 2 h later. A) Immunofluorescence withanti-Nur77 antibody (green) in brain sections of injected mice with the indicated drug/genotype combinations. Nuclei were stained with DAPI (blue). Scale bar: 100 mm. B)Quantification of the number of Nur77-positive cells in the dorsal striatum. Data aremeans þ SEM. Two-way ANOVA: Interaction F(1,10) ¼ 5.4, p ¼ 0.04; haloperidoltreatment effect, F(1,10) ¼ 8.7, p ¼ 0.01; genotype effect, F(1,10) ¼ 18, p ¼ 0.001. Post hocBonferroni test, haloperidol vs saline in wt mice, *p < 0.05, haloperidol in wt vs DARPP-32 T34A mice, ��p < 0.01.

3.5. DARPP-32 phosphorylation is also implicated in haloperidol-induced expression of Nur77 in D2 neurons of the nucleusaccumbens

To investigate whether the regulatory mechanisms controllingNur77 in the dorsolateral striatum are also operating in otherstriatal regions, we examined the effects of haloperidol in the nu-cleus accumbens core. Examination of sections in mice expressingGFP in D1 MSNs showed that Nur77 also occurred in GFP-negativeneurons (Fig. 5A). We then studied the response to haloperidol inwild type and T34A knock-in mice (Fig. 5B). The effects of halo-peridol were clearly visible in the nucleus accumbens core of wildtypemice whereas only a very small non-significant effect was seenin T34A mice (Fig. 5C). These experiments showed that Nur77 isregulated in the nucleus accumbens as in the dorsal striatum andthat the role of PP1 inhibition by phosphorylated DARPP-32 isimportant in both regions.

4. Discussion

Nur77 and other nuclear receptors have been suggested to beimportant players in the long-term adaptations in dopamine-regulated brain regions (Levesque and Rouillard, 2007). Since lit-tle is known about the molecular bases of the regulation of Nur77expression in vivo we have investigated the signaling mechanismsinvolved using pharmacological tools and transgenic mutant mice.Our data show that Nur77 protein expression is regulated inopposite directions in the dorsal striatum by D2R/D3R antagonistand agonist drugs. Haloperidol, as expected, increased the numberof Nur77-positive neurons, whereas quinpirole had an oppositeeffect. Using mice that express GFP under the control of the D1Rpromoter we found that in response to haloperidol Nur77 proteinwas very predominantly increased in MSNs not expressing GFP,therefore striatopallidal neurons (see Valjent et al., 2009 for a re-view). This result confirms and expands previous findings showingthat haloperidol-induced expression of Nur77 mRNA takes place inenkephalin-expressing cells of the dorsal striatum (Beaudry et al.,2000), whereas in basal conditions this transcript is colocalizedwith subsets of both preproenkephalin- and preprodynorphin-expressing neurons (Backman et al., 2001). The segregation ofdopamine receptors and neuropeptides between striatopallidal andstriatonigral neurons (Gerfen et al., 1990) has now been extensivelydocumented and extended to many other gene products (seeValjent et al., 2009). Haloperidol clearly increased Nur77 protein instriatopallidal MSNs that are known to express D2R, A2AR, andenkephalin. A recent report indicates an important role of pre-synaptic D2R in haloperidol-mediated induction of Nur77 (Maheuxet al., 2012). However, since different corticostriatal afferent fibersdo not appear to target a specific type of striatal neuron (Kress et al.,2013), the cell type specificity we observed suggests that specificfactors at the postsynaptic level are an important factor in the in-duction of Nur77 by dopamine selective antagonists.

How can the blockade of D2R induce the expression of Nur77?Since D2R are negatively coupled to adenylyl cyclase (Kebabian andCalne, 1979) a major aspect of the effects of D2R antagonists is thedisinhibition of the cAMP pathway. A2ARs stimulates cAMP pro-duction and is active in basal conditions due to the levels ofextracellular adenosine coming from multiple sources (Chen et al.,2013; Svenningsson et al., 1999b). Therefore A2ARs are involved inmany effects of D2R antagonists in vivo (Bertran-Gonzalez et al.,2009; Bonito-Oliva et al., 2013; Valjent et al., 2011). Caffeine, anA2AR antagonist, decreases Nur77 expression in enkephalin-containing neurons (Svenningsson et al., 1995). In contrastSCH58261, an antagonist of A2AR, had little effect by itself oneticlopride-induced Nur77 expression, although it potentiated the

Fig. 5. Haloperidol also induces Nur77 in the nucleus accumbens through regulation of DARPP-32. WT or DARPP-32 T34A knock-in mutant mice were treated with saline orhaloperidol (0.5 mg/kg) and perfused 2 h later. A) Confocal sections in the nucleus accumbens of Drd1a::EGFP mice showing immunofluorescence for Nur77 (red) or in combinationwith GFP fluorescence (green). A magnification of the area indicated by a white square is shown in the right panel (zoom). Scale bar: 100 mm. B) Immunofluorescence with anti-Nur77 antibody (green) in nucleus accumbens core sections of injected mice with the indicated drug/genotype combinations. Nuclei were stained with DAPI (blue). Scale bar:100 mm. C) Quantification of the number of Nur77-positive cells in the dorsal striatum. Data are means þ SEM. Two-way ANOVA: Interaction F(1,10) ¼ 10.4, p ¼ 0.009; haloperidoltreatment effect, F(1,10) ¼ 27.8, p ¼ 0.0004; genotype effect, F(1,10) ¼ 46.0, p < 0.0001. Post hoc Bonferroni test, haloperidol vs saline in wt mice, ***p < 0.001, haloperidol in wt vsDARPP-32 T34A mice, ���p < 0.001.

