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Neuropharmacology 93 (2015) 308e313

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Neuropharmacology

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

Postsynaptic D2 dopamine receptor supersensitivity in the striatum ofmice lacking TAAR1

Stefano Espinoza a, Valentina Ghisi b, Marco Emanuele a, Damiana Leo a, Ilya Sukhanov a,Tatiana D. Sotnikova a, c, Evelina Chieregatti a, Raul R. Gainetdinov a, c, d, *

a Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, 16163 Genova, Italyb Leibniz-Institut für Molekulare Pharmakologie (FMP), 13125 Berlin, Germanyc Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg 199034, Russiad Skolkovo Institute of Science and Technology (Skoltech) Skolkovo, Moscow Region 143025, Russia

a r t i c l e i n f o

Article history:Received 28 November 2014Received in revised form13 February 2015Accepted 16 February 2015Available online 24 February 2015

Keywords:TAAR1DopamineStriatumD2 receptor

Abbreviations: TAAR1, Trace Amine-AssociatedGPCR, G protein-coupled receptor; VTA, ventral tegm* Corresponding author. Department of Neuroscie

Italian Institute of Technology (IIT) Via Morego 30, G010 71781516; fax: þ39 010 720321.

E-mail address: raul.gainetdinov@iit.it (R.R. Gaine

http://dx.doi.org/10.1016/j.neuropharm.2015.02.0100028-3908/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) known to modulatedopaminergic system through several mechanisms. Mice lacking this receptor show a higher sensitivityto dopaminergic stimuli, such as amphetamine; however, it is not clear whether D1 or D2 dopaminereceptors and which associated intracellular signaling events are involved in this modulation. In thestriatum of TAAR1 knock out (TAAR1-KO mice) we found that D2, but not D1, dopamine receptors wereover-expressed, both in terms of mRNA and protein levels. Moreover, the D2 dopamine receptor-relatedG protein-independent AKT/GSK3 signaling pathway was selectively activated, as indicated by thedecrease of phosphorylation of AKT and GSK3b. The decrease in phospho-AKT levels, suggesting an in-crease in D2 dopamine receptor activity in basal conditions, was associated with an increase of AKT/PP2Acomplex, as revealed by co-immunoprecipitation experiments. Finally, we found that the locomotoractivation induced by the D2 dopamine receptor agonist quinpirole, but not by the full D1 dopaminereceptor agonist SKF-82958, was increased in TAAR1-KO mice. These data demonstrate pronouncedsupersensitivity of postsynaptic D2 dopamine receptors in the striatum of TAAR1-KO mice and indicatethat a close interaction of TAAR1 and D2 dopamine receptors at the level of postsynaptic structures hasimportant functional consequences.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) expressed in mammalian brain in majormonoaminergic regions, such as ventral tegmental area (VTA) anddorsal raphe, and their projections, including striatum, amygdala,hypothalamus and frontal cortex (Borowsky et al., 2001; Bunzowet al., 2001; Di Cara et al., 2011; Lindemann et al., 2008; Wolinskyet al., 2007). Many studies have focused on the role of TAAR1 inthe modulation of dopaminergic system, and it is evident thatgenerally TAAR1 exerts a negative control on dopaminergic activity

Receptor 1; DA, dopamine;ental area.nce and Brain Technologies,enova 16163, Italy. Tel.: þ39

tdinov).

(Di Cara et al., 2011; Lindemann et al., 2008; Revel et al., 2011, 2012,2013; Sukhanov et al., 2014). TAAR1 knock out (TAAR1-KO)mice aremore sensitive to the neurochemical and behavioral effect ofseveral amphetamine derivatives (Di Cara et al., 2011; Lindemannet al., 2008; Miller, 2012; Wolinsky et al., 2007). Moreover, VTAdopaminergic neurons of TAAR1-KO mice show an increase in thefiring rate (Lindemann et al., 2008) and dopamine release in thenucleus accumbens due to dysregulated function of D2 dopamineautoreceptors (Leo et al., 2014). Conversely, TAAR1 selective ago-nists decrease hyperdopaminergic manifestations induced bypharmacological tools or present in genetic mouse models (e.g.dopamine transporter knock out (DAT-KO) mice) (Espinoza andGainetdinov, 2014; Revel et al., 2011, 2012, 2013). Several reportshave indicated that TAAR1 is able to influence striatum-dependentbehaviors, such as amphetamine-induced locomotor hyperactivityand haloperidol-induced catalepsy (Espinoza et al., 2011; Wolinskyet al., 2007). However, the mechanism by which TAAR1 alters thedopamine system, dependent in major part on the D1-and D2-

