Introduction

Drugs that stimulate the trace amine-associated receptor 1 (TAAR1) have clinical potential for several neuropsychiatric disorders. Preclinical findings for addiction, schizophrenia, compulsive binge-like eating, and more have spurred clinical investigation [1,2,3,4,5,6,7,8,9]. For example, the TAAR1 partial agonist, RO5263397, reduced the motivation of rats to obtain methamphetamine (MA) and blocked the ablity of MA treatment to reinstate MA seeking; [6] the agonist, RO5256390, blocked binge-like eating of a highly palatable diet in rats; and the TAAR1 agonist Ulotaront had efficacy against cue-induced cocaine seeking in rats [7] and also had an antipsychotic-like behavioral profile in mice [3]. Ulotaront has progressed to Phase 3 clinical trials for the treatment of schizophrenia [10, 11]. Rather than in pursuit of drugs with TAAR1 activity, Ulotaront was identified in a broad search for drugs that lack dopamine (DA) D2 receptor and serotonin 5-HT2A receptor antagonist activity [11]. Taar1 was nominated as a genetic risk factor by an unbiased genome-wide search for genetic variants differing between selected lines of mice bred for high vs. low MA intake [12]. Subsequent directed research verified the significant role of Taar1 [13,14,15].

A complete understanding of the behavioral, physiological and molecular actions of TAAR1 activation is critical for the development of TAAR1 ligands for neuropsychiatric treatment. Although TAAR1 was cloned in 2001 [16, 17], the molecular consequences of its activation remain ill-defined. TAAR1 agonists reduce MA-induced DA elevations [18] and MA or amphetamine-induced DA elevations are lower in mice with functional TAAR1, compared to mice lacking TAAR1 function [6, 19,20,21]. TAAR1-specific agonists/partial agonists do not themselves support self-administration [6, 7]. They may reduce drug seeking and self-administration by preventing the rewarding/pleasurable effects of a drug that stimulates the dopaminergic system [22], or by substitution, in the case of MA, which is also a TAAR1 agonist [6].

Using Taar1 knockout mice and mice possessing a mutant Taar1 allele (Taar1m1J) that expresses a nonfunctional TAAR1, we demonstrated that TAAR1 function is important for experiencing aversive effects of MA [13, 15, 23]. Further, we found that in the absence of sensitivity to MA-induced aversion, MA reward and reinforcement are experienced [24,25,26,27]. Concurrent with our further exploration of the role of TAAR1 in aversive effects of MA in mice, a recent rat study [28] supported our view that stimulation of a functional TAAR1 with agonists induces an aversive state. Liu et al. [28]. found that the TAAR1 receptor-specific agonists, RO5263397 and RO5166017, induce conditioned taste aversion (CTA). For the work reported here, we conducted systematic dose-response studies in mouse models of MA use disorder, including the selectively bred MA high drinking (MAHDR) and MA low drinking (MALDR) lines and a Taar1 knock-in line. We report dose-related aversive, locomotor, and thermal effects of the potent TAAR1-specific agonist, RO5256390 [29]. We studied the hypothermic effect of RO5256390, based on evidence that sensitivity to MA-induced hypothermia may play a role in MA-induced aversion and intake [13, 30], and because MA induces acute hypothermia in mice with functional TAAR1 absent in mice lacking TAAR1 function [3, 15, 23]. We used a single nucleotide Taar1 knock-in model [15] to provide definitive evidence of a role for TAAR1 in the aversion, locomotor and hypothermia traits.

Materials and methods

Animals

All experiments involved MA- and experimentally-naïve adult male and female mice. Methods were consistent with guidelines from the National Institutes of Health and approved by the Institutional Animal Care and Use Committees of Minot State University (MSU), the VA Portland Health Care System (VAPORHCS), and Grand Valley State University (GVSU). For additional details, see Supplementary Information. Group sizes are in the figure captions.

Selected mouse lines for high and low MA intake

Adult (56–100 days old) MAHDR and MALDR mice were produced within the VAPORHCS animal facility and tested in Portland or Minot. Multiple replicates of these selectively bred lines were derived from independent populations of the F2 cross between the C57BL/6 J and DBA/2 J inbred strains. Reliable outcomes have been obtained [24, 27, 31]. For details about mice tested in the current studies, see Supplementary Information.

