Supplementary Online Information · In situ hybridization Radioactive in situ hybridization was...

Revel et al. Supplementary information

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Supplementary Information

Brain-specific overexpression of trace amine-associated receptor 1 alters

monoaminergic neurotransmission and decreases sensitivity to amphetamine

Florent G. Revel1, Claas A. Meyer2, Amyaouch Bradaia3, Karine Jeanneau1 *, Eleonora

Calcagno4, Cédric B. André1 *, Markus Haenggi1, Marie-Therese Miss1, Guido Galley5, Roger

D. Norcross5, Roberto W. Invernizzi4, Joseph G. Wettstein1, Jean-Luc Moreau1 & Marius C.

Hoener1

1 Neuroscience Research, 2 Discovery Technologies, 5 Discovery Chemistry, Pharmaceuticals Division, F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland 3 Neuroservice, Domaine de Saint-Hilaire, 595 rue Pierre Berthier, CS30531, 13593 Aix-en-Provence, France 4 Laboratory of Neurochemistry and Behaviour, Department of Neuroscience, Istituto di Ricerche Farmacologiche “Mario Negri”, Via La Masa 19, 20156 Milano, Italy

* Present address: Novartis Pharma AG, 4002 Basel, Switzerland

Supplementary Materials and Methods Supplementary References Tables S1-S2 Figures S1-S6


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SUPPLEMENTARY MATERIALS AND METHODS

In situ hybridization

Radioactive in situ hybridization was performed as described previously (Revel et al, 2007)

using riboprobes for mouse Taar1 (bases 200-999 from GenBank accession no.

NM_053205). Series of coronal brain sections (20 µm) were cut in a cryostat either through

the rostral brain (1.9 to 0.1 mm anterior to bregma; including the accumbens nucleus, Acb,

caudate putamen and medial prefrontal cortex, mPFC) for comparison of the B6-Tg(Taar1)19

and B6-Tg(Taar1)27 transgenic mice, or throughout the brain rostro-caudal axis (from +3.0

(olfactory nuclei) to -5.4 mm (locus coeruleus) relative to the bregma) for detailed

investigation of the B6-Tg(Taar1)27 mice. After they were fixed, acetylated, and dehydrated,

the sections were hybridized overnight at 54°C with 35S-UTP-labeled antisense or sense

riboprobes. The sections were then treated with RNase A (10 µg/ml), washed (saline sodium

citrate 0.1x, 60°C), dehydrated, exposed to a Fujifilm imaging plate (BAS-SR2025) for 3–5

days along with 14C radioactive standards to allow standardization of densitometric

measurements, and scanned (Fujifilm BAS-5000). Calibrated optical density bilateral

measurements of mRNA levels in the dorsal striatum and prefrontal cortex (cingulate cortex)

were performed on 2 consecutive sections using ImageJ (National Institutes of Health,

Bethesda, MD). For each animal (n=4/group), the signal measured with the sense probe was

subtracted to that measured with the antisense probe.

Real-time quantitative PCR

Total-RNA was extracted from the striatum, Acb, ventral tegmental area, substantia nigra

and dorsal raphe nucleus using the miRNeasy Mini kit (Qiagen), and treated with DNAse I.

Reverse transcription and polymerase chain reaction were performed in triplicates with 2 ng

total-RNA and TaqMan ABI probes as previously reported (Ozmen et al, 2009). A relative

quantification method based on GAPDH expression was used.

Autoradiography

Brains were rapidly removed from the skull, immediately frozen on dry ice, and stored at -

80°C. Series of coronal brain sections (14 µm) were cut in a cryostat through the striatum

(1.9 to 0.1 mm anterior to bregma), were thaw-mounted on Histobond glass slides

(Marienfeld Laboratories Glassware, Germany) (12 sections per slide) and stored at -20°C.

For each autoradiographic labeling, two series of slides were thawed and incubated with the


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relevant radioligand with or without a saturating concentration of competitor for determination

of non-specific binding (NSB) or total binding (TB), respectively.