S. Espinoza et al. / Neuropharmacology 93 (2015) 308e313 309

classes of dopamine receptor function, remains unclear. It has beenreported that TAAR1-KO mice have higher number of striatal D2dopamine receptors in high affinity state (Wolinsky et al., 2007).Another study showed that in vitro TAAR1 and the long isoform ofthe D2 dopamine receptor were able to form an heterodimer andthat the formation of this complex influences TAAR1-dependentcAMP signaling (Espinoza et al., 2011). However, it remained un-clarified how D2-related signaling and functions are altered in vivoin TAAR1-KO mice. Furthermore, no information is available on thestatus of D1 dopamine receptor activity in TAAR1-KOmice. The aimof our study was to evaluate the functionality of D1 and D2 dopa-mine receptors in the striatum of TAAR1-KO mice. We found that,while D1 dopamine receptors displayed no abnormalities, D2dopamine receptors were overexpressed and D2-related G protein-independent signaling pathway was activated. Furthermore, thesedata indicating selective increase of postsynaptic D2 dopaminereceptor activity in TAAR1-KO mice were directly confirmed inbehavioral experiments with selective agonists.

2. Materials and methods

2.1. Animals

TAAR1 knock out (TAAR1-KO) mice of mixed C57BL/6J � 129Sv/J backgroundwere generated as previously described (Wolinsky et al., 2007). Animal care andtreatments were performed in accordance with the Guide for the Care and Use ofLaboratory Animals (USA National Institutes of Health publication #865-23,Bethesda, MD), and the protocols were approved by the Italian Ministry of Health.All TAAR1-KO and WT littermates were obtained from heterozygous mating, andgenotyping was performed in all individuals. Mice of both sexes were used in allexperiments.

2.2. Western blot

Western blot analyses of striatal samples to evaluate dopamine signaling wereperformed as previously described (Beaulieu et al., 2004). The conditions of theprimary antibodies were as follows: anti phospho-AKT (Thr308) (1:3000), antiphospho-GSK3b (Ser9) (1:3000), anti b-catenin (1:1000), anti b-arrestin2 (1:2000),anti PP2A (1:5000), anti phospho-ERK1/2 (1:10,000), anti phospho-CREB (1:1000),anti phospho-DARPP32 (Thr74) (1:1000), anti-total AKT (1:5000) and anti-totalGSK3b (1:5000). All antibodies were purchased from Cell Signaling (Danvers, MA,USA). Total proteins were measured by the Bio-Rad Protein Assay (Bio-Rad Labo-ratories, Segrate, Milan, Italy). Twenty micrograms of proteins for each sample wererun on a SDS-10% polyacrylamide gel under reducing conditions and then electro-phoretically transferred onto nitrocellulose membranes (GE Healthcare, Milan,Italy). Blots were blocked for 1 h at room temperature with 10% non-fat dry milk inTBS þ 0.1% Tween-20 buffer, incubated with antibodies against the phosphorylatedforms of the proteins and then stripped and reprobed with antibodies against thecorresponding total proteins. Immunocomplexes were visualized by chem-iluminescence using the Chemidoc MP Imaging System (Bio-Rad Laboratories).Densitometric analysis was performed by normalizing the phosphoproteins level tothe corresponding total form. The total form of the proteins was then normalized tothe actin levels.

2.3. Immunoprecipitation

Immunoprecipitations were carried out as previously described (Beaulieu et al.,2005). Briefly, tissue was homogenized at 4 �C in 200 ml of lysis buffer (10 mMTriseHCl [pH 7.4], 1.0% Triton X-100) containing a cocktail of protease inhibitors(SigmaeAldrich). Immunoprecipitations were conducted overnight at 4 �C withanti-AKT antibody, followed by incubation with protein G sepharose (Ge-Healthcare).

