16928 • The Journal of Neuroscience, November 23, 2011 • 31(47):16928–16940 Behavioral/Systems/Cognitive Genetic Deletion of Trace Amine 1 Receptors Reveals Their Role in Auto-Inhibiting the Actions of Ecstasy (MDMA) Benjamin Di Cara,1 Roberto Maggio,5 Gabriella Aloisi,5 Jean-Michel Rivet,1 Ebba Gregorsson Lundius,6 Takashi Yoshitake,6 Per Svenningsson,6 Mauricette Brocco,1 Alain Gobert,1 Lotte De Groote,1 Laetitia Cistarelli,1 Sylvie Veiga,1 Catherine De Montrion,2 Marianne Rodriguez,2 Jean-Pierre Galizzi,2 Brian P. Lockhart,2 Francis Coge´,3 Jean A. Boutin,3 Philippe Vayer,4 P. Monika Verdouw,7 Lucianne Groenink,7 and Mark J. Millan1 Departments of 1Neurobiology, 2Molecular Pharmacology and Pathophysiology, 3Biotechnology and Cellular-Molecular Pharmacology, and 4Biopharmaceutical Research, Institut de Recherches Servier, 78290 Croissy-sur-Seine, France, 5Department of Experimental Medicine, University of L’Aquila, L-7100 L’Aquila, Italy, 6Department of Clinical Neuroscience, Karolinska Institute, SE-171 77 Stockholm, Sweden, and 7Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, 3584 Utrecht, The Netherlands “Ecstasy” [3,4-methylenedioxymetamphetamine (MDMA)] is of considerable interest in light of its prosocial properties and risks asso- ciated with widespread recreational use. Recently, it was found to bind trace amine-1 receptors (TA1Rs), which modulate dopaminergic transmission. Accordingly, using mice genetically deprived of TA1R(TA1-KO), we explored their significance to the actions of MDMA, which robustly activated human adenylyl cyclase-coupled TA1R transfected into HeLa cells. In wild-type (WT) mice, MDMA elicited a time-, dose-, and ambient temperature-dependent hypothermia and hyperthermia, whereas TA1-KO mice displayed hyperthermia only. MDMA-induced increases in dialysate levels of dopamine (DA) in dorsal striatum were amplified in TA1-KO mice, despite identical levels of MDMA itself. A similar facilitation of the influence of MDMA upon dopaminergic transmission was acquired in frontal cortex and nucleus accumbens, and induction of locomotion by MDMA was haloperidol-reversibly potentiated in TA1-KO versus WT mice. Con- versely, genetic deletion of TA1R did not affect increases in DA levels evoked by para-chloroamphetamine (PCA), which was inactive at hTA1 sites.TheTA1Ragonisto-phenyl-3-iodotyramine(o-PIT)bluntedtheDA-releasingactionsofPCAbothinvivo(dialysis)andinvitro (synaptosomes) in WT but not TA1-KO animals. MDMA-elicited increases in dialysis levels of serotonin (5-HT) were likewise greater in TA1-KO versus WT mice, and 5-HT-releasing actions of PCA were blunted in vivo and in vitro by o-PIT in WT mice only. In conclusion, TA1Rs exert an inhibitory influence on both dopaminergic and serotonergic transmission, and MDMA auto-inhibits its neurochemical and functional actions by recruitment of TA1R. These observations have important implications for the effects of MDMA in humans. Introduction It is thus important to elucidate the precise mechanisms of action The monoamine releaser and reuptake suppressor, “ecstasy” of MDMA. [3,4-methylenedioxymetamphetamine (MDMA)], facilitates MDMA displaces neuronal serotonin (5-HT) via reversal of prosocial behaviors in animals and humans (Bedi et al., 2009; vesicular and plasma membrane transporters (Rudnick and Wall, Johansen and Krebs, 2009), actions related to enhanced release of 1992; Mlinar and Corradetti, 2003). Further, it prevents 5-HT oxytocin in centers controlling social behavior, like amygdala and reuptake (Iravani et al., 2000) and inhibits the activity of the septum (McGregor et al., 2008). However, greater attention has catabolytic enzyme monoamine oxidase A (Leonardi and Azmi- been devoted to the psychostimulant properties of MDMA and tia, 1994). Concomitant elevations in dopamine (DA) and nor- its widespread recreational use (Vollenweider et al., 1999, 2002). adrenaline (NA) levels occur either via a similar pattern of direct In addition, MDMA can trigger a serotonergic syndrome and actions upon catecholaminergic pathways (Gough et al., 2002; disrupt thermoregulatory mechanisms, neurotoxic effects espe- Baumann et al., 2005, 2008) and/or events downstream of sero- cially prominent in young adults under conditions of dehydra- tonergic transmission, such as activation of 5-HT2A receptors tion (Schifano, 2004; Baumann et al., 2007; Karlsen et al., 2008). (Gobert et al., 2000). Moreover, at high concentrations, MDMA binds to 5-HT2A, histamine H1, and muscarinic M1/M2 receptors, though their putative roles in its functional actions remain poorly Received May 18, 2011; revised Sept. 8, 2011; accepted Sept. 10, 2011. characterized (Green et al., 2003). Author contributions: B.D.C., R.M., J.-M.R., P.S., M.B., A.G., L.D.G., C.D.M., M.R., J.