For labeling of the dopamine receptor D1, the slides were preincubated in assay

buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2-2H2O, 1 mM MgCl2*6H2O, 1 μM

ketanserin, pH 7.4) for 2 x 5 min at RT, then incubated at RT for 15 min in assay buffer

containing 1 nM [N-methyl-3H]SCH23390 plus (NSB) or minus (TB) 10 μM SFK38393.

For labeling of the dopamine receptor D2, the slides were preincubated in assay

buffer (50 mM Tris, 150 mM NaCl, 0.1% ascorbic acid, pH 7.4) for 2 x 10 min at RT, then

incubated at RT for 60 min in assay buffer containing 1 nM [methoxy-3H]raclopride plus

(NSB) or minus (TB) 10 μM butaclamol.

For labeling of the dopamine transporter (DAT), the slides were preincubated in assay

buffer (50 mM Tris, 300 mM NaCl, 0.2% bovine serum albumine, pH 7.5) for 2 x 15 min at RT,

then incubated overnight at 4°C in assay buffer containing 2 nM [propylene-2,3-3H]GBR12935 plus (NSB) or minus (TB) 50 μM mazindol.

The brains sections were then rinsed with ice-cold assay buffer (D1 and D2) or ice-

cold rinse buffer (DAT; assay buffer with 450 mM NaCl) consisting of 1 brief wash followed

by 2 x 2 min (D1, D2) or 2 x 5 min (DAT) soaking and distilled water (3 brief dips). The slides

were air dried overnight at 4°C and exposed together with [3H] microscales against tritium-

sensitive imaging plates (BAS-TR2025) for 6 days. The plates were scanned with a high

resolution phosphor imager device (Fujifilm BAS-5000) and calibrated measurements of

radioactivity (fmol/mg protein) were made using the MCID M2 image analysis system

(Imaging Research Inc., St. Catherines, Ontario, Canada). All analyses were performed blind

to treatment.

In vivo microdialysis

Microdialysis studies were performed as detailed previously (Calcagno and Invernizzi, 2010).

Briefly, 3-5-months old male WT and Tg mice were group caged upon shipment from Roche

and allowed to adapt to the new animal facility for at least one week. Concentric dialysis

probes were prepared as described (Robinson and Whishaw, 1988) with a dialysis

membrane made of Cuprophan (SorinBiomedica, Italy; 216 µm outer diameter; 3,000 Da

cutoff). The length of exposed membrane was 2 mm (Acb) and 3 mm (mPFC). The probes

were surgically implanted within the mPFC and Acb under general anesthesia. The

stereotaxic coordinates for the probe tip relative to the bregma and dura surface (Franklin

and Paxinos, 1997) were: mPFC, anteroposterior = +2.1 mm, lateral = ±0.3 mm, dorsoventral

= -2.5 mm; Acb, AP= +1.2 mm, L = ±1.1 mm and dorsoventral = -4.7 mm, with an angle of

20° from vertical in the mediolateral plane. Each animal was implanted with a single probe.

Correct positioning of the probe was examined at the end of the dialysis experiment by visual


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inspection of 30 µm coronal brain sections cut on a cryostat. Only animals with correct probe

placement were considered in the results.

Animals were allowed to recover from anesthesia one per cage with free access to

food and water. All microdialysis experiments were carried out 3-4 days after surgery in

awake, freely moving mice, and were performed in the middle of the light phase (3-9 h after

lights on). Each animal was placed in a cage and the inlet cannulae of the probe connected

by polyethylene tubing to a 2.5 mL plastic syringe containing artificial cerebrospinal fluid (in

mM: NaCl 145, KCl 3, CaCl2 1.26, 1 MgCl2 in distilled water, buffered at pH 7.4 with 2 mM

sodium phosphate buffer). Probes were perfused at a constant flow-rate of 1 µL/min with a

microinfusion pump (CMA/Microdialysis, Stockholm, Sweden). After 90 min washout,

consecutive 30-min samples of dialysate were collected in minivials containing 1.5 µL of

antioxidant solution (0.27 mM Na2EDTA, 3.3 mM cysteine HCl and 0.5 mM ascorbic acid in

0.1 M acetic acid). Mice were then treated intraperitoneally (ip) with d-amphetamine (2.5

mg/kg) and dialysate samples were collected for further 2.5 h. Dialysate samples were stored

frozen at -80°C until analysis of DA, NA and 5-HT concentrations. Samples were split into 2

portions of 15 µL each for the analysis of catecholamines and 5-HT according to the HPLC

methods described elsewhere (Invernizzi et al, 1992; Feenstra et al, 1998).