2.4. D1 and D2 dopamine receptor saturation binding assay on striatal membranes

Binding experiments were conducted according to Ghisi et al. (2009); Striataltissues from wild-type, DAT-KO and DAT-tg mice were rapidly dissected andimmediately homogenized, using a Teflonglass homogenizer, in 2 ml of lysis buffercontaining 50 mM TriseHCl (pH 7.4), 120 mM NaCl, 1 mM EDTA and a cocktail ofprotease inhibitors at 1:1000 dilution. The homogenatewas centrifuged at 1000 rpmfor 10 min at 4 �C to remove tissue debris and nuclei and the resulting supernatantcentrifuged twice at 40,000 g for 20 min at 4 �C. The final pellet was suspended inassay buffer containing 50 mM TriseHCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 2 mMCaCl2, 1 mM MgCl2. For D1 receptor saturation experiments, membranes (50 ml;1.2 mg/ml final proteins concentration) were incubated with six increasing concen-trations of [3H]-SCH23390 (50 ml; range: 0.5e16 nM) and 50 ml of ketanserin(100 nM), a specific antagonist of serotonin receptor, in order to prevent the bindingof [3H]-SCH23390 to these receptors. For D2 receptor saturation experiments both

[3H]-Spiperone and [3H]-Raclopride selective D2 antagonists were used. In the caseof [3H]-Spiperone, membranes (150 ml; 0.5 mg/ml final proteins concentration) wereincubated with six increasing doses of this radioactive ligand (50 ml; range:0.03e1 nM), while for [3H]-Raclopride, membranes (50 ml; 1.2 mg/ml final proteinsconcentration) were incubated with six increasing doses of [3H]-Raclopride (100 ml;range: 1.8e60 nM). The different reactions were carried out at RT for 1 h in the caseof [3H]-SCH23390 and [3H]-Raclopride in a total volume of 200 ml of assay bufferand at RT for 2 h in the case of [3H]-Spiperone in a total volume of 250 ml of assaybuffer. Nonspecific binding was measured using nonradioactive flupenthixol(10 mM) for [3H]-SCH23390 binding or haloperidol (6 mM for [3H]-Spiperonebinding, 50 mM for [3H]-Raclopride binding) in parallel assay tubes and was sub-tracted from total binding to obtain respectively specific [3H]-SCH23390 or [3H]-Spiperone/[3H]-Raclopride binding. The incubations were terminated by rapidfiltration over Brandel GF/C glass fiber filters washed with ice-cold assay buffer. Thefilters were incubated overnight in 5 ml high flash point scintillation cocktail (Lefko-Fluor) before their radioactivity content was counted by a liquid scintillationcounter.

2.5. Real time PCR

WT and TAAR1-KO mice of 3 months were sacrificed, striata were dissected andstriatal tissue was dissociated for 15 min at 37 �C with Pronase enzyme (Sigma) inHank's Balanced Salt Solution (HBSS, Invitrogen). Brain samples were trituratedwiththree glass pipettes of decreasing tip diameter and centrifuged at 900 rpm at roomtemperature for 5 min. To remove excess debris, cell pellets were resuspended inHBSS and filtered through a 70 mm mesh (BD Falcon, #352350). Cells-to-CT kit (LifeTechnology) was used to produce DNase I digested cell lysates and perform cDNAsynthesis, according to manufacturer's instructions. cDNA were used for Taqmansingleplex PCR. All reagents were supplied by Applied Biosystems. PCR master mixcontained 1x Taqman Universal PCR Master Mix, 1x Gene Expression Assay mix, and1 ml cDNA for a total volume of 20 ml. The following Gene Expression Assays wereused: Drd2 (Assay ID Mm00438545_m1), Drd1a (Assay ID Mm01353211_m1),Gapdh (Assay IDMm99999915_g1) and Hprt (Assay IDMm00446968_m1). Sampleswere run in three replicates for each Gene Expression Assay. PCR reactions werecarried out on a 7900 Thermal Cycler (Applied Biosystems) with 40 cycles of 95 �Cfor 15 s and 60 �C for 1min. CT values for each genewere normalized to CT values forGAPDH and HPRT to obtain a relative expression level for each replicate and the fivereplicates were averaged together. Gene expression data were normalized by themultiple internal control gene method with GeNorm algorithm available in qBase-Plus software (Biogazelle).

