Effects of repeated alcohol abstinence on within-subject prefrontal cortical gene expression in rhesus macaques.

Male rhesus monkeys (n = 24) had a biopsy of prefrontal cortical area 46 prior to chronic ethanol self-administration (n = 17) or caloric control (n = 7). Fourteen months of daily self-administration (water vs. 4% alcohol, 22 h access/day termed "open-access") was followed by two cycles of prolonged abstinence (5 weeks) each followed by 3 months of open-access alcohol and a final abstinence followed by necropsy. At necropsy, a biopsy of Area 46, contralateral to the original biopsy, was obtained. Gene expression data (RNA-Seq) were collected comparing biopsy/necropsy samples. Monkeys were categorized by drinking status during the final post-abstinent drinking phase as light (LD), binge (BD), heavy (HD) and very heavy (VHD drinkers). Comparing pre-ethanol to post-abstinent biopsies, four animals that converted from HD to VHD status had significant ontology enrichments in downregulated genes (necropsy minus biopsy n = 286) that included immune response (FDR < 9 × 10-7) and plasma membrane changes (FDR < 1 × 10-7). Genes in the immune response category included IL16 and 18, CCR1, B2M, TLR3, 6 and 7, SP2 and CX3CR1. Upregulated genes (N = 388) were particularly enriched in genes associated with the negative regulation of MAP kinase activity (FDR < 3 × 10-5), including DUSP 1, 4, 5, 6 and 18, SPRY 2, 3, and 4, SPRED2, BMP4 and RGS2. Overall, these data illustrate the power of the NHP model and the within-subject design of genomic changes due to alcohol and suggest new targets for treating severe escalated drinking following repeated alcohol abstinence attempts.

Modeling Brain Gene Expression in Alcohol Use Disorder with Genetic Animal Models.

Animal genetic models have and will continue to provide important new information about the behavioral and physiological adaptations associated with alcohol use disorder (AUD). This chapter focuses on two models, ethanol preference and drinking in the dark (DID), their usefulness in interrogating brain gene expression data and the relevance of the data obtained to interpret AUD-related GWAS and TWAS studies. Both the animal and human data point to the importance for AUD of changes in synaptic transmission (particularly glutamate and GABA transmission), of changes in the extracellular matrix (specifically including collagens, cadherins and protocadherins) and of changes in neuroimmune processes. The implementation of new technologies (e.g., cell type-specific gene expression) is expected to further enhance the value of genetic animal models in understanding AUD.

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

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.

Gait Abnormalities and Aberrant D2 Receptor Expression and Signaling in Mice Carrying the Human Pathogenic Mutation DRD2I212F.

A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. That allelic variant D2-I212F is a constitutively active and G protein-biased receptor. We now describe mice engineered using CRISPR-Cas9-mediated gene editing technology to carry the D2-I212F variant. Drd2I212F mice exhibited gait abnormalities resembling those in other mouse models of chorea and/or dystonia and had striatal D2 receptor expression that was decreased approximately 30% per Drd2I212F allele. Electrically evoked inhibitory postsynaptic conductances in midbrain dopamine neurons and striatum from Drd2I212F mice, caused by G protein activation of potassium channels, exhibited slow kinetics (e.g., approximately four- to sixfold slower decay) compared with Drd2 +/+ mice. Current decay initiated by photolytic release of the D2 antagonist sulpiride from CyHQ-sulpiride was also ∼fourfold slower in midbrain slices from Drd2I212F mice than Drd2 +/+ mice. Furthermore, in contrast to Drd2 +/+ mice, in which dopamine is several-fold more potent at neurons in the nucleus accumbens than in the dorsal striatum, reflecting activation of Gα o versus Gα i, dopamine had similar potencies in those two brain regions of Drd2I212F mice. Repeated cocaine treatment, which decreases dopamine potency in the nucleus accumbens of Drd2 +/+ mice, had no effect on dopamine potency in Drd2 I212F mice. The results demonstrate the pathogenicity of the D2-I212F mutation and the utility of this mouse model for investigating the role of pathogenic DRD2 variants in early-onset hyperkinetic movement disorders. SIGNIFICANCE STATEMENT: The first dopamine receptor mutation to cause a movement disorder, D2-I212F, was recently identified. The mutation makes receptor activation of G protein-mediated signaling more efficient. To confirm the pathogenesis of D2-I212F, this study reports that mice carrying this mutation have gait abnormalities consistent with the clinical phenotype. The mutation also profoundly alters D2 receptor expression and function in vivo. This mouse model will be useful for further characterization of the mutant receptor and for evaluation of potential therapeutic drugs.