Pharmacokinetics experiments

Experiments were performed as published previously (Revel et al, 2011). Male C57BL/6J

mice and Wistar rats were dosed with RO5073012 either intravenously (iv) via a jugular vein

or orally (per os; p.o.). In the mouse the compound was dosed at 10 or 5 mg/kg, p.o. and iv

respectively, and plasma and brain samples were collected after euthanasia of two animals

per group at 0.08, 0.33, 0.75, 1.5, 4.2 and 8.1 h (iv) or 0.33, 0.75, 1.5, 4.2 and 8.2 h (p.o.)

post dose. For the rat, two jugular vein canulated animals per dose route were administered

with 3.7 or 5 mg/kg of RO5073012, p.o. and iv respectively, and plasma samples taken at

0.083, 0.25, 0.75, 2, 4, 8 and 24 h (iv) or 0.25, 0.75, 1.5, 3, 5, 8 and 24 h (p.o.). To determine

the brain to plasma ratio in the rat, four further animals were dosed at 8 mg/kg p.o. and after

euthanasia at 0.5, 2, 4.4 and 7.2 h post dose, plasma and brain samples were taken.

Concentrations of RO5073012 were determined using quantitative liquid

chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Pharmacokinetic

parameters were calculated by non-compartmental analysis of plasma concentration-time

curves using WinNonlin, version 4.1 software (Pharsight Corporation, Mountain View, CA).


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Supplementary References

Feenstra MG, Botterblom MH, van Uum JF (1998). Local activation of metabotropic glutamate receptors inhibits the handling-induced increased release of dopamine in the nucleus accumbens but not that of dopamine or noradrenaline in the prefrontal cortex: comparison with inhibition of ionotropic receptors. J Neurochem 70: 1104-1113.

Franklin KBJ, Paxinos G (1997). The Mouse Brain in Stereotaxic Coordinates. Academic Press: San Diego.

Invernizzi R, Belli S, Samanin R (1992). Citalopram's ability to increase the extracellular concentrations of serotonin in the dorsal raphe prevents the drug's effect in the frontal cortex. Brain Res 584: 322-324.

Ozmen L, Albientz A, Czech C, Jacobsen H (2009). Expression of transgenic APP mRNA is the key determinant for beta-amyloid deposition in PS2APP transgenic mice. Neurodegener Dis 6: 29-36.

Revel FG, Herwig A, Garidou ML, Dardente H, Menet JS, Masson-Pevet M, et al. (2007). The circadian clock stops ticking during deep hibernation in the European hamster. Proc Natl Acad Sci U S A 104: 13816-13820.

Revel FG, Moreau JL, Gainetdinov RR, Bradaia A, Sotnikova TD, Mory R, et al. (2011). TAAR1 activation modulates monoaminergic neurotransmission, preventing hyperdopaminergic and hypoglutamatergic activity. Proc Natl Acad Sci U S A 108: 8485-8490.

Robinson TE, Whishaw IQ (1988). Normalization of extracellular dopamine in striatum following recovery from a partial unilateral 6-OHDA lesion of the substantia nigra: a microdialysis study in freely moving rats. Brain Res 450: 209-224.


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SUPPLEMENTARY TABLES

Table S1. Selectivity screen for RO5073012.

RO5073012 was tested in a CEREP selectivity screen consisting of receptor binding and

enzyme assays. In the receptor binding assays, the specific binding (SB) of a radioligand to

the target receptor was defined as the difference between the total binding and the

nonspecific binding determined in the presence of a cold competitor in excess. The results

are expressed as a percent inhibition of control SB obtained in the presence of RO5073012

at 10 μM. Shown are targets, radioligands, competitors as well as reference compounds

used to validate the accuracy of the radioligand binding assays. When SB was significantly

inhibited by RO5073012, the Ki of RO5073012 for such target was determined and compared

to the Ki at mouse TAAR1. When enzyme assays were used, the results are expressed as a

percent of control values obtained in the presence of RO5073012 (10 μM). Shown are

targets and substrates, stimulus or tracers as well as reference compounds used to validate

the accuracy of the enzyme and cell based assays. Details on the CEREP screen are

available at www.cerep.fr. Ratio, ratio of the target Ki over mouse TAAR1 Ki.