2.6. Locomotor activity

Locomotor activity after drug or saline treatment was assessed as previouslydescribed (Gainetdinov et al., 2003). Locomotion was evaluated in an automatedOmnitech Digiscan apparatus (AccuScan Instruments, Columbus, OH) under illu-minated conditions. Apparatus included 4 open field monitors. Each Open Fieldmonitor consisted of sets of 16 light beams arrayed in the horizontal X and Y axes.The hardware detected beams broken by the animal so that the software candetermine the location of the mouse within the cage. Cages were divided on 4compartments (20 cm � 20 cm). Animals were tested individually for defined pe-riods with 5-min intervals. Horizontal activities were expressed in terms of thenumber of beam breaks. Briefly, WTor mutant animals were injected i.p. with eithersaline, 2 mg/kg quinpirole or 0.5 mg/kg SKF-82958 (Chloro-APB) and placed in theactivity monitor. The experimenter conducting the behavioral test was blinded forgenotype/treatment of animals. Cumulative counts for 30 min (starting 10 min afterdrug or saline injection to avoid handling effects) were recorded for data analysis.We used both sexes in the locomotor tests, and initially analyzed male and femalesseparately. Since we did not observe differences in effects of drugs between malesand females we pooled the data together in the final analysis.

3. Results

3.1. Lack of TAAR1 modulates D2 dopamine receptor expression inthe striatum

Previous studies have documented TAAR1 expression in severalbrain regions, including the striatum, with some discrepancies re-ported likely due to the low level of TAAR1 expression inmost areas(Di Cara et al., 2011; Lindemann et al., 2008). Thus, we firstconfirmed TAAR1 expression in the striatum. Using RT-PCR, wedocumented that TAAR1 is expressed in the striatum with nodetection of transcript in TAAR1-KO mice (Fig. 1A). As a first step toevaluate the consequences of TAAR1 deletion on dopamine trans-mission in this area, we evaluated the levels of D1 and D2 dopaminereceptor mRNA expression in the striatum of mutant mice. We

Fig. 1. D2 but not D1 dopamine receptor expression is increased in the striatum of TAAR1-KO mice. (A) mRNA levels of TAAR1 in striatum of WT and KO mice relative to thehousekeeping genes GAPDH and HPRT as measured by RT-PCR. (B) mRNA levels measured with RT-PCR from striatal extracts. (C) D1 and D2 dopamine receptor levels weremeasured by saturation binding experiments. [3H]-SCH23390 and [3H]-Spiperone were used to quantify D1 and D2 dopamine receptors in the striatal membranes. Levels areexpressed as a percentage compared to the control. Data were analyzed by GraphPad Prism software using a one site binding (hyperbola) equation. N ¼ 5 mice per group; datarepresent means ± SEM. *, P < 0.05. Student's unpaired two-tailed t-test.

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extracted total RNA from dissected striata of WT and TAAR1-KOmice, and using qRT-PCR, we measured D1 and D2 dopamine re-ceptor mRNA levels. As shown in Fig. 1B, D2 dopamine receptormRNA levels were significantly increased, while D1 dopamine re-ceptor mRNA levels were not altered. To test if dopamine receptorlevels were also affected, we directly measured the number of D1and D2 dopamine receptors by using radioligand binding. [3H]-SCH23390 was utilized to selectively detect the levels of D1dopamine receptors, while [3H]-Spiperone was used to quantify D2dopamine receptors. As shown in Fig. 1C, similar to what wasobserved with mRNA, TAAR1-KO mice had a 2-fold increase in thelevels of D2 dopamine receptors in the striatum, while therewas noalteration in D1 dopamine receptor density.