Brain gene expression differences related to ethanol preference in the collaborative cross founder strains.

The collaborative cross (CC) founder strains include five classical inbred laboratory strains [129S1/SvlmJ (S129), A/J (AJ), C57BL/6J (B6), NOD/ShiLtJ (NOD), and NZO/HILtJ (NZO)] and three wild-derived strains [CAST/EiJ (CAST), PWK/PhJ (PWK), and WSB/EiJ (WSB)]. These strains encompass 89% of the genetic diversity available in Mus musculus and ∼10-20 times more genetic diversity than found in Homo sapiens. For more than 60 years the B6 strain has been widely used as a genetic model for high ethanol preference and consumption. However, another of the CC founder strains, PWK, has been identified as a high ethanol preference/high consumption strain. The current study determined how the transcriptomes of the B6 and PWK strains differed from the 6 low preference CC strains across 3 nodes of the brain addiction circuit. RNA-Seq data were collected from the central nucleus of the amygdala (CeA), the nucleus accumbens core (NAcc) and the prelimbic cortex (PrL). Differential expression (DE) analysis was performed in each of these brain regions for all 28 possible pairwise comparisons of the CC founder strains. Unique genes for each strain were identified by selecting for genes that differed significantly [false discovery rate (FDR) < 0.05] from all other strains in the same direction. B6 was identified as the most distinct classical inbred laboratory strain, having the highest number of total differently expressed genes (DEGs) and DEGs with high log fold change, and unique genes compared to other CC strains. Less than 50 unique DEGs were identified in common between B6 and PWK within all three brain regions, indicating the strains potentially represent two distinct genetic signatures for risk for high ethanol-preference. 338 DEGs were found to be commonly different between B6, PWK and the average expression of the remaining CC strains within all three regions. The commonly different up-expressed genes were significantly enriched (FDR < 0.001) among genes associated with neuroimmune function. These data compliment findings showing that neuroimmune signaling is key to understanding alcohol use disorder (AUD) and support use of these 8 strains and the highly heterogeneous mouse populations derived from them to identify alcohol-related brain mechanisms and treatment targets.

Midazolam, methamphetamine, morphine and nicotine intake in high-drinking-in-the-dark mice.

The high-drinking-in-the-dark (HDID) lines of mice were selectively bred for achieving high blood alcohol levels in the drinking-in-the-dark (DID) task and have served as a unique genetic risk model for binge-like alcohol intake. However, little is known about their willingness to consume other addictive drugs. Here, we examined (a) whether the HDID-1 and HDID-2 lines of mice would voluntarily consume midazolam, methamphetamine, morphine and nicotine in a DID test and (b) whether the HDID lines differ from their founders, heterogeneous stock/Northport (HS/NPT), in consumption levels of these drugs at the concentrations tested. Separate groups of HDID-1, HDID-2 and HS/NPT mice were given 4 days of access to each drug, using the single-bottle, limited-access DID paradigm. Male and female mice of both HDID lines consumed all four offered drugs. We observed no genotype differences in 40 µg/ml methamphetamine intake, but significant differences in nicotine, midazolam and morphine intake. Both HDID lines drank significantly more (150 µg/ml) midazolam than their founders, providing strong support for a shared genetic contribution to binge ethanol and midazolam intake. HDID-2 mice, but not HDID-1 mice, consumed more morphine (700 µg/ml) and more nicotine across a range of concentrations than HS/NPT mice. These results demonstrate that the HDID mice can be utilized for tests of voluntary drug consumption other than ethanol and highlight potentially important differences between HDID lines in risk for elevated drug intake.

Sex Differences in the Brain Transcriptome Related to Alcohol Effects and Alcohol Use Disorder.