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inhibitory effect of a mGluR5 antagonist (Maheux et al., 2012). Inour experimental conditions, SCH58261 did not prevent haloper-idol effects (data not shown) and another A2AR antagonist, KW6002had intrinsic effects, preventing its use to explore the role of A2AR(data not shown). Therefore we investigated the role of Gaolf, whichmediates the effects of A2AR on adenylyl cyclase in MSNs (Corvolet al., 2001; Hervé et al., 2001; Kull et al., 2000). The levels ofGaolf are a limiting factor for signaling downstream of receptorsin vivo, as indicated by the dramatic alterations of PKA responses inheterozygous Gaolf (Gnalþ/�) mutant mice (Alcacer et al., 2012;Corvol et al., 2007). Accordingly, several signaling pathways acti-vated by haloperidol through disinhibition of A2AR are markedlyimpaired in Gaolf heterozygous mutant mice (Bertran-Gonzalezet al., 2009; Valjent et al., 2011). In contrast, the effects of halo-peridol on Nur77 expression were not altered in Gnalþ/� mice.These results reveal that Gaolf is not a limiting factor for Nur77induction by D2R blockade. This may indicate that a limited acti-vation of the cAMP pathway, possibly through A2AR, is sufficient forNur77 induction and/or that alternative signaling mechanisms arealso operating.

Our results in DARPP-32 mutant mice provide at least a partialexplanation for this observation. In T34Amutant mice inwhich PP1inhibition by DARPP-32 is selectively abrogated, while the otherfunctions are intact, the basal expression of Nur77 was decreasedand the effects of haloperidol were completely prevented. Thisclearly implicates phosphatase regulation as an importantcomponent in the control of Nur77, as previously reported for othergenes (Fienberg et al., 1998).

Our results allow better understanding the regulation of Nur77expression. It had been shown that in mice lacking the long isoformof D2R, presumably the major postsynaptic D2R in MSNs, halo-peridol was still capable to induce Nur77 expression, leading to thesuggestion of a major role of presynaptic D2R (Maheux et al., 2012).Our results do not contradict this hypothesis. They underline,however, the importance of regulation of DARPP-32 phosphoryla-tion on T34, which results in PP1 inhibition. The combination of ablockade of haloperidol-induced Nur77 expression in T34A-DARPP-32 mice and its lack of impairment in Gnalþ/� mice indicates thatthe cAMP pathway plays a necessary permissive role through PP1inhibition, but that its strong activation is not required. In fact,

phosphatase inhibition is likely to potentiate the effects of otherkinases previously shown to be involved in the control of Nur77expression, including ERK and PKC (Bourhis et al., 2008). Themechanisms of activation of these kinases in haloperidol-treatedmice is not known but may involve the formation of heteromericreceptors including A2AR that may be favored when D2R areblocked. For example, mGluR5-A2AR complexes has been reportedto induce intracellular Ca2þ release via phospholipase C andinositol-trisphosphate pathways (Zezula and Freissmuth, 2008).Such effects, which are presumably independent of Gaolf, would becompatible with the present observations. However, further workis necessary to clarify the role of A2AR/mGluR5 heterodimers, if any,in Nur77 induction in the striatum.

In summary, our results clarify the regulation of Nur77 up-regulation by haloperidol, showing the critical role of phosphory-lation of DARPP-32 on T34 and of the resulting PP1 inhibition. Ourobservations also suggest that this regulatory mechanism maypotentiate other pathways including ERK and PKC that have beenpreviously suggested to be involved in Nur77 regulation. Nur77plays a key role in mediating the effects of haloperidol on theexpression of late genes such as enkephalin (Ethier et al., 2004a).Nur77 is also necessary for motor effects of antipsychotic agents(Ethier et al., 2004a, 2004b). In line with these results and with thepresent findings, DARPP-32 in striatopallidal neurons is alsonecessary for the cataleptic effects of haloperidol (Bateup et al.,2010). Therefore we suggest that the control of Nur77 expressionby both protein kinases and PP1 in striatopallidal neurons isimplicated in the extrapyramidal effects of typical antipsychoticdrugs. Whether similar pathways are also involved in otherdopamine-dependent neuro-adaptations and in the therapeuticeffects of antipsychotics will be the topic of further investigations.

Acknowledgments

The authors thank Pr. Paul Greengard, the Rockefeller University,New York, for kindly providing mutant mice used in this study. Thisstudy was supported in part by FONDECYT N� 1110352, MSI N� P10-063-F, CONICYT Doctoral Thesis Fellowship 24121352 to NS. Workin Girault’s lab was supported by Inserm, Université Pierre et MarieCurie (UPMC), the Michael Stern Parkinson’s Research Foundation,

N. Sánchez et al. / Neuropharmacology 79 (2014) 559e566 565

and European Research Council. Girault’s and Hervé’s group isaffiliated with the Paris school of Neuroscience (ENP) and the Bio-Psy laboratory of excellence.

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