A breeding strategy to identify modifiers of high genetic risk for methamphetamine intake.

Trace amine-associated receptor 1 (Taar1) impacts methamphetamine (MA) intake. A mutant allele (Taar1m1J ) derived from the DBA/2J mouse strain codes for a non-functional receptor, and Taar1m1J/m1J mice consume more MA than mice possessing the reference Taar1+ allele. To study the impact of this mutation in a genetically diverse population, heterogeneous stock-collaborative cross (HS-CC) mice, the product of an eight-way cross of standard and wild-derived strains, were tested for MA intake. HS-CC had low MA intake, so an HS-CC by DBA/2J strain F2 intercross was created to transfer the mutant allele onto the diverse background, and used for selective breeding. To study residual variation in MA intake existing in Taar1m1J/m1J mice, selective breeding for higher (MAH) vs lower (MAL) MA intake was initiated from Taar1m1J/m1J F2 individuals; a control line of Taar1+/+ individuals (MAC) was retained. The lines were also examined for MA-induced locomotor and thermal responses, and fluid and tastant consumption. Taar1m1J/m1J F2 mice consumed significantly more MA than Taar1+/+ F2 mice. Response to selection was significant by generation 2 and there were corresponding differences in fluid consumed. Fluid consumption was not different in non-MA drinking studies. Taar1m1J/m1J genotype (MAL or MAH vs MAC mice) was associated with heighted MA locomotor and reduced hypothermic responses. MAL mice exhibited greater sensitization than MAH mice, but the selected lines did not consistently differ for thermal or tastant phenotypes. Residual variation among high-risk Taar1m1J/m1J mice appears to involve mechanisms associated with neuroadaptation to MA, but not sensitivity to hypothermic effects of MA.

Confirmation of a Causal Taar1 Allelic Variant in Addiction-Relevant Methamphetamine Behaviors.

Sensitivity to rewarding and reinforcing drug effects has a critical role in initial use, but the role of initial aversive drug effects has received less attention. Methamphetamine effects on dopamine re-uptake and efflux are associated with its addiction potential. However, methamphetamine also serves as a substrate for the trace amine-associated receptor 1 (TAAR1). Growing evidence in animal models indicates that increasing TAAR1 function reduces drug self-administration and intake. We previously determined that a non-synonymous single nucleotide polymorphism (SNP) in Taar1 predicts a conformational change in the receptor that has functional consequences. A Taar1 m1J mutant allele existing in DBA/2J mice expresses a non-functional receptor. In comparison to mice that possess one or more copies of the reference Taar1 allele (Taar1 +/+ or Taar1 +/m1J ), mice with the Taar1 m1J/m1J genotype readily consume methamphetamine, express low sensitivity to aversive effects of methamphetamine, and lack sensitivity to acute methamphetamine-induced hypothermia. We used three sets of knock-in and control mice in which one Taar1 allele was exchanged with the alternative allele to determine if other methamphetamine-related traits and an opioid trait are impacted by the same Taar1 SNP proven to affect MA consumption and hypothermia. First, we measured sensitivity to conditioned rewarding and aversive effects of methamphetamine to determine if an impact of the Taar1 SNP on these traits could be proven. Next, we used multiple genetic backgrounds to study the consistency of Taar1 allelic effects on methamphetamine intake and hypothermia. Finally, we studied morphine-induced hypothermia to confirm prior data suggesting that a gene in linkage disequilibrium with Taar1, rather than Taar1, accounts for prior observed differences in sensitivity. We found that a single SNP exchange reduced sensitivity to methamphetamine conditioned reward and increased sensitivity to conditioned aversion. Profound differences in methamphetamine intake and hypothermia consistently corresponded with genotype at the SNP location, with only slight variation in magnitude across genetic backgrounds. Morphine-induced hypothermia was not dependent on Taar1 genotype. Thus, Taar1 genotype and TAAR1 function impact multiple methamphetamine-related effects that likely predict the potential for methamphetamine use. These data support further investigation of their potential roles in risk for methamphetamine addiction and therapeutic development.

A Spontaneous Mutation in Taar1 Impacts Methamphetamine-Related Traits Exclusively in DBA/2 Mice from a Single Vendor.

Major gene effects on traits associated with substance use disorders are rare. Previous findings in methamphetamine drinking (MADR) lines of mice, bred for high or low voluntary MA intake, and in null mutants demonstrate a major impact of the trace amine-associated receptor 1 (Taar1) gene on a triad of MA-related traits: MA consumption, MA-induced conditioned taste aversion and MA-induced hypothermia. While inbred strains are fundamentally genetically stable, rare spontaneous mutations can become fixed and result in new or aberrant phenotypes. A single nucleotide polymorphism in Taar1 that encodes a missense proline to threonine mutation in the second transmembrane domain (Taar1m1J ) has been identified in the DBA/2J strain. MA is an agonist at this receptor, but the receptor produced by Taar1m1J does not respond to MA or endogenous ligands. In the present study, we used progeny of the C57BL/6J × DBA/2J F2 cross, the MADR lines, C57BL/6J × DBA/2J recombinant inbred strains, and DBA/2 mice sourced from four vendors to further examine Taar1-MA phenotype relations and to define the chronology of the fixation of the Taar1m1J mutation. Mice homozygous for Taar1m1J were found at high frequency early in selection for high MA intake in multiple replicates of the high MADR line, whereas Taar1m1J homozygotes were absent in the low MADR line. The homozygous Taar1m1J genotype is causally linked to increased MA intake, reduced MA-induced conditioned taste aversion, and reduced MA-induced hypothermia across models. Genotype-phenotype correlations range from 0.68 to 0.96. This Taar1 polymorphism exists in DBA/2J mice sourced directly from The Jackson Laboratory, but not DBA/2 mice sourced from Charles River (DBA/2NCrl), Envigo (formerly Harlan Sprague Dawley; DBA/2NHsd) or Taconic (DBA/2NTac). By genotyping archived samples from The Jackson Laboratory, we have determined that this mutation arose in 2001-2003. Our data strengthen the conclusion that the mutant Taar1m1J allele, which codes for a non-functional receptor protein, increases risk for multiple MA-related traits, including MA intake, in homozygous Taar1m1J individuals.

Differential network analysis reveals genetic effects on catalepsy modules.

We performed short-term bi-directional selective breeding for haloperidol-induced catalepsy, starting from three mouse populations of increasingly complex genetic structure: an F2 intercross, a heterogeneous stock (HS) formed by crossing four inbred strains (HS4) and a heterogeneous stock (HS-CC) formed from the inbred strain founders of the Collaborative Cross (CC). All three selections were successful, with large differences in haloperidol response emerging within three generations. Using a custom differential network analysis procedure, we found that gene coexpression patterns changed significantly; importantly, a number of these changes were concordant across genetic backgrounds. In contrast, absolute gene-expression changes were modest and not concordant across genetic backgrounds, in spite of the large and similar phenotypic differences. By inferring strain contributions from the parental lines, we are able to identify significant differences in allelic content between the selected lines concurrent with large changes in transcript connectivity. Importantly, this observation implies that genetic polymorphisms can affect transcript and module connectivity without large changes in absolute expression levels. We conclude that, in this case, selective breeding acts at the subnetwork level, with the same modules but not the same transcripts affected across the three selections.