-P.G., B.P.L., F.C., J.A.B., P.V., L.G.,andM.J.M.designedresearch;G.A.,E.G.L.,T.Y.,L.C.,S.V.,andP.M.V.performedresearch;B.D.C.,R.M.,andP.S. A potentially novel dimension to the neurobiology of MDMA analyzed data; B.D.C., R.M., P.S., and M.J.M. wrote the paper. was unveiled by the suggestion that it recognizes Gs-coupled TA1 The authors declare no competing financial interests. receptors [TA1Rs (sometimes called TAAR1s)] (Bunzow et al., Correspondence should be addressed to Mark J. Millan, Institut de Recherche Servier, Department of Neurobiol- 2001; Lindemann and Hoener, 2005; Maguire et al., 2009). While ogy, 125, Chemin de Ronde, 78290 Croissy-sur-Seine, France. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.2502-11.2011 most subtypes of trace amine (TA) receptor are preferentially Copyright © 2011 the authors 0270-6474/11/3116928-13$15.00/0 expressed in olfactory epithelium (Liberles and Buck, 2006), • Di Cara et al. TA1R Blunt Actions of MDMA J. Neurosci., November 23, 2011 • 31(47):16928–16940 • 16929 TA1Rs are mainly distributed in limbic structures and regions containing mono- aminergic perykarias (Borowsky et al., 2001; Wolinsky et al., 2007; Lindemann et al., 2008). The TAs, tyramine, tryptamine, and ␤-phenethylamine are implicated in psychiatric and neurological disorders as- sociated with monoaminergic dysfunc- tion (Branchek and Blackburn, 2003; Berry, 2004; Burchett and Hicks, 2006; Sotnikova et al., 2008), yet it remains un- proven that they act via TA1Rs in vivo. Conversely, the thyroxine derivatives 3-iodothyronamine (T1AM) and o-phenyl-3- iodotyramine (o-PIT) behave as agonists at TA1Rs, and their hypothermic actions are blunted in TA1 knock-out (TA1-KO) mice (Scanlan et al., 2004; Hart et al., 2006; Doyle et al., 2007) (M.J. Millan, un- published observations). Likewise of pertinence to MDMA, TA1Rs inhibit the electrical and synthetic activity of nigrostriatal dopaminergic neurons (Geracitano et al., 2004; Linde- mann et al., 2008; Bradaia et al., 2009; Le- donne et al., 2010). Moreover, functional interactions have been reported between colocalized TA1R and DA transporters (Xie and Miller, 2008; Xie et al., 2008). Interestingly, T1AM and o-PIT suppress locomotor activity in mice (Scanlan et al., 2004), actions opposite to those of MDMA (Baumann et al., 2008). Collectively, these observations raise the intriguing possibility that TA1R may modulate the actions of MDMA. Though EPPTB (N-(3-ethoxy-phenyl)-4-pyrroli- din-1-yl-3-trifluoromethyl-benzamide) was described as a selective TA1R antagonist (Bradaia et al., 2009; Stalder et al., 2011), it is not centrally active upon systemic adminis- tration. The present study addressed the role of TA1R in the physiological, neurochemi- cal, and behavioral actions of MDMA by ex- ploiting TA1-KO mice. Materials and Methods Construction of the targeting vector. Targeting Ϫ/Ϫ vector construction and knockout strategy Figure 1. Generation of TA1R knock-out mice. A, Strategy for targeted deletion of the Taar1 gene in mouse ES cells. The have been designed and performed by genO- Taar1codingsequencewaspartiallyreplacedbyacDNAencodingIRES-LacZ-NeoTkcassette.TheIRES-LacZ-Neowasfusedinframe way. Genomic clones containing the murine with the endogenous Taar1 start codon. B, BgIII site; E, EcoRI site; H, HindIII site; N, NsiI site; S, SpeI site; X, XbaI site; CDS, coding Taar1 locus were isolated from a 129S6/ Ј domainsequenceofuniqueexonofTaar1gene.ThearrowheadcorrespondstotheloxPsites.B,C,PCRscreeningonthe3 (B)and SvEvTacRPCI-22 bacterial artificial chromo- Ј 5 (C) end of homologous recombination event. The 1505 bp (primers GW224/GW475 corresponding to PGKneo-Taar1 locus) and some (BAC) genomic library using a probe 5374 bp (primers GW587/GW114 corresponding to Taar1 locus-IRES/LacZ) bands identified appropriated targeted disruption of corresponding to the murine Taar1 (nucleo- Ј Ј Ј Ј murinemTaar1locuson3 and5 end-targetingevents,respectively.ThePCRscreeningonthe5 and3 endsofthehomologous tides 45–950, GenBank accession #AF380187). ϩ recombinationeventisshownforEScellclones1A2and1D2.M,NEBBiolabs1kbpDNAladder;C ,genomicDNAfromEScellclone Two BAC clones (37K19 and 393O10) con- transfected with positive vector. D, Southern blot analysis of positive and wild-type ES cell clones. Digestion of genomic DNA with XbaI resulted in the following fragments: wild-type allele of 5.4 kb band, appropriate targeting of the Taar1 locus of 10.5 kb band. E, F, PCR screening analysis of the offspring of Taar1-mutated mice. Heterozygous and homozygous F2 mice were discriminate 4 from wild type by PCR using couple primers specific for targeted (GW224/GW475, 1505 bp band, E) and wild-type allele (GW264/ GW265, 886 bp band, F). G, Southern blot analysis of the offspring