Knock-in MAHDR-Taar1+/+ and control MAHDR-Taar1m1J/m1J mice

Adult (68–115 days old) mice were produced and tested within the VAPORHCS animal facility or in Allendale. CRISPR-Cas9 technology [32, 33] was used to generate the lines. To create the knock-in mice, the Taar1m1J single nucleotide polymorphism, encoding a non-synonymous proline to threonine mutation at amino acid position 77 (found in all MAHDR mice) was excised and replaced to convert their genotype at position 77 back to proline, consistent with the reference Taar1+ allele [13, 15]. The control line was derived from mice that underwent the CRISPR-Cas9 procedures, for which their Taar1 genotype was not successfully altered.

Drugs

The selective TAAR1 agonist, RO5256390 [(S)-4-((S)-2-phenyl-butyl)-4,5-dihydro-oxazol-2-ylamine], was the generous gift of F. Hoffmann-La Roche, Ltd., Basel Switzerland. RO5256390 was dissolved in 3% Tween 80 in distilled water and injected intraperitoneally (IP; 10 ml/kg). For details about efficacy and doses chosen for study, see Supplementary Information.

Conditioned taste aversion (CTA) procedure

Isolate housed MALDR mice were tested in Minot, using established procedures for measuring MA-induced CTA to a novel 0.2 M sodium chloride (NaCl) solution [26, 27]. The dependent variable was NaCl intake across drug conditioning trials. For procedural details, see Supplementary Information.

Place conditioning and testing procedure

Place conditioning experiments used methods consistent with those established in Portland for MA [26, 27]. For conditioning, a compound tactile/wall color cue was used. A cue preference pre-test was conducted, then the cues were associated with vehicle or RO5256390 across 6 alternating trials each. Drug-free and drug-present cue preference tests were conducted 2 and 5 days after conditioning, respectively. Thirty-minute test trials measured percent time (%Time) spent with the cues previously paired with RO5256390, first in the absence of treatment during the test (drug-free), and then after drug treatment (drug-present). %Time was calculated as sec on the drug-paired floor divided by 1800 s (total test period) x 100. Locomotor activity was measured during conditioning and test trials. For procedural details, see Supplementary Information.

Four studies were conducted; the first 3 in Minot and the last in Allendale. The first experiment tested MALDR mice at RO5256390 doses of 0.5 and 4 mg/kg. Based on robust effects, two additional studies were conducted to determine the effective dose range; the first at doses of 0.05 and 0.1 mg/kg; the second at doses of 0.005 and 0.01 mg/kg. The fourth experiment tested knock-in and control mice at doses of 0.05 and 0.1 mg/kg.

Drug-induced hypothermia

The effect of RO5256390 on core body temperature was measured on the day after the drug-present test in the mice that were tested in the place conditioning studies. Methods were similar to those previously established [13]; see Supplementary Information. Rectal temperatures were recorded prior to RO5256390 treatment (T0) and then at several times up to 180 min (T180) post-treatment. For the second two RO5256390 dose studies (0.005, 0.01, 0.05, and 0.1 mg/kg), body temperature data after vehicle treatment were also collected by obtaining thermal data on 2 consecutive days: RO5256390 on 1 day and vehicle on the other, in counterbalanced order.

Two additional experiments measured thermal effects of RO5256390 in mice that had no prior drug exposure. MALDR mice were tested in Minot after vehicle, 0.01, 0.05 or 0.1 mg/kg RO5256390, and had the goal of determining the approximate lowest RO5256390 dose able to produce significant hypothermia in the MALDR mice. A second experiment, performed in Portland, compared MAHDR, MALDR, knock-in and control mice for thermal response to a single treatment with vehicle, 0.05 and 0.1 mg/kg RO5256390.

Data analysis

Data were analyzed with IBM SPSS (IBM Corp., Armonk, NY, USA), using repeated measures analysis of variance (ANOVA) with test day, conditioning trial or time as the within-subjects factor. Dose, sex and mouse line (genotype) were potential between-group factors. Sex was included as a factor in initial analyses, and then excluded if no significant sex effects were found. Sex is mentioned in the results when it had a significant effect. Data were checked for normality and sphericity. We applied Huynh-Feldt corrections to reduce Type I error, when sphericity was violated. Three-way interactions were investigated using two-way ANOVA within each level of a relevant factor (e.g., for effects of dose and time within each mouse line). Two-way interactions were examined for significant simple effects, and for thermal data, post hoc mean comparisons with Bonferroni corrections were used to identify mean differences. To reduce Type I error, mean comparisons were restricted to those appropriate for evaluating specific hypotheses, rather than testing for all possible differences. Statistical results were considered significant at p < 0.05.

