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, 106 (47), 20081-6

The Selective Antagonist EPPTB Reveals TAAR1-mediated Regulatory Mechanisms in Dopaminergic Neurons of the Mesolimbic System

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The Selective Antagonist EPPTB Reveals TAAR1-mediated Regulatory Mechanisms in Dopaminergic Neurons of the Mesolimbic System

Amyaouch Bradaia et al. Proc Natl Acad Sci U S A.

Abstract

Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) that is nonselectively activated by endogenous metabolites of amino acids. TAAR1 is considered a promising drug target for the treatment of psychiatric and neurodegenerative disorders. However, no selective ligand to identify TAAR1-specific signaling mechanisms is available yet. Here we report a selective TAAR1 antagonist, EPPTB, and characterize its physiological effects at dopamine (DA) neurons of the ventral tegmental area (VTA). We show that EPPTB prevents the reduction of the firing frequency of DA neurons induced by p-tyramine (p-tyr), a nonselective TAAR1 agonist. When applied alone, EPPTB increases the firing frequency of DA neurons, suggesting that TAAR1 either exhibits constitutive activity or is tonically activated by ambient levels of endogenous agonist(s). We further show that EPPTB blocks the TAAR1-mediated activation of an inwardly rectifying K(+) current. When applied alone, EPPTB induces an apparent inward current, suggesting the closure of tonically activated K(+) channels. Importantly, these EPPTB effects were absent in Taar1 knockout mice, ruling out off-target effects. We additionally found that both the acute application of EPPTB and the constitutive genetic lack of TAAR1 increase the potency of DA at D2 receptors in DA neurons. In summary, our data support that TAAR1 tonically activates inwardly rectifying K(+) channels, which reduces the basal firing frequency of DA neurons in the VTA. We hypothesize that the EPPTB-induced increase in the potency of DA at D2 receptors is part of a homeostatic feedback mechanism compensating for the lack of inhibitory TAAR1 tone.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Chemical structure of the selective TAAR1 antagonist N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide (EPPTB).
Fig. 2.
Fig. 2.
Effects of p-tyr and EPPTB on the spontaneous firing rate of DA neurons in the VTA. (A) In neurons of WT mice, the firing frequency was assessed before (control) and during the application of p-tyr alone and in combination with EPPTB. EPPTB not only inhibited the p-tyr-mediated decrease in firing frequency but increased the firing frequency over control levels (control, 2.1 ± 0.8 Hz; p-tyr, 0.5 ± 0.1 Hz; p-tyr + EPPTB, 15.1 ± 1.0 Hz; n = 10; ***, P < 0.001 for p-tyr + EPPTB versus p-tyr). (B) In neurons of Taar1−/− mice the firing frequency was not significantly affected by application of p-tyr or EPPTB (control, 16.0 ± 1.0 Hz; p-tyr, 15.0 ± 1.3 Hz; p-tyr + EPPTB, 16.1 ± 1.1 Hz; n = 10). (C) In neurons of WT mice the firing frequency was significantly increased by EPPTB in the absence of p-tyr application. After wash-out of EPPTB for 1 h (wash) the firing frequency reverted close to control levels (control, 1.9 ± 0.2 Hz; EPPTB, 15.1 ± 1.0 Hz; wash, 3.5 ± 0.8 Hz; n = 4; ***, P < 0.001 for EPPTB versus control). (D) In neurons of Taar1−/− mice, EPPTB had no effect on the firing frequency (control, 14.3 ± 1.1 Hz; EPPTB, 14.8 ± 0.9 Hz; wash, 15.2 ± 0.9 Hz; n = 6). In A–D, representative recordings are shown on the left and summary bar graphs on the right. (Scale bar, 20 mV/2 s.)
Fig. 3.
Fig. 3.
TAAR1-evoked currents in DA neurons of the VTA. (A) Representative current traces recorded from DA neurons of WT (black trace) and Taar1−/− (gray trace) mice. Application of p-tyr (10 μM) induced an outward current in WT neurons that was antagonized by EPPTB (10 nM). (B) Summary bar graph illustrating that application of EPPTB reduced the current below baseline in WT but not in Taar1−/− neurons (WT: p-tyr, 21.2 ± 1.5 pA, EPPTB, −9.4 ± 0.6 pA; Taar1−/−: p-tyr, 0.40 ± 0.01 pA, EPPTB, −0.05 ± 0.02 pA; n = 7, ***, P < 0.001). The D2 antagonist sulpiride (sulp) did not significantly inhibit the outward current induced by p-tyr application (WT: p-tyr + sulp, 18.8 ± 0.8 pA, n = 7; P > 0.05 versus p-tyr). Likewise, infusion of the PKA inhibitor H8 via the recording pipette did not significantly alter the