1. Binding to receptors, ion channels and transporters

Target Radioligand Competitor Reference Compound

% Inhibition of Control SB Ki (nM) Ratio

5-HT transporter (h) (SERT) [3H]imipramine imipramine imipramine 40 20 926 6 539

5-HT (non-selective) [3H]serotonin serotonin serotonin 38

5-HT1A (h) [3H]8-OH-DPAT) 8-OH-DPAT 8-OH-DPAT > 10 000 > 3 125

5-HT1D [3H]serotonin serotonin serotonin 556 174

5-HT2A (h) [3H]ketanserin ketanserin ketanserin > 10 000 > 3 125

5-HT2B (h) [125I](±)DOI (±)DOI (±)DOI > 10 000

5-HT3 (h) [3H]BRL 43694 MDL 72222 MDL 72222 1 744 545

5-HT5A (h) [3H]LSD serotonin serotonin 3 516 1 099

5-HT6 (h) [3H]LSD serotonin serotonin 3 496 1 092

5-HT7 (h) [3H]LSD serotonin serotonin 654 204

A1 (h) [3H]DPCPX DPCPX DPCPX 5

A2A (h) [3H]CGS 21680 NECA NECA 8

A3 (h) [125I]AB-MECA IB-MECA IB-MECA 48

alpha 1 (non-selective) [3H]prazosin prazosin prazosin 76 4 029 1 259


7

alpha 2 (non-selective) [3H]RX 821002 (-)epinephrine yohimbine 95 484 151

AMPA [3H]AMPA L-glutamate L-glutamate 4

Androgen (h) (AR) [3H]methyltrienolone mibolerone methyltrienolone, mibolerone

9

AT1 (h) [125I][Sar1,Ile8]-AT II angiotensin II saralasin 4

AT2 (h) [125I]CGP 42112A angiotensin II saralasin, angiotensine II

-2

B1 (h) [3H](-)CGP 12177 alprenolol desArg10-KD -2

B2 (h) [3H](-)CGP 12177 alprenolol NPC 567 -19

beta 1 (h) [3H]desArg10-KD desArg9[Leu8]-BK

atenolol 13

beta 2 (h) [3H]bradykinin bradykinin ICI 118551 7

BLT1 (LTB4) (h) [3H]LTB4 LTB4 LTB4 0

BZD (central) [3H]flunitrazepam diazepam diazepam 9

Ca2+ channel (L, dihydropyridine site)

[3H](+)PN 200-110 nifedipine nitrendipine 18

Ca2+ channel (L, diltiazem site)

[3H]diltiazem diltiazem diltiazem 39

Ca2+ channel (L, verapamil site)

[3H](-)D 888 D 600 D 600 43

CB1 (h) [3H]CP 55940 WIN 55212-2 CP 55940 15

CB2 (h) [3H]WIN 55212-2 WIN 55212-2 WIN 55212-2 -8

CCK1 (CCKA) (h) [125I]CCK-8 CCK-8 CCK-8s 15

CCK2 (CCKB) (h) [125I]CCK-8 CCK-8 CCK-8s -60

Choline transporter (CHT1) (h)

[3H]hemicholinium-3 hemicholinium-3

hemicholinium-3

14

Cl- channel (GABA-gated) [35S]TBPS picrotoxinin picrotoxinin -4

CRF1 (h) [125I]sauvagine sauvagine sauvagine 8

D1 (h) [3H]SCH 23390 SCH 23390 SCH 23390 49 > 10 000 > 3125

D2S (h) [3H]spiperone (+)butaclamol (+)butaclamol 51 > 10 000 > 3125

D3 (h) [3H]spiperone (+)butaclamol (+)butaclamol 23 > 1 329 > 415

D4.4 (h) [3H]spiperone (+)butaclamol clozapine 15

Dopamine transporter (h) (DAT)

