Improved prevention and treatment of drug addiction will require deeper understanding of genetic factors contributing to susceptibility to excessive drug use. Intravenous operant self-administration methods have greatly advanced understanding of behavioral traits related to addiction. However, these methods are not suitable for large-scale genetic experiments in mice. Selective breeding of mice can aggregate 'addiction alleles' in a model that has the potential to identify coordinated effects of multiple genes. We produced mouse lines that orally self-administer high (MAHDR) or low (MALDR) amounts of methamphetamine, representing the first demonstration of selective breeding for self-administration of any psychostimulant drug. Conditioned place preference and taste aversion results indicate that MAHDR mice are relatively more sensitive to the rewarding effects and less sensitive to the aversive effects of methamphetamine, compared to MALDR mice. These results validate the oral route of self-administration for investigation of the motivational effects of methamphetamine and provide a viable alternative to intravenous self-administration procedures. Gene expression results for a subset of genes relevant to addiction-related processes suggest differential regulation by methamphetamine of apoptosis and immune pathways in the nucleus accumbens of MAHDR and MALDR mice. In each line, methamphetamine reduced an allostatic state by bringing gene expression back toward 'normal' levels. Genes differentially expressed in the drug-naïve state, including Slc6a4 (serotonin transporter), Htr3a (serotonin receptor 3A), Rela [nuclear factor κB (NFκB)] and Fos (cFos), represent candidates whose expression levels may predict methamphetamine consumption and susceptibility to methamphetamine reward and aversion.

Amphetamines have rewarding and aversive effects. Relative sensitivity to these effects may be a better predictor of vulnerability to addiction than sensitivity to one of these effects alone. We tested this hypothesis in a dose-response study in a second replicate set of mouse lines selectively bred for high vs low methamphetamine (MA) drinking (MADR). Replicate 2 high (MAHDR-2) and low (MALDR-2) MA drinking mice were bred based on MA consumption in a two-bottle choice procedure, and examined for novel tastant drinking. Sensitivities to the rewarding and aversive effects of several doses of MA (0.5, 2, and 4 mg/kg) were measured using a place conditioning procedure. After conditioning, mice were tested in a drug-free and then drug-present state for time spent in the saline- and MA-paired contexts. Similar to the first set of MADR lines, by the end of selection, MAHDR-2 mice consumed about 6 mg MA/kg/18 h, compared to nearly no MA in MALDR-2 mice, but had similar taste preference ratios. MAHDR-2 mice exhibited place preference in both the drug-free and drug-present tests, and no significant place aversion. In contrast, MALDR-2 mice exhibited no place preference or aversion during the drug-free test, but robust place aversion in the drug-present test. These data extend our preliminary findings from the first set of MADR lines, and support the hypothesis that the combination of greater sensitivity to the rewarding effects of MA and insensitivity to the aversive effects of MA is genetically associated with heightened risk for MA consumption.

In an effort to identify genes that may be important for drug-abuse liability, we mapped behavioral quantitative trait loci (bQTL) for sensitivity to the locomotor stimulant effect of methamphetamine (MA) using two mouse lines that were selectively bred for high MA-induced activity (HMACT) or low MA-induced activity (LMACT). We then examined gene expression differences between these lines in the nucleus accumbens, using 20 U74Av2 Affymetrix microarrays and quantitative polymerase chain reaction (qPCR). Expression differences were detected for several genes, including Casein Kinase 1 Epsilon (Csnkle), glutamate receptor, ionotropic, AMPA1 (GluR1), GABA B1 receptor (Gabbr1), and dopamine- and cAMP-regulated phosphoprotein of 32 kDa (Darpp-32). We used the www.WebQTL.org database to identify QTL that regulate the expression of the genes identified by the microarrays (expression QTL; eQTL). This approach identified an eQTL for Csnkle on Chromosome 15 (LOD = 3.8) that comapped with a bQTL for the MA stimulation phenotype (LOD = 4.5), suggesting that a single allele may cause both traits. The chromosomal region containing this QTL has previously been associated with sensitivity to the stimulant effects of cocaine. These results suggest that selection was associated with (and likely caused) altered gene expression that is partially attributable to different frequencies of gene expression polymorphisms. Combining classical genetics with analysis of whole-genome gene expression and bioinformatic resources provides a powerful method for provisionally identifying genes that influence complex traits. The identified genes provide excellent candidates for future hypothesis-driven studies, translational genetic studies, and pharmacological interventions.

