Neuropsychopharmacology. 2015 Aug;40(9):2175–84.
Trace amine-associated receptor 1 regulation of methamphetamine intake and related traits.
Harkness JH, Shi X, Janowsky A, Phillips TJ.
Veterans Affairs Portland Health Care System, Methamphetamine Abuse Research Center, Department of Behavioral Neuroscience, and Department of Psychiatry, Oregon Health & Science University, Portland, Oregon 97239, USA.
Continued methamphetamine (MA) use is dependent on a positive MA experience and is likely attenuated by sensitivity to the aversive effects of MA. Bidirectional selective breeding of mice for high (MAHDR) or low (MALDR) voluntary consumption of MA demonstrates a genetic influence on MA intake. Quantitative trait locus (QTL) mapping identified a QTL on mouse chromosome 10 that accounts for greater than 50% of the genetically-determined differences in MA intake in the MAHDR and MALDR lines. The trace amine-associated receptor 1 gene (Taar1) is within the confidence interval of the QTL and encodes a receptor (TAAR1) that modulates monoamine neurotransmission and at which MA serves as an agonist. We demonstrate the existence of a non-functional allele of Taar1 in the DBA/2J mouse strain, one of the founder strains of the selected lines, and show that this non-functional allele co-segregates with high MA drinking and with reduced sensitivity to MA-induced conditioned taste aversion (CTA) and hypothermia. The functional Taar1 allele, derived from the other founder strain, C57BL/6J, segregates with low MA drinking and heightened sensitivity to MA-induced CTA and hypothermia. A role for TAAR1 in these phenotypes is corroborated in Taar1 transgenic mice: Taar1 knockout mice consume more MA and exhibit insensitivity to MA-induced CTA and hypothermia, compared with Taar1 wild-type mice. These are the first data to show that voluntary MA consumption is, in part, regulated by TAAR1 function. Behavioral and physiological studies indicate that TAAR1 function increases sensitivity to aversive effects of MA, and may thereby protect against MA use.
In addition to its commonly known action as a substrate for dopamine and other monoamine transporters, MA is a trace amine-associated receptor 1 (TAAR1) agonist. Multiple TAAR subtypes are found across species and are thought to be involved in synaptic transmission. However, TAAR1 is unique. It is the only member of this G-protein-coupled subfamily that is conserved across all species and is not expressed in the main olfactory epithelium (1). TAAR1 is expressed in the brain, and exerts inhibitory control on monoamine activity (2). Thus, it is not surprising that amphetamine-induced release of monoamines is enhanced in null mutant mice that lack TAAR1. Gene mapping studies using DNA samples from lines of mice that were bred for differences in voluntary MA consumption identified a region of chromosome 10 and indicated that a gene or genes at this location accounted for more than 50% of the variation in MA intake in the MA drinking (MADR) lines. This finding and multiple characteristics of these mice have recently been summarized (3). A review of mouse sequence databases, focusing specifically on chromosome 10 genes within the confidence interval of the mapped location, identified a Taar1 sequence difference between the DBA/2J and C57BL/6J mouse strains, which served as the progenitor strains of the MADR selected lines. This non-synonymous single nucleotide polymorphism (SNP), which causes substitution of a threonine for a proline residue at the cytoplasmic/luminal interface of one transmembrane domain, was predicted to reduce receptor function. We hypothesized that greater MA consumption is associated with reduced TAAR1 function and that reduced TAAR1 function is associated with low sensitivity to aversive effects of MA.
First, we determined the frequency of the DBA/2J Taar1 allele in our MA high drinking (MAHDR) and MA low drinking (MALDR) mice. All 10 of 10 MAHDR mice were homozygous for the DBA/2J allele, so that the frequency was 1. The frequency in the 10 MALDR mice was 0.2, with 6 animals homozygous for the C57BL/6J allele and 4 heterozygous for the two Taar1 allele types. Thus, homozygosity for the DBA/2J allele was found only in mice bred for high MA drinking. This suggests that the C57BL/6J allele has a dominant effect and is associated with lower MA intake. We next examined whether the absence of Taar1 is associated with heightened MA drinking using a knockout mouse approach. We measured voluntary MA intake using a two-bottle choice procedure (Figures 1 and 2) in mice that were homozygous for a targeted mutation in the Taar1 gene (knockout mice), along with their heterozygous and wild-type littermates. Knockout mice consumed more MA than heterozygous or wild-type mice and the latter two genotypes consumed similar amounts of MA. Thus, the knockout phenotype corresponds with the higher MA drinking phenotype of the MAHDR mice and the heterozygous and wild-type phenotype corresponds with the lower MA drinking phenotype of the MALDR mice.
Figure 1. Mouse in a two-bottle choice procedure showing how the two drinking tubes are oriented on the cage top with food placed near both tubes to avoid a food-associated preference.
Figure 2. Mouse consuming fluid from one of the drinking tubes. The white material in the cage is nesting material that is provided to singly-housed mice. Single housing is necessary to obtain accurate drinking data for each individual.
