JPET Fast Forward. Published on December 14, 2007 as DOI:10.1124/jpet.107.132647 JPET #132647 Trace Amine-Associated Receptor 1 (TAAR1) Modulates Dopaminergic Activity Lothar Lindemann, Claas Aiko Meyer, Karine Jeanneau, Amyaouch Bradaia, Laurence Ozmen, Horst Bluethmann, Bernhard Bettler, Joseph G. Wettstein, Edilio Borroni, Jean-Luc Moreau & Marius C. Hoener F. Hoffmann-La Roche Ltd., Pharmaceuticals Division, Preclinical CNS Research, CH-4070 Basel, Switzerland (LL, KJ, LO, JGW, EB, JLM, MCH) F. Hoffmann-La Roche Ltd., Roche Center for Medical Genomics, CH-4070 Basel, Switzerland (CAM, HB) Department of Biomedicine, Institute of Physiology, Pharmacenter, University of Basel, CH-4056 Basel, Switzerland (AB, BB) 1 Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics. JPET #132647 Running title: TAAR1 modulates dopaminergic activity Author for correspondence: Marius C. Hoener, Ph.D. F. Hoffmann-La Roche Ltd. Pharmaceuticals Division, CNS Research Dept. PRBD-N, Bldg. 70/331 CH-4070 Basel, Switzerland phone: +41 61 68 84192 fax: +41 61 68 81720 e-mail: [email protected] Number of Text pages: 34 Number of Tables: 1 Number of Figures: 5 Number of References: 40 Words in abstract: 156 Words in introduction: 480 Words in discussion: 1150 2 JPET #132647 Abbreviations: TAAR, trace amine-associated receptor; GPCR, G protein-coupled receptor; NLS, nuclear localization signal; DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HIAA, 5- hydroxyindoleacetic acid; ES cell, embryonic stem cell; GAPDH, glyceraldehyde 3- phosphate dehydrogenase; PBS, phosphate buffered saline; ACSF, artificial cerebrospinal fluid Section assignment: Neuropharmacology 3 JPET #132647 Abstract The recent identification of the trace amine associated receptor 1 (TAAR1) provides an opportunity to dissociate the effects of trace amines on the dopamine transporter from receptor- mediated effects. In order to separate both effects on a physiological level, a Taar1 knock-out mouse line was generated. Taar1 knock-out mice display increased sensitivity to amphetamine as revealed by enhanced amphetamine-triggered increases in locomotor activity and augmented striatal release of dopamine as compared to wild-type animals. Under baseline conditions, locomotion as well as extracellular striatal dopamine levels were similar between Taar1 knock- out and wild-type mice. Electrophysiological recordings revealed an elevated spontaneous firing rate of dopaminergic neurons in the ventral tegmental area of Taar1 knock-out mice. The endogenous TAAR1 agonist p-tyramine specifically decreased the spike frequency of these neurons in wild-type but not in Taar1 knock-out mice, consistent with the prominent expression of Taar1 in the ventral tegmental area. Taken together, the data reveal TAAR1 as regulator of dopaminergic neurotransmission. 4 JPET #132647 Introduction Trace amines like p-tyramine, β-phenylethylamine, octopamine and tryptamine are endogenous amine compounds related to the classical neurotransmitters dopamine, serotonin and noradrenaline by structure, metabolism and tissue distribution (Philips, 1984). However, trace amines are found in the mammalian brain at concentrations about 1000-fold lower than catecholamines (Berry, 2004). Trace amines have been postulated to function as co-transmitters in classical neurotransmitter systems (Saavedra and Axelrod, 1976), as neurotransmitters in their own right (Sabelli et al., 1978) or as neuromodulators of catecholamines (for review see Berry, 2004). The identification of trace amine-specific receptors (Borowsky et al., 2001; Bunzow et al., 2001) and detailed characterization of the trace amine-associated receptor (TAAR) family in various species (Lindemann et al., 2005; Lindemann and Hoener, 2005) provided the basis to further elucidate the interplay between trace amines and catecholamines and to understand the physiological roles of trace amines at the molecular level. The TAAR family consists of three subgroups (TAAR1-4, TAAR5 and TAAR6-9) and is phylogenetically and functionally distinct from other G protein-coupled receptor (GPCR) families as well as from invertebrate octopamine or tyramine receptors (Lindemann and Hoener, 2005). With the exception of TAAR1 and TAAR4, none of the other TAARs are sensitive to one of the classical trace amines p-tyramine, β- phenylethylamine, octopamine and tryptamine (Borowsky et al., 2001; Lindemann et al., 2005; Liberles and Buck, 2006). All TAARs except TAAR1 were recently detected in mouse olfactory sensory neurons, and mouse TAAR5, TAAR7, and TAAR3 have been shown to be activated by small molecular weight volatile amines suggesting a potential role of TAARs as odorant receptors in rodents (Liberles and Buck, 2006). However, the