[3H]BTCP BTCP BTCP 98 6 964 2 176

Estrogen (ER, non-selective) (h)

[3H]estradiol 17-beta-estradiol

17-beta -estradiol

-5

ETA (h) [125I]endothelin-1 endothelin-1 endothelin-1 -1

ETB (h) [125I]endothelin-1 endothelin-1 endothelin-3 4

GABA (non-selective) [3H]GABA GABA GABA 8

GABA transporter [3H]GABA (+ 10 µM isoguvacine + 10 µM baclofen)

GABA nipecotic acid 7

Glucocorticoid (h) (GR) [3H]dexamethasone triamcinolone dexamethasone -20

H1 (h) [3H]pyrilamine pyrilamine pyrilamine 25

H2 (h) [125I]APT tiotidine cimetidine 17


8

H3 (h) [3H]Na-Me-histamine (R)alpha-Me-histamine

(R)alpha -Me-histamine

61

I1 [3H]clonidine (+ 10 µM RX821002)

rilmenidine rilmenidine 89 770 241

I2 [3H]idazoxan cirazoline idazoxan 80 4 000 1 250

Kainate [3H]kainic acid L-glutamate kainic acid -10

KATP channel [3H]glibenclamide glibenclamide glibenclamide 2

KV channel [125I]alpha-dendrotoxin

alpha-dendrotoxin

alpha -dendrotoxin

-3

LTD4 (h) (CysLT1) [3H]LTD4 LTD4 LTD4 -33

M (non-selective) [3H]QNB atropine atropine 69

M1 (h) [3H]pirenzepine atropine pirenzepine 2 100 656

M3 (h) [3H]4-DAMP atropine 4-DAMP 4 800 1 500

MC4 (h) [125I]NDP-alpha-MSH NDP-alpha-MSH

NDP-alpha -MSH

5

N neuronal alpha -BGTX-insensitive (alpha 4beta 2)

[3H]cytisine nicotine nicotine -14

Na+ channel (site 2) [3H]batrachotoxinin veratridine veratridine 42

NK1 (h) [125I]BH-SP [Sar9,Met(O2)1

1]-SP [Sar9,Met(O2)11]-SP

14

NK2 (h) [125I]NKA [Nle10]-NKA (4-10)

[Nle10]-NKA(4-10)

33

NK3 (h) [3H]SR 142801 SB 222200 SB 222200 2

NMDA [3H]CGP 39653 L-glutamate CGS 19755 2

NOP (ORL1) (h) [3H]nociceptin nociceptin nociceptin -4

Norepinephrine transporter (h) (NET)

[3H]nisoxetine desipramine protriptyline 33 24 350 7 609

Opioid (non-selective) [3H]naloxone naloxone naloxone 7

P2X [3H]alpha, beta-MeATP

alpha, beta-MeATP

alpha ,beta -MeATP

2

P2Y [35S]dATPalpha S dATPalpha S dATPalpha S 6

PCP [3H]TCP MK 801 MK 801 4

Progesterone (h) (PR) [125I]PDGF BB PDGF BB R 5020, promegestone

-1

Sigma (non-selective) [3H]DTG haloperidol haloperidol 21

SKCa channel [125I]apamin apamin apamin 10

TRH1 (h) [3H]Me-TRH TRH TRH 3

V1a (h) [3H]AVP AVP [d(CH2)51,Tyr(Me)2]-AVP

6

V2 (h) [3H]AVP AVP AVP -2

Y (non-selective) [125I]peptide YY NPY NPY 5


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2. Enzymes & cell-based assays

Target Substrate/ Stimulus/ Tracer Reference compound

% of control values

Acetylcholinesterase (h) AMTCh (50 µM) neostigmine 8

Adenylyl cyclase (basal) ATP (0.5 mM) cAMP 2

ATPase (Na+/K+) ATP (2 mM) ouabain -2

COMT (Catechol- O-methyl transferase) esculetin (1 µM) scopoletin 8

constitutive NOS (h) (endothelial) [3H]arginine (28 nM) + arginine (50 nM) diclofenac -4