Our previously published research found that MAHDR mice are insensitive to conditioned aversive effects of MA, whereas MALDR mice are highly sensitive (3). To determine whether TAAR1 impacts this MA effect, the Taar1 transgenic mice were tested for sensitivity to MA-induced conditioned taste aversion. In this test, mice are introduced to a novel taste, in this case a sodium chloride solution, are then treated with saline or MA immediately after consuming the novel taste, and then the novel solution is again offered 24 hours later. This procedure is repeated several times, with intake of the sodium chloride solution measured each time it is offered. This procedure is repeated across multiple trials of consumption and saline/MA treatment. If MA treatment has adverse consequences, sodium chloride solution consumption will decline across trials, because the adverse MA effects have been associated with the novel taste, producing a conditioned aversion. Knockout mice resembled MAHDR mice and formed no taste aversion, whereas heterozygous and wild-type mice resembled MALDR mice and formed a robust taste aversion. These data support a role for TAAR1 in sensitivity to the conditioned aversive effects of MA.
Existing data indicated a role for TAAR1 in the effects of amphetamines on body temperature. We examined sensitivity to MA-induced body temperature change in the MADR lines for doses of MA from 1 to 16 mg/kg. Hypothermic effects found for doses up to 4 mg/kg in the MALDR line were completely absent in MAHDR mice. When the middle 2 mg/kg dose of MA was used to test the Taar1 transgenic mice, a similar outcome was obtained with the knockout mice being completely resistant to the MA-induced hypothermia found in the other two genotypes. There were no differences between the MADR lines or among the Taar1 transgenic genotypes in body temperature response to ethanol (2 or 4 g/kg). The period during which aversion can be conditioned can be prolonged by low body temperature. Thus, MA-induced hypothermia may enhance MA-conditioned taste aversion in MALDR and Taar1 wild-type and heterozygous mice and play a role in the difference in sensitivity to aversive effects of MA in our genetic mouse models.
Finally, both the position of the SNP and the threonine for proline residue change associated with the Taar1 polymorphism in DBA/2J and MAHDR mice predicted that the TAAR1 expressed in these mice would have reduced or absent function; however, it was important to directly test this prediction. Thus, the DBA/2J-like and C57BL/6J-like constructs were transfected into cells grown in culture, receptor expression analysis was used to verify that both alleles expressed a receptor at largely equivalent levels, and the cells were treated with MA across a wide range of concentrations. The accumulation of cAMP was used as a measure of function for this stimulatory G-protein-coupled receptor. There was no cAMP response in cells transfected with the DBA/2J-like construct, whereas an MA dose-dependent cAMP response was found in the cells transfected with the C57BL/6J-like construct. This cAMP response was blocked by a TAAR1-specific antagonist. These data indicate that the receptor expressed by the DBA/2J-like allele is non-functional.
Importantly, the results of this research indicate that aversive effects of MA may not be experienced in the absence of TAAR1 function. Moreover, the strong genetic correlation between level of MA intake and sensitivity to conditioned aversive effects of MA supports an important role for aversion sensitivity in genetically-determined risk for continued MA use. Because resistance to MA-induced hypothermia is associated with higher levels of MA intake, this physiological response could serve as a biomarker of risk for the development of a MA use disorder. Although, we have identified human SNPs that impact TAAR1 function (4), data are needed to confirm an impact on MA use. Such an impact would suggest an important new treatment mechanism for MA addiction to explore.
- Eyun SI, Moriyama H, Hoffmann FG, Moriyama EN 2016 Molecular evolution and functional divergence of trace amine-associated receptors. PLOS One 11:e0151023
- Khan MZ, Nawaz W 2016 The emerging roles of human trace amines and human trace amine-associated receptors (hTAARs) in central nervous system. Biomedicine & Pharmacotherapy 83:439-449
- Phillips TJ, Shabani S 2015 An animal model of differential genetic risk for methamphetamine intake. Frontiers in Neuroscience 9:327
- Shi X, Walter NA, Harkness JH, Neve KA, Williams RW, Lu L, Belknap JK, Eshleman AJ, Phillips TJ, Janowsky A (2016) Genetic polymorphisms affect mouse and human trace amine-associated receptor 1 function. PLoS One 11:e0152581
Acknowledgements: This work was supported by NIH grant T32 DA007262, The Methamphetamine Abuse Research Center (P50 DA018165), the Portland Alcohol Research Center (P60 AA010760), a VA Merit Review grant (I01 BX002106) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service, and the VA Research Career Scientist Program. We extend our thanks to Cheryl Reed and John Mootz for photography.
Tamara J. Phillips, Ph.D.
Professor and Vice-Chair of Behavioral Neuroscience
Director, Portland Alcohol Research Center
Scientific Director, Methamphetamine Abuse Research Center
VA Senior Research Career Scientist
3710 SW US Veterans Hospital Rd.
R&D 32, Portland, OR 97239