There is compelling evidence that sex and gender have crucial roles in excessive alcohol (ethanol) consumption. Here, we review some of the data from the perspective of brain transcriptional differences between males and females, focusing on rodent animal models. A key emerging transcriptional feature is the role of neuroimmune processes. Microglia are the resident neuroimmune cells in the brain and exhibit substantial functional differences between males and females. Selective breeding for binge ethanol consumption and the impacts of chronic ethanol consumption and withdrawal from chronic ethanol exposure all demonstrate sex-dependent neuroimmune signatures. A focus is on resolving sex-dependent differences in transcriptional responses to ethanol at the neurocircuitry level. Sex-dependent transcriptional differences are found in the extended amygdala and the nucleus accumbens. Telescoping of ethanol consumption is found in some, but not all, studies to be more prevalent in females. Recent transcriptional studies suggest that some sex differences may be due to female-dependent remodeling of the primary cilium. An interesting theme appears to be developing: at least from the animal model perspective, even when males and females are phenotypically similar, they differ significantly at the level of the transcriptome.

On the Use of Heterogeneous Stock Mice to Map Transcriptomes Associated With Excessive Ethanol Consumption.

We and many others have noted the advantages of using heterogeneous (HS) animals to map genes and gene networks associated with both behavioral and non-behavioral phenotypes. Importantly, genetically complex Mus musculus crosses provide substantially increased resolution to examine old and new relationships between gene expression and behavior. Here we report on data obtained from two HS populations: the HS/NPT derived from eight inbred laboratory mouse strains and the HS-CC derived from the eight collaborative cross inbred mouse strains that includes three wild-derived strains. Our work has focused on the genes and gene networks associated with risk for excessive ethanol consumption, individual variation in ethanol consumption and the consequences, including escalation, of long-term ethanol consumption. Background data on the development of HS mice is provided, including advantages for the detection of expression quantitative trait loci. Examples are also provided of using HS animals to probe the genes associated with ethanol preference and binge ethanol consumption.

Combined and sequential effects of alcohol and methamphetamine in animal models.

Comorbid drug use, often alcohol with other drugs, poses significant health and societal concerns. Methamphetamine is among the illicit drugs most often co-used with alcohol. The current review examines the animal literature for impacts of comorbid alcohol and methamphetamine exposure. We found evidence for additive or synergistic effects of combined or sequential exposure on behavior and physiology. Dopaminergic, serotonergic, and glutamatergic systems are all impacted by combined exposure to alcohol and methamphetamine and cyclooxygenase-2 activity plays an important role in their combined neurotoxic effects. Adverse consequences of comorbid exposure include altered brain development with prenatal exposure, impaired learning and memory, motor deficits, gastrotoxicity, hepatotoxicity, and augmented intake under some conditions. Given high susceptibility to drug experimentation in adolescence, studies of co-exposure during the adolescent period and of how adolescent exposure to one drug impacts later use or sensitivity to the other drug should be a priority. Further, to gain traction on prevention and treatment, additional research to identify motivational and neurobiological drivers and consequences of comorbid use is needed.

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 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.

Genetic and Brain Mechanisms of Addictive Behavior and Neuroadaptation.

Genetic differences play a role in the susceptibility to addictive drug use, the probability that the use of these drugs will escalate and result in a drug use disorder, and whether relapse to use will occur during or after treatment [...].

Phenotypic and gene expression features associated with variation in chronic ethanol consumption in heterogeneous stock collaborative cross mice.

Of the more than 100 studies that have examined relationships between excessive ethanol consumption and the brain transcriptome, few rodent studies have examined chronic consumption. Heterogeneous stock collaborative cross mice freely consumed ethanol vs. water for 3 months. Transcriptional differences were examined for the central nucleus of the amygdala, a brain region known to impact ethanol preference. Early preference was modestly predictive of final preference and there was significant escalation of preference in females only. Genes significantly correlated with female preference were enriched in annotations for the primary cilium and extracellular matrix. A single module in the gene co-expression network was enriched in genes with an astrocyte annotation. The key hub node was the master regulator, orthodenticle homeobox 2 (Otx2). These data support an important role for the extracellular matrix, primary cilium and astrocytes in ethanol preference and consumption differences among individual female mice of a genetically diverse population.

Differential genetic risk for methamphetamine intake confers differential sensitivity to the temperature-altering effects of other addictive drugs.

