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Comparative Study
. 2011 Nov 23;31(47):16928-40.
doi: 10.1523/JNEUROSCI.2502-11.2011.

Genetic deletion of trace amine 1 receptors reveals their role in auto-inhibiting the actions of ecstasy (MDMA)

Benjamin Di Cara  1 , Roberto Maggio, Gabriella Aloisi, Jean-Michel Rivet, Ebba Gregorsson Lundius, Takashi Yoshitake, Per Svenningsson, Mauricette Brocco, Alain Gobert, Lotte De Groote, Laetitia Cistarelli, Sylvie Veiga, Catherine De Montrion, Marianne Rodriguez, Jean-Pierre Galizzi, Brian P Lockhart, Francis Cogé, Jean A Boutin, Philippe Vayer, P Monika Verdouw, Lucianne Groenink, Mark J Millan
Affiliations
Comparative Study

Genetic deletion of trace amine 1 receptors reveals their role in auto-inhibiting the actions of ecstasy (MDMA)

Benjamin Di Cara et al. J Neurosci. .

Abstract

"Ecstasy" [3,4-methylenedioxymetamphetamine (MDMA)] is of considerable interest in light of its prosocial properties and risks associated with widespread recreational use. Recently, it was found to bind trace amine-1 receptors (TA(1)Rs), which modulate dopaminergic transmission. Accordingly, using mice genetically deprived of TA(1)R (TA(1)-KO), we explored their significance to the actions of MDMA, which robustly activated human adenylyl cyclase-coupled TA(1)R transfected into HeLa cells. In wild-type (WT) mice, MDMA elicited a time-, dose-, and ambient temperature-dependent hypothermia and hyperthermia, whereas TA(1)-KO mice displayed hyperthermia only. MDMA-induced increases in dialysate levels of dopamine (DA) in dorsal striatum were amplified in TA(1)-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 TA(1)-KO versus WT mice. Conversely, genetic deletion of TA(1)R did not affect increases in DA levels evoked by para-chloroamphetamine (PCA), which was inactive at hTA(1) sites. The TA(1)R agonist o-phenyl-3-iodotyramine (o-PIT) blunted the DA-releasing actions of PCA both in vivo (dialysis) and in vitro (synaptosomes) in WT but not TA(1)-KO animals. MDMA-elicited increases in dialysis levels of serotonin (5-HT) were likewise greater in TA(1)-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, TA(1)Rs exert an inhibitory influence on both dopaminergic and serotonergic transmission, and MDMA auto-inhibits its neurochemical and functional actions by recruitment of TA(1)R. These observations have important implications for the effects of MDMA in humans.