Results

Conditioned taste aversion (CTA) test in MALDR mice

The 2 and 4 mg/kg RO5256390 doses induced comparable levels of CTA (Fig. 1). Repeated measures ANOVA identified a significant dose x test day interaction (F6,115 = 3.6, p < 0.01). There were significant changes in NaCl consumption across day for each RO5256390 dose (p < 0.001 and p < 0.01, respectively), but not for vehicle, with RO5256390-treated mice consuming less NaCl on all trials subsequent to treatment, compared to their initial NaCl consumption. In addition, NaCl consumption was lower in RO5256390 than vehicle-treated mice on several trials, reflecting Taar1 agonist-induced CTA (see Fig. 1).

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

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.

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Place conditioning in MALDR mice

Locomotor activity on conditioning days

During conditioning, locomotor activity was suppressed by RO5256390 doses of 0.05 to 4 mg/kg, compared to vehicle, but not by 0.005 or 0.01 mg/kg (Fig. 2A–C). Statistical outcomes are in Supplementary Information-Results for locomotor activity on conditioning days in MALDR mice.

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

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.

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Percent time on drug-paired floor

Data for %Time on the drug-paired floor for each of the 3 studies (Fig. 2D–F) were collected and analyzed separately. Doses of 0.05–4 mg/kg induced similar levels of conditioned place aversion (CPA). Repeated measures ANOVA identified a significant effect of test day for the 0.5 and 4 mg/kg (Fig. 2D; F1.3,60 = 61.8, p < 0.001), and the 0.05 and 0.1 mg/kg (Fig. 2E; F1.5,69 = 50.2, p < 0.001) studies, but no interaction with dose in either study. Therefore, data were combined for the two doses in each study and means compared. In both studies, mice spent significantly less %Time on the drug-paired floor during the drug-free and drug-present tests, compared to the pre-test. They also spent significantly less %Time on the drug-paired floor during the drug-present test, compared to the drug-free test, indicating that more profound aversion was expressed when mice were tested under the drug-conditioning state (drug-present). There were no significant statistical outcomes for RO5256390 doses of 0.005 and 0.01 mg/kg (Fig. 2F).

Locomotor activity on test days

Locomotor activity was suppressed during the drug-present tests for the 0.1–4 mg/kg doses. For the 0.5 and 4 mg/kg study (Fig. 2G), there was a significant main effect of test day (F1.9,90.9 = 83.0, p < 0.001), but no significant effect of dose. Mean comparisons for data combined across dose identified higher activity during the drug-free test, compared to the pre-test, and significantly lower activity during the drug-present test, compared to the other two tests. For the 0.05 and 0.1 mg/kg study (Fig. 2H), there was a significant test day x dose interaction (F2.0,90 = 4.0, p < 0.05), with a significant effect of test day for both doses (ps < 0.001). For 0.05 mg/kg, activity was higher on the drug-free test, compared to the pre-test and drug-present test. For the 0.1 mg/kg dose, activity was higher on the drug-free test compared to pre-test, and lower on the drug-present test compared to the other tests. Finally, for the lowest two doses (Fig. 2I), there was a significant main effect of test day (F1.3,62.4 = 15.3, p < 0.001), but no significant effect of RO5256390 dose. Mean comparisons for data combined across dose identified higher activity during the drug-free and drug-present tests, compared to the pre-test, and higher activity during the drug-free test than drug-present test.

Post-place conditioning test of RO5256390 thermal effects

For temperature data after each of the place conditioning studies, repeated measures ANOVA, identified significant RO5256390-induced hypothermia at doses of 0.05–4 mg/kg (Fig. 3A–C). For the 0.5 and 4 mg/kg doses (Fig. 3A), there was a significant effect of time (F2.2,93.8 = 140.6, p < 0.001), and a time x sex interaction (F2.2,93.8 = 4.7, p < 0.01). Temperature decreased from T0 to T60 and returned toward baseline from T60 to T180. A significant sex difference (p < 0.01) was found only at T180; mean temperature was higher in females than males by 0.6 °C.