endogenous ligands have not yet been identified for TAARs other than TAAR1 and TAAR4. Taar1 is encoded by a single exon in all species analyzed, couples to the G protein Gαs, and responds to p-tyramine and β- 5 JPET #132647 phenylethylamine with an EC between 0.2 – 1.4 µM and with much lower sensitivity also to 50 octopamine and tryptamine (Borowsky et al., 2001; Lindemann et al., 2005). In human, all Taar genes are located in a narrow region of about 109 kb comprised in the locus 6q23.1 (Lindemann et al., 2005), which has been genetically linked to schizophrenia and bipolar disorder (Cao et al., 1997; Cichon et al., 2001; Vladimirov et al., 2007). The interest in trace amines and their target receptors is fueled by their proposed link to highly prevalent psychiatric disorders, most notably depression and schizophrenia (for review, see Lindemann and Hoener, 2005; Branchek and Blackburn, 2003). It has been reported that trace amines exhibit some amphetamine-like properties through inhibition of the dopamine transporter (Berry, 2004; Sotnikova et al., 2004; Parker and Cubeddu, 1988), but little is known about the effects of trace amines mediated directly by activation or inhibition of TAAR1. The Taar1 knock- out mouse line allows to dissociate the specific contributions of dopamine transporter and of TAAR1 to the physiological effects of trace amines. 6 JPET #132647 Methods Animals All experiments involving living animals performed at F. Hoffmann-La Roche Ltd. were performed in compliance with Swiss Federal and Cantonal laws on animal research and approved by the cantonal veterinary office. Two 4.7 kb and 1.7 kb genomic fragments located 5’ and 3’ from the Taar1 coding sequence were amplified from C57BL/6 genomic DNA by PCR with the oligonucleotides F3 and R1, and with the oligonucleotides F4 and R2, respectively (Fig. 1). The targeting vector was assembled from these genomic fragments, a LacZ coding sequence fused to a nuclear localization sequence (NLS; Kalderon et al., 1984), a PgK-NeoR (Galceran et al., 2000) and a diphteria toxin cassette (Gabernet et al., 2005). The targeting vector linearized with Sac II was electroporated into C57BL/6 embryonic stem (ES) cells (Eurogentech, Seraing, Belgium) and G418-resistant ES cell clones were selected as described previously (Gabernet et al., 2005). An ES cell clone carrying a homologous recombination event was identified by PCR (data not shown) and used to generate chimeras according to standard protocols (Joyner, 1999). The recombinant allele was maintained in a pure C57BL/6 background (Charles River Laboratories France, Les Oncins, France) in a specific pathogen free facility with a 12:12 day/ night cycle and ad libitum access to food and water. The correct homologous recombination was further confirmed by PCR amplification of fragments PCR 1-3 (Fig. 1) from genomic DNA derived from tail biopsies of Taar1-/- animals and subsequent DNA sequence analysis (details regarding PCR conditions and oligonucleotide sequences in supplemental methods). Standard genotyping was performed by PCR using oligonucleotides detecting the disrupted- and the intact Taar1 coding sequence, respectively (details regarding PCR conditions and oligonucleotide sequences in supplemental methods). 7 JPET #132647 The purity of the C57BL/6 genetic background of the knock-out mouse line was confirmed by microsatellite analysis of Taar1+/- animals of the F1 generation employing a total of 37 markers (details regarding the panel of microsatellite markers and oligonucleotides used in the microsatellite analysis in supplemental methods). Genomic DNA of 6 individual animals was analyzed along with samples of C57BL/6, DBA and SV129 mice (Jackson Laboratories, Bar Harbor, ME, USA). Whole brain cDNA of two weeks old and adult Taar1+/+ and Taar1-/- mice was analyzed for the presence of transcripts encoding glyceraldehyde 3-phosphate dehydrogenase (Gapdh), Taar1 and NLSLacZ by means of RT-PCR. Whole brain cDNAs were prepared essentially as described by Lindemann et al. (2005), and PCR was performed using the following oligonucleotides for the individual transcripts (oligonucleotide sequences in supplemental methods): GAPDH: GAPDHU and GAPDHD (452 bp PCR fragment); NLSLacZ: LacZ U1 and LacZ D1 (631 bp PCR fragment); TAAR1: mTAAR U1 and mTAAR D1 (936 bp PCR fragment). Drugs Drugs were purchased from Sigma Chemie (Buchs, Switzerland) at the highest purity available and d-amphetamine was synthesized by F. Hoffmann-La Roche Ltd. Drugs were dissolved in 0.9% NaCl and administered i.p. before the behavioral testing or the collection of microdialysis samples, as indicated for the individual experiments. In each experiment, drugs were administered by