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Figures

Figure 1.
Figure 1.
Generation of TA1R−/− knock-out mice. A, Strategy for targeted deletion of the Taar1 gene in mouse ES cells. The Taar1 coding sequence was partially replaced by a cDNA encoding IRES-LacZ-NeoTk cassette. The IRES-LacZ-Neo was fused in frame 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 domain sequence of unique exon of Taar1 gene. The arrowhead corresponds to the loxP sites. B, C, PCR screening on the 3′ (B) and 5′ (C) end of homologous recombination event. The 1505 bp (primers GW224/GW475 corresponding to PGKneo-Taar1 locus) and 5374 bp (primers GW587/GW114 corresponding to Taar1 locus-IRES/LacZ) bands identified appropriated targeted disruption of murine mTaar1 locus on 3′ and 5′ end-targeting events, respectively. The PCR screening on the 5′ and 3′ ends of the homologous recombination event is shown for ES cell clones 1A2 and 1D2. M, NEB Biolabs 1 kbp DNA ladder; C+, genomic DNA from ES cell clone 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 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 of Taar1-mutated mice. Southern blot was performed on XbaI-digested genomic DNA using an XbaI/HindIII probe specific for targeted region. The wild-type allele corresponds to a 5.4 kb band, and the appropriate targeting of the Taar1 locus to a 10.5 kb band. A nonspecific background signal due to the 5′ probe used, was observed ∼7 kb for all of the samples analyzed. In E and G, wild type is depicted by square, heterozygous by triangle, and homozygous by disc. MW, Molecular weight. H, Sagittal sections illustrating the histoenzymological staining of β-galactosidase in the substantia nigra (left) and ventral tegmental area (right) of TA1-KO mice.
Figure 2.
Figure 2.
Effect of MDMA, o-PIT, and PCA on cAMP accumulation in a cell line expressing hTA1R. cAMP accumulation was assessed by the α screen method (5.104 cells/well) in HeLa cells transiently transfected with human TA1Rs. Data are expressed as a percentage, with 100% being the inhibition elicited by 1 μm cAMP and 0% being the value with no cAMP (blank). All data are means ± SEM. N = 3 per concentration.
Figure 3.
Figure 3.
Thermoregulatory response of MDMA in TA1-KO mice. Body temperature was assessed at T30, T60, and T120 after injection of MDMA (0.63–20 mg/kg, i.p.), and its effect was expressed as the change from vehicle values. A–C, Effect of MDMA on body temperature of WT and TA1-KO mice, under standard condition (ambient temperature of 21°C). n = 6–10; p < 0.05, influence of treatment (★) or genotype (☆). D, Summary graph illustrating the effect of ambient temperature upon the thermoregulatory response of MDMA (10 mg/kg) at 30 min postinjection. Body temperature was measured under conditions of elevated temperature (27°C; Elev. temp.) and “standard” conditions (21°C; Std. temp.). For simplification of the graph, body temperature changes induced by vehicle are not illustrated (Std. temp.: 0.01 ± 0.14 and −0.34 ± 0.13 in WT and TA1-KO, respectively; Elev. temp.: 0.25 ± 0.19 and −0.31 ± 0.45 in WT and TA1-KO, respectively). n = 5–11; p < 0.05, influence of treatment (★), genotype (☆), or ambient temperature (•).
Figure 4.
Figure 4.
Effect of MDMA on extracellular levels of DA in the dorsal striatum of freely moving TA1-KO mice. A, Time course of the effect of MDMA in the striatum of WT and TA1-KO mice. Injection (vertical dotted line) of MDMA (10 mg/kg, i.p.) elicited a long-lasting overflow of DA. B, Dose–response relationship of the effect of MDMA on striatal DA overflow represented by AUC analysis (percentage × minute × 10−3; arbitrary units) of observations from 0 to 180 min. C, Time course of the effect of MDMA (10 mg/kg) in the nucleus accumbens of WT and TA1-KO mice. D, Graph illustrating the effect of MDMA represented by AUC. In A and C, data were analyzed using repeated measurements over the 180 min postinjection period. n = 5–7, p < 0.05, influence of treatment (★) or genotype (☆).
Figure 5.
Figure 5.
Influence of the TA1R agonist o-PIT upon the PCA-induced release of DA in the dorsal striatum in vivo (A–C) and in vitro (D). In freely moving WT mice, but not TA1-KO mice, o-PIT significantly reduced the action of PCA. A, B, Time course of the effect of o-PIT in combination with PCA upon dialysate levels of DA in the dorsal striatum. o-PIT (10 mg/kg, i.p.) was administered 20 min before PCA (5 mg/kg, i.p.) (vertical dotted lines) in WT and TA1-KO mice. Changes in DA levels were expressed compared with basal values (100%). C, Summary graph showing the action of o-PIT in combination with PCA, using AUC (percentage × minute × 10−3; arbitrary units) of observations from 20 to 180 min. D, Histogram of the o-PIT/PCA interaction upon the [3H]-DA release in synaptosomal preparation from dorsal striatum and nucleus accumbens. Perfusion of o-PIT (100 nm) significantly reduced the PCA (1 μm)-induced release of [3H]-DA (expressed as a percentage of total [3H]-DA overflow over 6 min of perfusion) in WT but not in TA1-KO mice. n = 6–10; p < 0.05, influence of treatment (★), pretreatment (•), or genotype (☆).
