Revel et al. Supplementary information 1 Supplementary Information Brain-specific overexpression of trace amine-associated receptor 1 alters monoaminergic neurotransmission and decreases sensitivity to amphetamine Florent G. Revel 1 , Claas A. Meyer 2 , Amyaouch Bradaia 3 , Karine Jeanneau 1 *, Eleonora Calcagno 4 , Cédric B. André 1 *, Markus Haenggi 1 , Marie-Therese Miss 1 , Guido Galley 5 , Roger D. Norcross 5 , Roberto W. Invernizzi 4 , Joseph G. Wettstein 1 , Jean-Luc Moreau 1 & Marius C. Hoener 1 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
17
Embed
Supplementary Online Information · In situ hybridization Radioactive in situ hybridization was performed as described previously (Revel et al, 2007) using riboprobes for mouse Taar1
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Revel et al. Supplementary information
1
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
parameters were calculated by non-compartmental analysis of plasma concentration-time
curves using WinNonlin, version 4.1 software (Pharsight Corporation, Mountain View, CA).
Revel et al. Supplementary information
5
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.
Revel et al. Supplementary information
6
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
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
Revel et al. Supplementary information
11
SUPPLEMENTARY FIGURES
Figure S1
Revel et al. Supplementary information
12
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).
Revel et al. Supplementary information
13
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.
Revel et al. Supplementary information
14
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).
Revel et al. Supplementary information
15
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).
Revel et al. Supplementary information
16
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).
Revel et al. Supplementary information
17
Figure S6
Figure S6. Chemical structure of the selective TAAR1 partial agonist RO5073012 ((4-Chloro-phenyl)-(3H-imidazol-4-ylmethyl)-isopropyl-amine).