Robust aversive effects of trace amine-associated receptor 1 activation in mice

Abstract

Drugs that stimulate the trace amine-associated receptor 1 (TAAR1) are under clinical investigation as treatments for several neuropsychiatric disorders. Previous studies in a genetic mouse model of voluntary methamphetamine intake identified TAAR1, expressed by the Taar1 gene, as a critical mediator of aversive methamphetamine effects. Methamphetamine is a TAAR1 agonist, but also has actions at monoamine transporters. Whether exclusive activation of TAAR1 has aversive effects was not known at the time we conducted our studies. Mice were tested for aversive effects of the selective TAAR1 agonist, RO5256390, using taste and place conditioning procedures. Hypothermic and locomotor effects were also examined, based on prior evidence of TAAR1 mediation. Male and female mice of several genetic models were used, including lines selectively bred for high and low methamphetamine drinking, a knock-in line in which a mutant form of Taar1 that codes for a non-functional TAAR1 was replaced by the reference Taar1 allele that codes for functional TAAR1, and their matched control line. RO5256390 had robust aversive, hypothermic and locomotor suppressing effects that were found only in mice with functional TAAR1. Knock-in of the reference Taar1 allele rescued these phenotypes in a genetic model that normally lacks TAAR1 function. Our study provides important data on TAAR1 function in aversive, locomotor, and thermoregulatory effects that are important to consider when developing TAAR1 agonists as therapeutic drugs. Because other drugs can have similar consequences, potential additive effects should be carefully considered as these treatment agents are being developed.

Fig. 1. The TAAR1 agonist, RO5256390, induces robust conditioned taste aversion in MALDR mice.

Shown are means ± SEM for NaCl consumption (ml) in MALDR mice across vehicle and RO5256390 conditioning trials. Treatments were administered by IP injection on days 7, 9, 11 and 13, immediately following 1 h access to 0.2 M NaCl. n = 7–8/sex/dose. *p < .05, **p < .01, ***p < 0.001 vs day 7; ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 vs the 2 and 4 mg/kg groups. MALDR methamphetamine low drinking selected line, NaCl sodium chloride, TAAR1 trace amine-associated receptor 1.

Fig. 2. RO5256390 induces robust conditioned place aversion in MALDR mice.

Shown in A–C are means ± SEM for locomotor activity (Total beam breaks) during the 15-min alternating vehicle and drug conditioning trials. RO5256390 doses for each experiment are indicated in legends within each panel. For A–C *p < 0.05, **p < 0.01, ***p < 0.001 for the difference from the previous trial collapsed on vehicle group or RO5256390 dose (because the vehicle groups and dose groups were not significantly different) or in C, collapsed across all groups (because there were no significant group or treatment effects); ⁺⁺⁺p < 0.001 for the difference between vehicle vs. RO5256390 on the indicated conditioning trial (A) or for the main effect (B). In both A and B, data are collapsed on vehicle group or RO5256390 dose (because the vehicle groups and dose groups were not significantly different). Shown in D–F are means ± SEM for percent time (%Time) on the drug-paired floor during the 30-min pre-test, drug-free test after IP vehicle injection, and drug-present test after IP RO5256390 injection for the 0.5 and 4 mg/kg (D), 0.05 and 0.1 mg/kg (E), and 0.005 and 0.01 mg/kg (F) RO5256390 studies. Shown in G–I are means ± SEM for locomotor activity (Total beam breaks) during the three tests. For D–I ***p < 0.001 for the comparison with pre-test collapsed on RO5256390 dose (because the dose groups were not significantly different); ⁺⁺⁺p < 0.001 for the comparison with drug-free collapsed on RO-5256390 dose (because the dose groups were not significantly different); for H symbols on each side of the slash (/) indicate significance results for 0.05 mg/kg to the left and 0.1 mg/kg to the right. n = 11–12/sex/dose. MALDR methamphetamine low drinking selected line.

Fig. 3. RO5256390 induces robust hypothermia in MALDR mice.