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

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.

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A vehicle response day was added to the subsequent two studies. Data for each study were analyzed by a nested (treatment within day) repeated measures ANOVA. For vehicle vs. RO5256390 0.05 and 0.1 mg/kg (Fig. 3B), there were significant treatment x time (F2.1,92.2 = 35.7, p < 0.001), and time x sex (F2.3,100.5 = 5.7, p < 0.01) interactions. Effects were similar for the two RO5256390 doses, but mice had lower body temperatures after RO5256390, compared to vehicle, at T60 and T120. For the vehicle data, there were declining temperatures across time, typical of isolate-housed mice. Females had a slightly lower mean temperature than males of 0.5 °C at T0 only. For vehicle vs. RO5256390 0.005 and 0.01 mg/kg (Fig. 3C), there was a significant main effect of time (F 2.5, 111.1 = 52.3, p < 0.001), and time x sex interaction (F 2.5, 100.5 = 3.5, p < 0.05), but no significant effects involving treatment or dose. These doses of RO5256390 did not alter body temperature. Follow-up analysis of the time x sex interaction did not identify a significant sex difference at any time point.

RO5256390 thermal effects in drug-naïve MALDR mice

Data are presented in Fig. 3D. A repeated measures ANOVA identified a significant time x dose interaction (F5.3, 84.7 = 8.0, p < 0.001). Simple effects analysis of the effect of dose at each time point identified significant dose effects at T60 (p < 0.001) and T120 (p < 0.05). Mean rectal temperatures were significantly lower for the 0.1 mg/kg treatment group compared to vehicle at T60 and T120. The mean temperature of the 0.1 mg/kg group was also significantly lower than that of the 0.05 and 0.01 mg/kg dose groups at T60. Simple effects analysis of the effect of time for vehicle and each dose group identified a significant effect of time in each case (ps < 0.001), with significantly lower body temperatures at T60-T180, compared to T0 for all groups.

Place conditioning in knock-in and control mice

Locomotor activity on conditioning days

During conditioning, the locomotor activity of the knock-in mice was equivalently suppressed by RO5256390 doses of 0.05 and 0.1 mg/kg, compared to vehicle-treated mice (Fig. 4A, B). Statistical outcomes are in Supplementary Information-Results for locomotor activity on conditioning days in knock-in and control mice.

Fig. 4: Sensitivity to RO5256390-induced conditioned place aversion and hypothermia is reinstated by excision of the Taar1m1J mutation and knock-in of the wild-type Taar1+ allele.
figure 4

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; Taar1m1J: trace amine-associated receptor 1 mutant allele coding for a non-functional receptor.

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Percent time on drug-paired floor

RO5256390 had conditioned aversive effects in knock-in, but not control mice (Fig. 4C). The repeated measures ANOVA for %Time on the drug-paired floor identified a significant test day x mouse line interaction (F1.4,126.8 = 6.4, p < 0.001), but no significant dose effects. Separate analyses of data for each mouse line, combined across dose, revealed no test day effect for MAHDR-Taar1m1J/m1J mice, but a significant effect of test day (F1.4,62.7 = 11.1, p < 0.001) for MAHDR-Taar1+/+ mice. MAHDR-Taar1+/+ mice spent less %Time on the drug-paired floor during the drug-free test than pre-test and during the drug-present test than drug-free test.

Locomotor activity on test days

Overall, locomotor activity was lower in knock-in mice, compared to control mice, on all test days, regardless of treatment; however, RO5256390 treatment produced significant locomotor suppression only in knock-in mice (Fig. 4D). Repeated measures ANOVA detected a significant effect of mouse line (F1,90 = 77.4, p < 0.001), and a significant mouse line x test day interaction (F1.8,160.8 = 29.0, p < 0.001), but no significant dose effects. There was a significant effect of test day for knock-in (p < 0.001) and control (p < 0.05) mice. Knock-in mice were less active during the drug-present test compared to the pre-test and drug-free test; control mice were more active during the drug-free than pre-test.