3.2. Striatal D2 dopamine receptor signaling is enhanced in TAAR1-KO mice

Evidence in the literature indicates a higher proportion ofstriatal D2 dopamine receptors in the high affinity state in TAAR1-KOmice compared toWTmice (Wolinsky et al., 2007). An increasedD2 high affinity state and an increase in amphetamine sensitivitywere also found in animal models of psychosis, as well as in clinicalstudies of schizophrenic patients (Seeman et al., 2005). Thesestudies, together with the present observations showing selectiveup-regulation of striatal D2 dopamine receptors in TAAR1-KOmice,led us to analyze the status of D2 dopamine receptor-relatedsignaling in TAAR1-KO mice. It is well known that D2 dopaminereceptors signal through the Gi proteins to reduce cAMP levels. D2dopamine receptors can also signal in a G-protein independentmanner through the multifunctional protein b-arrestin2, leading tothe dephosphorylation of AKT and the subsequent activation ofGSK3b (Beaulieu and Gainetdinov, 2011). Both of these pathwayshave been shown to be important for dopamine-mediated behav-iors (Beaulieu and Gainetdinov, 2011). Thus, by using western blot,we analyzed the basal phosphorylation state of proteins associatedwith D1 and D2 dopamine receptor activation. We observed a basaldecrease in the phosphorylation of AKT and GSK3b in the striatumof TAAR1-KO mice (Fig. 2A, B), two downstream targets of b-arrestin2-dependent D2 dopamine receptor signaling (Beaulieuet al., 2005) with no alterations in the levels of the proteins(Fig. 2F). It has been demonstrated that D2 dopamine receptor uponits activation forms a complex with b-arrestin2, AKT and PP2A,leading to the dephosphorylation of AKT and the subsequentdephosphorylation of GSK3b thereby increasing GSK3b activity(Beaulieu et al., 2009, 2005, 2004). In agreement, we observed thatthe levels of b-catenin, a target of GSK3b that is degraded by the

increased GSK3 activity, were also decreased in mutant mice(Fig. 2C). Interestingly, we observed no alterations for other effec-tors, such as pERK1/2, pCREB and pDARPP32 (Fig. 2F), which areregulated by both the D1 and D2 dopamine receptors via G protein-related PKA/cAMP signaling cascade. Thus, the activation of theAKT/GSK3 pathway correlates well with the enhanced D2 dopa-mine receptor function that was found in TAAR1-KO mice by ourgroup and others. To unveil the putative mechanism for the acti-vation of this pathway, we also evaluated the levels of PP2A that isknown to form the complex with AKT and the D2 receptor upon itsactivation. No alterations were observed in the total levels of thisprotein in TAAR1-KO mice (Fig. 2D). However, immunoprecipita-tion experiments have revealed an increase in the formation ofcomplex between AKT and PP2A in the striatal tissue from TAAR1-KO mice (Fig. 2E), suggesting that the increased interaction be-tween these proteins may underlie the increased dephosphoryla-tion of AKTand GSK3b (Beaulieu et al., 2005) observed in TAAR1-KOmice.

3.3. TAAR1-KO mice are hypersensitive to the locomotor effects ofD2 but not D1 dopamine receptor agonist

To test whether the locomotor supersensitivity to dopaminergicstimulation observed in TAAR1-KO mice is related to D2 receptorsupersensitivity, we assessed the locomotor activity of animalsfollowing treatment with the selective D2-class dopamine receptoragonist quinpirole at a high “postsynaptic” dose, i.e., 2 mg/kg, i.p.(Usiello et al., 2000). At this dose, quinpirole activates not onlypresynaptic autoreceptors, leading to a decrease in DA transmissionand global locomotor suppression, but also provides sufficientstimulation of post-synaptic D2 dopamine receptors, inducing aminor increase in forward locomotion over the levels caused bypresynaptic effects but not achieving the levels observed in saline-controls (Gainetdinov et al., 2003). It has been previously docu-mented that mice with an enhanced D2 dopamine receptor sensi-tivity due to lack of GPCR kinase GRK-6 display a higher levels oflocomotor activity in response to this dose of quinpirole(Gainetdinov et al., 2003). Similarly, as shown in Fig. 3A, locomotoractivity following quinpirole treatment was significantly higher inTAAR1-KO compared to WT mice, directly demonstrating theincreased D2 dopamine receptor functionality. By comparison, thelocomotor stimulating effect of the selective D1 dopamine receptoragonist SKF-82958 at 1 mg/kg was not altered in mutant micecompared to WT animals (Fig. 3B). Taken together, these datademonstrate a significant over-activity of post-synaptic D2receptor-mediated transmission in the striatum of TAAR1-KO mice.