Methamphetamine (MA) and cocaine induce behavioral effects primarily through modulation of dopamine neurotransmission. However, the genetic regulation of sensitivity to these two drugs may be similar or disparate. Using selective breeding, lines of mice were produced with extreme sensitivity (high MA activation; HMACT) and insensitivity (low MA activation; LMACT) to the locomotor stimulant effects of acute MA treatment. Studies were performed to determine whether there is pleiotropic genetic influence on sensitivity to the locomotor stimulant effect of MA and to other MA-and cocaine-related behaviors. The HMACT line exhibited more locomotor stimulation in response to several doses of MA and cocaine, compared to the LMACT line. Both lines exhibited locomotor sensitization to 2 mg/kg of MA and 10 mg/kg of cocaine; the magnitude of sensitization was similar in the two lines. However, the lines differed in the magnitude of sensitization to a 1 mg/kg dose of MA, a dose that did not produce a ceiling effect that may confound interpretation of studies using higher doses. The LMACT line consumed more MA and cocaine in a two-bottle choice drinking paradigm; the lines consumed similar amounts of saccharin and quinine, although the HMACT line exhibited slightly elevated preference for a low concentration of saccharin. These results suggest that some genes that influence sensitivity to the acute locomotor stimulant effect of MA have a pleiotropic influence on the magnitude of behavioral sensitization to MA and sensitivity to the stimulant effects of cocaine. Further, extreme sensitivity to MA may protect against MA and cocaine self-administration.

A common expression of neuroadaptations induced by repeated exposure to addictive drugs is a persistent sensitized behavioral response to their stimulant properties. Neuroplasticity underlying drug-induced sensitization has been proposed to explain compulsive drug pursuit and consumption characteristic of addiction. The hypothalamic-pituitary-adrenal (HPA) axis-activating neuropeptide, corticotropin-releasing factor (CRF), may be the keystone in drug-induced neuroadaptation. Corticosterone-activated glucocorticoid receptors (GRs) mediate the development of sensitization to ethanol (EtOH), implicating the HPA axis in this process. EtOHinduced increases in corticosterone require CRF activation of CRF1 receptors. We posited that CRF1 signaling pathways are crucial for EtOH-induced sensitization. We demonstrate that mice lacking CRF1 receptors do not show psychomotor sensitization to EtOH, a phenomenon that was also absent in CRF 1 ؉ 2 receptor doubleknockout mice. Deletion of CRF2 receptors alone did not prevent sensitization. A blunted endocrine response to EtOH was found only in the genotypes showing no sensitization. The CRF1 receptor antagonist CP-154,526 attenuated the acquisition and prevented the expression of EtOH-induced psychomotor sensitization. Because CRF1 receptors are also activated by urocortin-1 (Ucn1), we tested Ucn1 knockout mice for EtOH sensitization and found normal sensitization in this genotype. Finally, we show that the GR antagonist mifepristone does not block the expression of EtOH sensitization. CRF and CRF1 receptors, therefore, are involved in the neurobiological adaptations that underlie the development and expression of psychomotor sensitization to EtOH. A CRF/CRF1-mediated mechanism involving the HPA axis is proposed for acquisition, whereas an extrahypothalamic CRF/CRF1 participation is suggested for expression of sensitization to EtOH.addiction ͉ CP-154,526 ͉ HPA axis ͉ knockout mice ͉ psychomotor sensitization

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.

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Lines of mice were created by selective breeding for the purpose of identifying genetic mechanisms that influence magnitude of the selected trait and to explore genetic correlations for additional traits thought to be influenced by shared mechanisms. DNA samples from high and low methamphetamine drinking (MADR) and high and low methamphetamine sensitization lines were used for quantitative trait locus (QTL) mapping. Significant additive genetic correlations between the two traits indicated common genetic influence, and a QTL on chromosome X was detected for both traits, suggesting one source of this commonality. For MADR mice, a QTL on chromosome 10 accounted for more than 50% of the genetic variance in that trait. Microarray gene expression analyses were performed for 3 brain regions for methamphetamine-naïve MADR line mice: nucleus accumbens, prefrontal cortex and ventral midbrain. Many of the genes that were differentially expressed between the high and low MADR lines were shared in common across the 3 brain regions. A gene network highly enriched in transcription factor genes was identified as being relevant to genetically-determined differences in methamphetamine intake. When the mu opioid receptor gene (Oprm1), located on chromosome 10 in the QTL region, was added to this top ranked transcription factor network, it became a hub in the network. These data are consistent with previously published findings of opioid response and intake differences between the MADR lines and suggest that Oprm1 or a gene that impacts activity of the opioid system, plays a role in genetically–determined differences in methamphetamine intake.