Post-place conditioning test of RO5256390 thermal effects

When temperature data collected after the conditioning study were examined by repeated measures ANOVA, we found significant RO5256390-induced hypothermia in knock-in, but not control mice (Fig. 4E, F). There was a significant mouse line x time x vehicle versus RO5256390 treatment interaction (F2.1,204.9 = 24.2, p < 0.001), so separate analyses were conducted for each mouse line. For control mice (Fig. 4E), there was a significant main effect of time (F2.6,136.9 = 50.6, p < 0.001), reflecting decreasing temperature characteristic of isolate-housed mice, and a vehicle versus drug treatment x time interaction (F3.0, 136.9 = 4.0, p < 0.01). Mean comparisons between vehicle and RO5256390 treatment at each time point identified no significant differences. For knock-in mice (Fig. 4F), there was a significant vehicle versus drug treatment x time interaction (F2.0,90.6 = 22.5, p < 0.001) that did not interact with dose. Mean comparisons between vehicle and RO5256390 treatment (collapsed on dose) at each time point identified significantly lower temperatures after RO5256390 treatment at T60 and T120, compared to vehicle treatment. Significant results for the effect of time within the different treatment groups are indicated in Fig. 4E, F.

Thermal effects of RO5256390 comparing MALDR, MAHDR, knock-in and control mice

These results supported RO5256390-induced hypothermia only in mice with functional TAAR1: MALDR and knock-in mice (Fig. 5A–D). The initial repeated measures ANOVA, including data from all four mouse lines, identified a significant mouse line x time x dose interaction (F17.5,769.0 = 6.3, p < 0.001). There was also a significant time x sex interaction (F17.5,769.0 = 2.8, p < 0.05); however, when further examined, no significant sex differences were found at any timepoint. Statistical analyses comparing data from the MAHDR and control lines, both Taar1m1J/m1J genotype, identified a significant time x mouse line interaction (F2.8,387.6 = 3.8, p < 0.05), but no significant effects of dose. MAHDR, compared to control, had higher temperatures by 0.2 °C at T30, T60 and T120 (ps < 0.05); however, there were no differences in temperature between vehicle and RO5256390-treated mice (Fig. 5A, C). Analyses comparing data from the MALDR and knock-in lines (Fig. 5B, D), which possess the Taar1+ allele, identified significant time x line (F5.4,360.2 = 6.4, p < 0.001) and dose x time (F5.2,360.2 = 19.6, p < 0.001) interactions. The interaction of line, dose and time was not significant, indicating that dose effects were similar in the two lines. We examined data for each line separately to verify significant hypothermic effects of RO5256390. There were significant time x dose interactions (p < 0.001) in both mouse lines and significant differences between vehicle and RO5256390 at T30 and T60 for both lines.

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

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; Taar1m1J trace amine-associated receptor 1 mutant allele coding for a non-functional receptor.

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Discussion

We provide novel evidence that a selective TAAR1 receptor agonist has aversive, locomotor suppressant and hypothermic effects in mice. These effects were not found in mice with the Taar1m1J allele that expresses a nonfunctional TAAR1 receptor, and knock-in of the wild-type Taar1+ allele rescued aversion, locomotor and thermal phenotypes. Because selective TAAR1 agonists are under consideration as treatments for several psychiatric disorders, including addiction [34,35,36], aversive, hypothermic and locomotor suppressant effects and potential drug x drug interactions must be considered in the development of these drugs as therapeutics.

Conditioned drug effects

Taste and place cues were readily conditioned to aversive effects of RO5256390. The lowest dose of RO5256390 that induced drug-free CPA in MALDR mice (0.05 mg/kg) was 80 times lower than the highest dose of MA we have tested (4 mg/kg), which did not induce drug-free CPA in MALDR mice [24]. The EC50 for RO5256390 for the mouse TAAR1 receptor expressed in HEK293 cells was 2 nM [29], whereas for MA it was 133 nM [37]. The place conditioning procedure is strong for identifying whether rewarding or aversive drug effects are present, but less sensitive for linear dose effects [24, 38, 39]. Nonetheless, we were able to determine that a RO5256390 dose as low as 0.05 mg/kg induced aversion, but 0.01 mg/kg did not. The more potent aversive effects of RO5256390, compared to MA, are consistent with the greater potency and selectivity of RO5256390 for TAAR1, compared to amphetamines [34, 37, 40], and with the opposing rewarding effects of MA, via non-TAAR1 mechanisms. We also note that the 0.01 and 0.005 mg/kg doses of RO5256390 had no locomotor depressant effects during conditioning and that the mice spent ~50% of their time on the drug-paired and vehicle-paired floors, suggesting that these doses were perceived by the mice to be similar to vehicle. Higher levels of activity on the drug-free test day could not be attributed to a latent effect of prior depression of locomotor behavior because they were observed even for doses of RO5256390 that did not impact locomotor activity.