Fig. 2. D2R-related AKT/GSK3 signaling pathway is activated in TAAR1-KO mice. Densitometric western blot analysis of phosphoprotein relative levels in striatal extracts from WTand TAAR1-KO mice. Antibodies against p-Thr-308-AKT (A), p-Ser-9-GSK3b (B), b-catenin (C), PP2A (D, E) and pERK1/2, pDARPP-32, pCREB, totAKT and totGSK3 (F) were used.Antibodies for the total form of AKT and GSK3b were used as the loading controls for densitometric analysis. Immunoprecipitation of AKT revealed coimmunoprecipitation of thecatalytic subunit C of PP2A from the striatal extracts of TAAR1-KO mice (E, right panel). Quantification of the interaction between AKT and PP2A, as measured by densitometricanalysis (E, left panel). Average ratios of PP2Awere normalized to AKT total levels and were labeled 1 in WT animals. Data represent means ± SEM. N ¼ 10e15 per group. *, p < 0.05;**, p < 0.01. Student's unpaired two-tailed t-test and repeated t-test for (F).

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4. Discussion

In this study, we provided the molecular mechanism respon-sible for the striatal dopamine hypersensitivity that has beenobserved in TAAR1-KOmice (Espinoza et al., 2011; Lindemann et al.,2008; Sotnikova et al., 2010; Wolinsky et al., 2007). Specifically, wedemonstrated that in the striatum of TAAR1-KOmice, D2 dopaminereceptors are up-regulated, and the related G protein-independentpathway (AKT/GSK3) is activated with a concomitantly increasedD2-dependent locomotor activation. Taken together, these dataprovide further support for the close functional TAAR1-D2 dopa-mine receptor interaction mediated likely via receptor hetero-dimerization (Espinoza et al., 2011).

TAAR1 function has been characterized extensively in previousyears, with particular attention paid to its role in the modulation of

the dopamine system and its possible implications in psychiatricdiseases (Borowsky et al., 2001; Bunzow et al., 2001; Espinoza andGainetdinov, 2014; Lindemann and Hoener, 2005; Sotnikova et al.,2009). On one side, TAAR1-KO mice display a higher sensitivity todopaminergic stimuli such as amphetamine and other psychosti-mulants (Lindemann et al., 2008; Wolinsky et al., 2007) andgenerally have a supersensitive dopaminergic system (Di Cara et al.,2011; Lindemann et al., 2008; Wolinsky et al., 2007) making theman interesting model relevant for schizophrenia (Wolinsky et al.,2007). On the other side, the recently developed TAAR1 selectiveagonists reduce manifestations of excessive dopaminergic tonecaused by either pharmacological (amphetamine, cocaine) or ge-netic (DAT-KO mice) manipulations, further supporting the ideathat TAAR1 could represent a new target for psychiatric diseases(Sotnikova et al., 2009).

Fig. 3. D2 dopamine receptor agonist quinpirole induces an increased locomotorresponse in TAAR1-KO mice. Locomotor activity following 2 mg/kg quinpirole (A) and1 mg/kg SKF-82958 (B) in WT and TAAR1-KO mice. Cumulative total distance traveledduring the session (30 min, 10e40 min after injection) was calculated. Time course ofquinpirole effect on locomotor activity of WT and TAAR1-KO mice is shown in theinsert. Data represent means ± SEM. N ¼ 10 per group. **, p < 0.01, ***, p < 0.001. One-way ANOVA with Dunnett's post hoc test was used.