Shown are mean ± SEM rectal temperatures at baseline (T0) and T60-180 min after IP injection of vehicle or RO5256390 after the 0.5 and 4 mg/kg (A), 0.05 and 0.1 mg/kg (B), and 0.005 and 0.01 mg/kg (C) RO5256390 place conditioning studies. Vehicle was not tested in the first study (A), but was included in the other two studies (B, C). n = 11–12/sex/dose. ***p < 0.001 for the difference from T0, collapsed on vehicle or RO5256390 dose (because the vehicle groups and dose groups were not significantly different), or in C, collapsed across all groups (because there were no significant group or treatment effects); ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 for the difference from vehicle collapsed on RO5256390 dose (because the dose groups were not significantly different). Shown in D are mean ± SEM temperature data for experiment- and drug-naïve MALDR mice after a single IP injection of vehicle or RO5256390. n = 7/sex/dose. ***p < 0.001 for the difference at T60-T180 from T0 for all groups; ⁺⁺p < 0.01, for the difference between RO5256390 0.1 mg/kg and vehicle at T120; ⁺⁺⁺p < 0.001 for the difference between RO5256390 0.1 mg/kg and all other groups at T60. MALDR methamphetamine low drinking selected line.

Fig. 4. Sensitivity to RO5256390-induced conditioned place aversion and hypothermia is reinstated by excision of the Taar1^m1J mutation and knock-in of the wild-type Taar1⁺ allele.

Shown are means ± SEM for locomotor activity (Total beam breaks) during the 15-min alternating vehicle and drug conditioning trials in control (A) and knock-in (B) mice. For A and B, *p < 0.05, for the difference from the previous trial collapsed on RO5256390 dose (because the dose groups were not significantly different); ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 for the difference between vehicle vs. RO5256390 treatment for the indicated conditioning trial, collapsed on vehicle group and RO5256390 dose (because the vehicle groups and dose groups were not significantly different). Also shown are means ± SEM for percent time (%Time) on the drug-paired floor during the 30-min pre-test, drug-free test after IP vehicle injection, and drug-present test after IP RO5256390 injection (C), and locomotor activity (Total beam breaks) during the three tests (D). For C and D, *p < 0.05, ***p < 0.001 for the comparison with pre-test collapsed on RO5256390 dose (because the dose groups were not significantly different); ⁺⁺⁺p < 0.001 for the comparison with drug-free collapsed on RO5256390 dose (because the dose groups were not significantly different); ^###p < 0.001 for the main effect of line. Also shown are mean ± SEM for rectal temperatures at baseline (T0) and T60–180 min after IP injection of vehicle or RO5256390 for control (E) and knock-in (F) mice. For E and F, **p < 0.01, ***p < 0.001 for the difference from T0 collapsed on vehicle group or RO5256390 dose (because the vehicle groups and dose groups were not significantly different); ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 for the difference from vehicle collapsed on RO5256390 dose (because the dose groups were not significantly different). n = 11–12/sex/line/dose. MAHDR methamphetamine high drinking selected line; Taar1⁺: trace amine-associated receptor 1 reference allele coding for a functional receptor; Taar1^m1J: trace amine-associated receptor 1 mutant allele coding for a non-functional receptor.

Fig. 5. Sensitivity to the hypothermic effect of RO5256390 is dependent on Taar1 genotype.

Shown are mean ± SEM rectal temperatures at baseline (T0) and T30-180 min after IP injection of vehicle or RO5256390 in experiment- and drug-naïve MAHDR mice (A), n = 11–12/sex/dose; MALDR mice (B), n = 12/sex/dose; control mice (C), n = 11–13/sex/dose; and knock-in mice (D), n = 12–13/sex/dose. ***p < 0.001 for the difference from T0 for the vehicle group or collapsed on RO5256390 dose (because the dose groups were not significantly different); ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 for the difference from vehicle collapsed on RO5256390 dose (because the dose groups were not significantly different). MAHDR methamphetamine high drinking selected line, MALDR methamphetamine low drinking selected line, Taar1⁺ trace amine-associated receptor 1 reference allele coding for a functional receptor; Taar1^m1J trace amine-associated receptor 1 mutant allele coding for a non-functional receptor.