COX1 (h) arachidonic acid (4 µM) NS398 -9

COX2 (h) arachidonic acid (2 µM) L-NMMA 4

GABA transaminase GABA (9 mM) + α-ketoglutarate (9 mM) succinic semialdehyde -31

Guanylyl cyclase (basal) (h) GTP (0.1 mM) cGMP -1

MAO-A (h) kynuramine (0.15 mM) clorgyline -6

MAO-B (h) benzylamine (0.5 mM) deprenyl 11

PDE1 [3H]cAMP + cAMP (1 µM) 8-methoxy-IBMX 9

PDE2 (h) [3H]cAMP + cAMP (1 µM) EHNA -3

PDE3 (h) [3H]cAMP + cAMP (1 µM) milrinone 5

PDE4 (h) [3H]cAMP + cAMP (1 µM) rolipram 8

PDE5 (h) [3H]cGMP + cGMP (1 µM) dipyridamole 3

PKCalpha (h) ATP + biotinyl-neurogranin 28-43 peptide (60 nM)

Bis 10 8

PNMT (Phenylethanolamine- N-methyl transferase)

[14C]SAM (4 µM) + normetanephrine (28 mM)

[14C]metanephrine 2

Tyrosine hydroxylase [3H]tyrosine (10 µM) [3H]H2O 2


10

Table S2. Pharmacokinetic assessment of RO5073012 after iv and p.o. administration to mouse and rat.

The TAAR1 partial agonist RO5073012 has a moderate clearance, half-life, and volume of

distribution as well as good oral bioavailability. Pharmacokinetic values are the mean for two

animals per dose route. Cmax, maximum concentration; Tmax, time at which maximum

concentration was observed; AUC, area under the plasma concentration vs. time curve; CL,

clearance; Vss, volume of distribution at steady state; T1/2, terminal half-life; F, bioavailability;

Fu, Fraction unbound in plasma.

Species Mouse Rat

Route iv p.o. iv p.o.

Dose mg/kg 5 10 5 3.8

Cmax/Dose ng/mL 414 174 509 138

Tmax h 0 1.5 0 0.5

AUC/Dose ng/h/mL 704 601 418 277

T1/2 h 0.82 1.8 0.7 1.0

Vss L/kg 2.0 4.1

CL mL/min/kg 24 40

F % 86 66

Fu % 8.0 6.3

Brain/Plasma ratio 1.5 0.6 6.6


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SUPPLEMENTARY FIGURES

Figure S1


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Figure S1. Taar1 expression in wild-type and transgenic mice. (a,b) Comparison of Taar1 expression in the brain of wild-type (WT) and 2 lines of Taar1 transgenic mice, as revealed by radioactive in situ hybridization. (a) Representative autoradiograms for Taar1 expression in the rostral brain (striatum level) showing signal detected with the sense (S) and antisense (AS) probes. No specific signal could be observed in the brain of WT animals, indicating that endogenous Taar1 mRNA is present at very low levels. In the transgenic mice (Tg), Taar1 was expressed at high levels throughout the brain, with higher signal in B6-Tg(Taar1)19 mice (line 19) as compared to B6-Tg(Taar1)27 mice (line 27). Scale: 1 mm. (b) Quantification of Taar1 expression in the dorsal striatum and prefrontal cortex (Cingulate cortex) showing ~4.5 and 3 more expression in line 19 as compared to line 27. a.u., arbitrary units. ***P < 0.001 vs WT same line; ###P < 0.001 vs Tg line 27 (2-way ANOVA, followed by Student t test). (c) Rostro-caudal distribution (from top to bottom) of Taar1 expression in the brain of wild-type (WT) and B6-Tg(Taar1)27 mice (Tg27). Brain sections were taken at the following levels relative to the bregma (β, in mm): +3 (olfactory nuclei), +1.6 (accumbens nucleus), +0.3 (Caudate/Putamen, anterior commissure), -0.35 (Anterior hypothalamic area, paraventricular thalamic nucleus, anterodorsal preoptic nucleus), -1.6 (rostral hippocampus, dorsomedial / ventromedial hypothalamic nuclei), -2.2 (median eminence, arcuate nucleus), -2.8 (mammillary nuclei), -3.8 (ventral tegmental area), -4.5 (dorsal raphe nucleus), -5.4 (locus coeruleus).