Figure 6.
Figure 6.
Phosphorylation state and activity of TH in slices from the dorsal striatum of TA1-KO mice. A, Levels of p-Ser19-TH, p-Ser31-TH, and p-Ser40-TH, adjusted to actin, compared with WT value (100%) and illustrated by representative immunoblots (inserts), were increased in TA1-KO mice. Levels of total TH were unchanged in TA1-KO mice. B, Measurement of the enzymatic activity of TH in the dorsal striatum of TA1-KO mice. Compared with WT value (100%), TH activity was significantly increased in TA1-KO mice. C, Influence of MDMA on levels of p-Ser40-TH in the dorsal striatum. The perfusion of MDMA (100 nm) significantly reduced the levels of p-Ser40-TH (% of WT value) in TA1-KO mice but not in WT mice. n = 4–5; p < 0.05, influence of treatment (★) or genotype (☆).
Figure 7.
Figure 7.
Influence of MDMA on locomotor activity in TA1-KO mice. A, Time course of the action of MDMA over a 180 min period. The effect of MDMA (10 mg/kg, i.p.; vertical dotted line) on locomotor activity was measured by 10 min bin in WT and TA1-KO mice. B, Histogram showing the abrogation of the action of MDMA (10 mg/kg) by the dopamine D2 antagonist haloperidol. The increase in locomotor activity (total count over the 180 min) was dose-dependently reduced by haloperidol (0.01–0.16 mg/kg, i.p.), administered 30 min later. n = 6–12; p < 0.05, influence of treatment (★), pretreatment (•), or genotype (☆).
Figure 8.
Figure 8.
Effect of MDMA on extracellular levels of DA in the frontal cortex, in vivo (A) and influence of o-PIT upon the PCA-induced released of DA in the frontal cortex, in vitro (B). A, Administration of MDMA (10 mg/kg, i.p.; vertical dotted line) elevated DA dialysate levels from basal levels (expressed as 100%). The action of MDMA was more pronounced in TA1-KO than in WT mice. AUC (percentage × minute × 10−3; arbitrary units) values are as follows: vehicle, 17.9 ± 1.4 and MDMA, 231.6 ± 40.8, in WT mice; vehicle, 19.5 ± 1.3 and MDMA, 374.2 ± 74.7, in TA1-KO mice. B, Histogram showing the o-PIT/PCA interaction on [3H]-DA release in synaptosomal preparation from frontal cortex. Perfusion of o-PIT (100 nm) significantly reduced the PCA (1 μm)-induced release of [3H]-DA (expressed as percentage of total [3H]-DA overflow over 6 min of perfusion) in WT but not in TA1-KO mice. n = 6–10; p < 0.05, influence of treatment (★), pretreatment (•), or genotype (☆).
Figure 9.
Figure 9.
Action of MDMA on extracellular levels of 5-HT in the dorsal striatum, nucleus accumbens, and frontal cortex of freely moving WT and TA1-KO mice. A, Time course of the effect of MDMA on 5-HT levels in the dorsal striatum. Administration of MDMA (10 mg/kg, i.p.; vertical dotted line) robustly increased 5-HT levels. B, Dose–response relationship of the effect of MDMA on striatal 5-HT levels. Values are AUC (percentage × minute × 10−3; arbitrary units) calculated from 0 to 180 min. C, Time course of the effect of MDMA on 5-HT levels in the nucleus accumbens. AUC values in WT mice: 19.3 ± 2.0, vehicle; and 239.6 ± 18.8, MDMA. AUC values in TA1-KO mice: 16.5 ± 1.3, vehicle; and 336.2 ± 29.1, MDMA. D, Time course of the effect of MDMA on 5-HT levels in the frontal cortex. AUC values in WT mice: 15.3 ± 1.0, vehicle; and 250.6 ± 17.6, MDMA. AUC values in TA1-KO mice: 14.9 ± 0.7, vehicle; and 212.1 ± 67.4, MDMA. n = 5–8; p < 0.05, influence of treatment (★) or genotype (☆).
Figure 10.
Figure 10.
Influence of o-PIT upon the PCA-elicited release of 5-HT in the dorsal striatum in vivo (A–C) and in vitro (D). A, B, Time course of the effect of o-PIT in combination with PCA upon dialysate levels of 5-HT in the dorsal striatum. o-PIT (10 mg/kg, i.p.), administered 20 min before PCA (5 mg/kg, i.p.) (vertical dotted lines), significantly reduced the action of PCA on 5-HT levels (expressed in percentage from basal) in WT but not in TA1-KO mice. C, Summary graph showing the action of o-PIT in combination with PCA, using AUC (percentage × minute × 10−3; arbitrary units) of observations from 20 to 180 min. D, Histogram of the o-PIT/PCA interaction upon the [3H]-5-HT release in synaptosomal preparation from dorsal striatum and nucleus accumbens. Perfusion of o-PIT (100 nm) reduced the PCA (1 μm)-induced release of [3H]-5-HT (expressed as percentage of total [3H]-5-HT overflow over 6 min perfusion) in WT but not in TA1-KO mice. n = 4–10; p < 0.05, influence of treatment (★), pretreatment (•), or genotype (☆).
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