outward current induced by p-tyr (WT: p-tyr + H8, 20.7 ± 1.4, n = 5; P > 0.05 versus p-tyr). (C) Without preceding stimulation by p-tyr, EPPTB induced an apparent inward current in neurons from WT but not Taar1−/− mice. (D) Summary bar graph illustrating changes in the holding current following EPPTB application. The amplitude of the EPPTB-induced apparent inward current was significantly increased in the presence of the nonspecific monoamine oxidase inhibitor pargylin, which increases the extracellular TA concentration (WT: EPPTB, −11.5 ± 1.1 pA, EPPTB + pargylin, −25.9 ± 1.7 pA; Taar1−/−: EPPTB, −0.04 ± 0.01 pA, EPPTB + pargylin, −0.15 ± 0.02 pA; n = 5; ***, P < 0.001). (E) Bar graph representing maximal p-tyr currents in the presence or absence of GDPβS (2 mM for 40 min), which was infused via the recording pipette (WT: control, 20.0 ± 1.0 pA; GDPβS, 3.6 ± 0.9 pA; Taar1−/−: control, 0.10 ± 0.02 pA, GDPβS, 0.25 ± 0.01 pA; n = 10; ***, P < 0.001). [Scale bar, 5 min/10 pA in (A) and (C).]
Fig. 4.
Fig. 4.
TAAR1 activates an inwardly rectifying K+ current in DA neurons of the VTA. The p-tyr-induced currents were obtained by calculating the difference between the I–V curves before and after addition of p-tyr (10 μM), both in the absence (black traces) and presence of EPPTB (10 nM, gray traces). (A) An inwardly rectifying, EPPTB-sensitive current was induced by p-tyr in neurons from WT but not Taar1−/− mice. At physiological extracellular [K+] (2.5 mM) the polarity of the p-tyr-induced current reversed at −101 mV. (B) Raising extracellular [K+] to 12.5 mM shifted the reversal potential of the p-tyr-induced current to −60 mV.
Fig. 5.
Fig. 5.
TAAR1 activates Kir3 channels in Xenopus oocytes via PTX-insensitive G-proteins. (A) Current traces were recorded from oocytes clamped to −50 mV, which were injected with mRNAs encoding mouse TAAR1 and human Kir3.1/3.2. In the presence of 45 mM K+ the application of p-tyr (10 μM, black bars) induced an inward current that was of similar amplitude with a second application of p-tyr (top trace). The p-tyr-induced current was reduced by EPPTB (bottom trace). (B) Bar graph depicting the ratio of the second-to-first p-tyr current response in the presence or absence of EPPTB (control). On average, EPPTB inhibited the p-tyr-induced current by 72 ± 2% (n = 4). (C) Bar graph depicting that PTX pretreatment did not significantly alter the p-tyr response of TAAR1 expressing oocytes. In contrast, PTX significantly reduced the response to acetylcholine (ACh, 10 μM) in oocytes expressing the human muscarinic ACh receptor 2 (hM2), which couples to Kir3 via Gi/o-type G-proteins. (D) Bar graph depicting the average amplitudes of oocytes injected with mRNAs for TAAR1 and Kir3.1/3.2 without (control) and with mRNA for human Gαs (20 pg/nL). Coexpression of Gαs drastically increased the p-tyr-mediated current. (Scale bar, 1 min/100 nA in A.)
Fig. 6.
Fig. 6.
TAAR1 activity modulates D2 receptor desensitization rate and agonist potency in DA neurons of the VTA. (A and B) Representative traces of quinpirole-induced currents in the presence and absence of EPPTB. (Scale bar, 5 min/20 pA.) Bar graphs represent the desensitization rate, which is expressed as the residual current after continuous quinpirole application for 15 min [I(t)], normalized to the initial maximal current (Imax). (A) Preincubation of WT slices with EPPTB (10 nM) prevents the D2 receptor desensitization seen in control slices (control, I(t)/Imax = 0.31 ± 0.04; EPPTB, I(t)/Imax = 1.08 ± 0.05; n = 9, ***, P < 0.001). (B) Taar1−/− slices exhibit non-desensitizing D2-mediated currents in the absence and presence of EPPTB (control, I(t)/Imax = 1.03 ± 0.14; EPPTB, I(t)/Imax = 1.09 ± 0.08; n = 6). Following application of the D2 antagonist sulpiride, the holding current was reduced below baseline (dotted line) in WT slices preincubated with EPPTB, similar as in Taar1−/− slices. (C) Dose-response relationships of the quinpirole-induced current in WT slices, WT slices preincubated with EPPTB (10 nM), WT slices preincubated with p-tyr (40 nM) and Taar1−/− slices. Current amplitudes were normalized to the maximal current obtained with a saturating concentration of quinpirole (10 μM). EC50 values: WT, 109.5 ± 3.8 nM, n = 9; WT + EPPTB, 27.2 ± 0.4 nM, n = 9; WT + p-tyr, 754.4 ± 6.6 nM, n = 4; Taar1−/−, 23.3 ± 0.4 nM, n = 9.

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