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Here, we extended these observations to an in vivo model byshowing that postsynaptic D2, but not D1, dopamine receptors areup-regulated in the striatum of TAAR1-KO mice. This evidence,together with the previously reported increase in the proportion of

D2 dopamine receptors in high affinity states (Wolinsky et al.,2007), suggests that D2 dopamine receptor-mediated signalingand behavior could bemarkedly altered by the absence of TAAR1. Totest this possibility directly, we evaluated the phosphorylation stateof several proteins that are regulated by D1 and D2 receptors. It iswell known that D2 dopamine receptors can signal through twomajor signaling pathways, the G protein-dependent, cAMP-medi-ated pathway and the G protein-independent, b-arrestin2-dependent pathway (Beaulieu et al., 2014; Beaulieu andGainetdinov, 2011), with both pathways being important fordopamine-related functions and behaviors (Beaulieu et al., 2008,2005, 2004). We found that the levels of pAKT and pGSK3b weredecreased, revealing the activation of the G protein-independent,b-arrestin2-dependent pathway. It has been demonstrated previ-ously that this pathway is activated following treatment withamphetamine and in drug-naïve hyperdopaminergic DAT-KO micein a D2 dopamine receptoredependent manner (Beaulieu et al.,2009, 2004). Moreover, we observed an increased level of thePP2A/AKT complex in the striatum of TAAR1-KO mice, suggestingthat the increased proportion of PP2A, which is complexed withAKT in response to D2 dopamine receptor activation, may beresponsible for AKT dephosphorylation (Beaulieu et al., 2008). Thedecrease in GSK3b phosphorylation typically leads to an increase inGSK3b activity, and accordingly, the expression level of a GSK3bsubstrate b-catenin was reduced in TAAR1-KO mice.

To directly test the functionality of D1 and D2 dopamine re-ceptors in vivo, we measured the locomotor response to selectiveD1 and D2 dopamine receptors agonists in mutant and controlmice. In agreement with the enhanced D2-related signaling instriatum, quinpirole at high “post-synaptic” dose induced anincreased locomotor response in TAAR1-KO mice, while the selec-tive D1 dopamine receptor agonist SKF-82958 effects showed nodifference between genotypes. Quinpirole is known to cause abiphasic locomotor response, by stimulating mainly presynaptic D2dopamine receptors and inhibiting locomotor activity at low doses,and stimulating also postsynaptic receptors and promoting loco-motor activity at high doses (Anzalone et al., 2012; Usiello et al.,2000). At relatively high dose affecting both the presynaptic andpostsynaptic dopamine D2 receptors quinpirole enhanced loco-motor stimulation in TAAR1-KO mice. Importantly, similar re-sponses to quinpirole were observed in mice with D2 dopaminereceptor supersensitivity due to GRK6 or norepinephrine trans-porter deficiency (Gainetdinov et al., 2003; Manago et al., 2012; Xuet al., 2000).

It should be noted, that TAAR1 is known to modulate presyn-aptic D2-like dopamine receptors autoreceptors, generally pro-ducing an enhancement of autoreceptor regulation (Leo et al.,2014). It seems that in case of postsynaptic receptors we have anopposite regulation revealed by an enhanced sensitivity of post-synaptic D2-like receptors in the absence of TAAR1. Furthermore, asin the case of presynaptic regulation (Leo et al., 2014), a potentialrole of postsynaptic D3 dopamine receptors in the effects observedin this study cannot be excluded and future work will be necessaryto address this question.

Taken together, these observations strongly support the hy-pothesis that TAAR1-KO mice have a supersensitive dopaminesystem not only due to deficient D2 dopamine autoreceptorregulation (Leo et al., 2014) but also in part via an up-regulationof postsynaptic striatal D2 dopamine receptor levels andfunctions.

Acknowledgment

This study was supported in part by the research award from F.Hoffmann La-Roche, Basel, Switzerland and by the Russian Science

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Foundation (project N14-25-00065). We are grateful to LundbeckA/G and Lundbeck USA for generously providing TAAR1 knockoutmice. We thank Dr. M. Morini, D. Cantatore and F. Piccardi for theirexcellent technical assistance.

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