Locomotor and hypothermic drug effects

The doses used by Liu et al. [28]. to induce CTA in rats were 1.6 to 5-fold higher than the lowest dose of RO5256390 we found to induce CTA in mice, and at least 64-fold higher than the lowest dose of RO5256390 we found to induce CPA. Our work was ongoing at the time the results in rats were reported; we chose to use RO5256390, based on its high selectivity and potency (see Supplementary Information). Although differences could be found among agonists, based on our results, it is likely that after treatment with the higher doses of TAAR1 agonists used in the rat studies, body temperature and locomotor activity were reduced, though there may be species differences.

DA levels are increased by addictive drugs in brain circuits associated with reward to a greater extent when TAAR1 function is absent [6, 18,19,20,21]. This likely plays a role in greater MA reward sensitivity and MA intake in the absence of TAAR1 function [14, 15, 23,24,25, 27, 41]. However, TAAR1 is expressed in multiple tissues outside the central nervous system [2, 42, 43] and TAAR1 agonists could peripherally induce an aversive state and hypothermia and/or activation of peripheral receptors could alter brain circuits involved in aversive behaviors. Another potential factor to consider is potential tolerance to the impact of TAAR1 activation. We collected hypothermia data in MALDR mice that had previously been treated with RO5256390 and in drug-naïve mice, and the data suggest a larger hypothermic response in the drug-naïve animals. This could be due to tolerance to the thermal effects of the agonist. However, to determine this, additional data are needed in which repeated and single exposure mice are tested simultaneously. Previous work for MA suggests that aversive MA effects are elicited and retained after prior MA exposure. For example, on the first day of voluntary MA intake, the MALDR and MAHDR mice consume similar amounts of MA, but exhibit a precipitous drop in intake on the subsequent day [25, 44]. In addition, when MALDR mice, and other mice with functional TAAR1, are repeatedly treated with MA in a conditioned taste aversion procedure, similar to results shown for RO5256390 in Fig. 1, their aversion grows stronger, rather than abating [24, 27]. Finally, forced consumption of MA by MALDR mice, by placing MA in their sole source of drinking water, does not subsequently result in higher MA intake, compared to mice without pre-exposure to MA [45].

Knock-in of the Taar1+ allele on the MAHDR background rescued sensitivity to RO5256390-induced CPA, hypothermia, and locomotor suppression. In a direct comparison, RO5256390 induced almost identical levels of hypothermia in MALDR and knock-in mice, indicating complete rescue of this phenotype by the single nucleotide change in Taar1. Such a direct comparison with MALDR mice has not yet been performed for the CPA and locomotor traits. In the dose-response place conditioning studies in MALDR mice, the level of locomotor suppression during conditioning was considerably more modest for the 0.05 and 0.1 mg/kg doses, compared to the 0.5 and 4 mg/kg doses, yet the magnitude of CPA was similar for all doses during the drug-free test. During the drug-present test, the magnitude of locomotor suppression was larger for the 0.5 and 4 mg/kg doses, compared to the 0.05 and 0.1 mg/kg doses, but the mice in all dose groups clearly expressed CPA. Therefore, locomotor suppression did not likely impact the ability to choose a particular location in the test chamber; i.e., to express aversion.

Conclusions

Our data indicate that TAAR1 agonists being considered as clinical treatments for multiple neuropsychiatric disorders have profound aversive, hypothermic and locomotor suppressant effects in mice. Similarly, a study in rats demonstrated aversive effects of two TAAR1 agonists [28]. It is possible that TAAR1 agonists reduce drug taking and seeking, as well as other motivational behaviors, like eating, by changing the balance of reward and aversion circuitry or by having behavioral suppressant or direct aversive interoceptive effects. Potential aversive, hypothermic and locomotor suppressant effects in humans should be examined. These effects could be particularly concerning if used in combination with other drugs that have CNS depressant and hypothermic effects.