pseudorandomized design over one to several treatment cycles, with a minimum period of 10 – 14 days between two cycles. Histochemistry on tissue sections Adult mice were transcardially perfused under terminal isoflurane anesthesia consecutively with phosphate buffered saline (PBS) and fixative (2% w/v paraformaldehyde and 0.2% w/v glutaraldehyde in PBS). Brains were post-fixed for 4 h in fixative at 4 °C, immersed over night in 8 JPET #132647 0.5 M sucrose in PBS at 4 °C, embedded in OCT compound (Medite Medizintechnik, Nunningen, Switzerland) in Peel-A-Way tissue embedding molds (Polysciences Inc., Warrington, USA) and frozen on liquid nitrogen. Tissue sections were cut on a cryostat (Leica Microsystems AG, Glattbrugg, Switzerland) at 10 - 50 µm, thaw-mounted on gelatin-coated glass slides (Fisher Scientific, Wohlen, Switzerland), air-dried at room temperature for 4 h and processed immediately for LacZ staining. For LacZ stainings, tissue sections were washed 5x for 10 min in PBS and incubated for 16- 24 h in LacZ staining solution (1 mg/ml 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, 5 mM K Fe(CN) , 5 mM K Fe(CN) , 2 mM MgCl in PBS) at 37 °C. The staining was stopped by 3 6 4 6 2 washing the tissue sections 5x for 10 min in PBS. Tissue sections were dehydrated through an ascending ethanol series, equilibrated to xylene, coverslipped with DePex (Serva GmbH, Heidelberg, Germany) and analyzed on an Axioplan I microscope equipped with an Axiocam CCD camera system (Carl Zeiss AG, Feldbach, Switzerland). Behavioral phenotyping Locomotor activity. A computerized Digiscan 16 Animal Activity Monitoring System (Omnitech Electronics, Colombus, OH) was used to quantify spontaneous locomotor activity. Data were obtained simultaneously from eight Digiscan activity chambers placed in a soundproof room with a 12 hr light/ dark cycle. All tests were performed during the light phase (6 a.m. to 6 p.m.). Each activity monitor consisted of a Plexiglas box (20 x 20 x 30.5 cm) with sawdust bedding on the floor surrounded by invisible horizontal and vertical infrared sensor beams. The cages were connected to a Digiscan Analyzer linked to a PC that constantly collected the beam status information. With this system, different behavioral parameters could be measured, such as horizontal and vertical activity, total distance travelled (in cm) and stereotypies. The mice were tested via a pseudo-Latin squares design with at least a 10 day interval between two consecutive test sessions. Animals were habituated for 30 min. Vehicle (saline 0.9%) or d- 9 JPET #132647 amphetamine (1, 2.5 mg/kg, i.p.) was then administered to wild-type (n = 24) and Taar1 knock- out (n = 24) mice. Locomotor activity was recorded during 30 min habituation and 90 min after treatment starting immediately after the mice were placed in the test compartment. Mice were additionally assessed for body temperature, body weight, grip strength, and general motor coordination (rotarod test; protocols and data regarding this additional behavioral testing in supplemental methods and data). Statistics. Behavioral observations were recorded as mean values +/- S.E.M. and analyzed with an unpaired t test. Locomotor activity data (total distance) were analyzed with a two-factor (genotype and dose) ANOVA with repeated measures. Comparisons of dose effects in each genotype were undertaken with a repeated measures ANOVA, followed in significant cases by paired t tests. A P value of 0.05 was accepted as statistically significant. In vivo microdialysis assessment of extracellular biogenic amine neurotransmitter levels Four months old male mice were used for these experiments. Surgery and implantation of the microdialysis probe Forty-five min before anesthesia mice received subcutaneous injections of 0.075 mg/kg buprenorfine. Mice were then anesthetized with isoflurane and placed in a stereotaxic device equipped with dual manipulator arms and an anesthetic mask. Anesthesia was maintained with isoflurane 0.8-1.2% (v/v; support gas oxygen/air, 2:1). The head was shaved and the skin was cut along the midline to expose the skull. A small bore hole was made in the skull to allow the stereotaxical insertion of the microdialysis probe (vertical probe carrying a 2 mm polyacrilonitrile dialysis membrane; Brains On-line, Groningen, The Netherlands) in the striatum (coordinates: A 0.9 mm, L -1.8 mm, V -4.6 mm). The probe was fixed using binary dental cement. Once the cement was firm, the wound was closed with silk thread for suture (Silkam), the animal was removed from the stereotaxic instrument and returned to its cage. At the end of the surgery and 24 h later mice were treated with meloxicam 1 mg/kg s.c. The body weight of the animals was 10