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Figure S2

Figure S2. General physical properties of the Taar1 transgenic mice. No statistical significant differences were observed between B6-Tg(Taar1)27 mice (Tg) and wild-type littermates (WT) in pooled animals in the body weight (a), the rectal body temperature (b), the performance in the rotarod test at 16 rpm / min (c) and 32 rpm / min (d), indicative of motor coordination and balance, the physical strength as revealed by the horizontal wire test (e) and the grip strength (f). Animals were housed under standard conditions with free access to food and water and tested at 11 weeks of age. *P < 0.05, **P < 0.01 versus WT (Student t test). Data represent the mean ± SEM of the males and females (n = 12 animals / group), either pooled or separated.


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Figure S3

Figure S3. Effect of p-tyramine (pTyr) on the firing rate of dopaminergic, serotonergic and noradrenergic neurons of Taar1 transgenic mice. (a, c, e) Representative current-clamp recordings from (a) dopaminergic neurons of the ventral tegmental area (VTA), (c) serotonergic neurons of the dorsal raphe nucleus (DRN), and (e) noradrenergic neurons of the locus coeruleus (LC) in brain slices from either Taar1 transgenic mice (Tg) or wild-type littermates (WT), together with respective quantification bar graphs (b, d, f) (blue bars, WT; red bars, Taar1 Tg mice). Application of p-Tyr (10 µM) reduced the firing rates in all structures, an effect that was revered by co-application of the TAAR1 antagonist EPPTB (10 nM; pTyr+EPPTB) in the VTA and LC (a-b, e-f) or upon washout (Wash) in the DRN (c-d). Scale bars, 20 mV / 1 s. Data represent the mean ± SEM (n = 5 neurons from 3-5 animals per condition). ***P < 0.001 versus preceding condition (Kolmogoroff-Smirnov test).


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Figure S4

Figure S4. Quantitative PCR analyses from selected brain regions of Taar1 transgenic and wild-type mice. Expression of the dopamine receptor 2 (Drd2), the dopamine transporter (Dat; Slc6a3), the tyrosine hydroxylase (Th), the serotonin transporter (Sert; Slc6a4) and the tryptophan hydroxylase 2 (Tph2) genes were quantified by real-time quantitative PCR (TaqMan ABI probes) in selected brain regions of Taar1 transgenic (Tg) and wild-type littermate (WT) mice. The brain samples were punched from the caudate putamen (CPu; dorsal striatum), accumbens nucleus (Acb; ventral striatum), ventral tegmental area (VTA), brain stem (including the locus ceruleus) and the dorsal raphe nucleus (DRN). A relative quantification method based on GAPDH expression was used. *P < 0.05 vs WT (Student t test). Data represent the mean ± SEM (n = 4 in WT, 3 in Tg).


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Figure S5

Figure S5. Autoradiography analysis of dopamine receptors and transporter in the dorsal striatum of Taar1 transgenic mice. Representative autoradiograms showing [N-methyl-3H]SCH23390 (1 nM) binding to dopamine D1 receptors, [methoxy-3H]raclopride (1 nM) binding to dopamine D2-like receptors and [propylene-2,3-3H]GBR12935 (2 nM) binding to the dopamine transporters (DAT) in brains of Taar1 transgenic (Tg) and wild-type littermate (WT) mice. There was no significant change in receptor density in the Tg mice compared to the WT animals, as measured in the dorsal striatum (quantification on the right panel). Similarly, no significant change in binding to DAT was measured. Data represent the ± SEM (n = 5-6). In all cases, P > 0.05 versus WT (Student t test).


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Figure S6

Figure S6. Chemical structure of the selective TAAR1 partial agonist RO5073012 ((4-Chloro-phenyl)-(3H-imidazol-4-ylmethyl)-isopropyl-amine).

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