Mice selectively bred for high methamphetamine (MA) drinking (MAHDR), compared with mice bred for low MA drinking (MALDR), exhibit greater sensitivity to MA reward and insensitivity to aversive and hypothermic effects of MA. Previous work identified the trace amine-associated receptor 1 gene (Taar1) as a quantitative trait gene for MA intake that also impacts thermal response to MA. All MAHDR mice are homozygous for the mutant Taar1 m1J allele, whereas all MALDR mice possess at least one copy of the reference Taar1 + allele. To determine if their differential sensitivity to MA-induced hypothermia extends to drugs of similar and different classes, we examined sensitivity to the hypothermic effect of the stimulant cocaine, the amphetamine-like substance 3,4-methylenedioxymethamphetamine (MDMA), and the opioid morphine in these lines. The lines did not differ in thermal response to cocaine, only MALDR mice exhibited a hypothermic response to MDMA, and MAHDR mice were more sensitive to the hypothermic effect of morphine than MALDR mice. We speculated that the µ-opioid receptor gene (Oprm1) impacts morphine response, and genotyped the mice tested for morphine-induced hypothermia. We report genetic linkage between Taar1 and Oprm1; MAHDR mice more often inherit the Oprm1 D2 allele and MALDR mice more often inherit the Oprm1 B6 allele. Data from a family of recombinant inbred mouse strains support the influence of Oprm1 genotype, but not Taar1 genotype, on thermal response to morphine. These results nominate Oprm1 as a genetic risk factor for morphine-induced hypothermia, and provide additional evidence for a connection between drug preference and drug thermal response.

The Role of Biogenic Amine Transporters in Trace Amine-Associated Receptor 1 Regulation of Methamphetamine-Induced Neurotoxicity.

Methamphetamine (MA) impairs vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT) function and expression, increasing intracellular DA levels that lead to neurotoxicity. The trace amine-associated receptor 1 (TAAR1) is activated by MA, but when the receptor is not activated, MA-induced neurotoxicity is increased. To investigate interactions among TAAR1, VMAT2, and DAT, transporter function and expression were measured in transgenic Taar1 knockout (KO) and wild-type (WT) mice 24 hours following a binge-like regimen (four intraperitoneal injections, 2 hours apart) of MA (5 mg/kg) or the same schedule of saline treatment. Striatal synaptosomes were separated by fractionation to examine the function and expression of VMAT2 localized to cytosolic and membrane-associated vesicles. DAT was measured in whole synaptosomes. VMAT2-mediated [3H]DA uptake inhibition was increased in Taar1 KO mice in synaptosomal and vesicular fractions, but not the membrane-associated fraction, compared with Taar1 WT mice. There was no difference in [3H]dihydrotetrabenazine binding to the VMAT2 or [125I]RTI-55 binding to the DAT between genotypes, indicating activation of TAAR1 does not affect VMAT2 or DAT expression. There was also no difference between Taar1 WT and KO mice in DAT-mediated [3H]DA uptake inhibition following in vitro treatment with MA. These findings provide the first evidence of a TAAR1-VMAT2 interaction, as activation of TAAR1 mitigated MA-induced impairment of VMAT2 function, independently of change in VMAT2 expression. Additionally, the interaction is localized to intracellular VMAT2 on cytosolic vesicles and did not affect expression or function of DAT in synaptosomes or VMAT2 at the plasmalemmal surface, i.e., on membrane-associated vesicles. SIGNIFICANCE STATEMENT: Methamphetamine stimulates the G protein-coupled receptor TAAR1 to affect dopaminergic function and neurotoxicity. Here we demonstrate that a functional TAAR1 protects a specific subcellular fraction of VMAT2, but not the dopamine transporter, from methamphetamine-induced effects, suggesting new directions in pharmacotherapeutic development for neurodegenerative disorders.

Taar1 gene variants have a causal role in methamphetamine intake and response and interact with Oprm1.

We identified a locus on mouse chromosome 10 that accounts for 60% of the genetic variance in methamphetamine intake in mice selectively bred for high versus low methamphetamine consumption. We nominated the trace amine-associated receptor 1 gene, Taar1, as the strongest candidate and identified regulation of the mu-opioid receptor 1 gene, Oprm1, as another contributor. This study exploited CRISPR-Cas9 to test the causal role of Taar1 in methamphetamine intake and a genetically-associated thermal response to methamphetamine. The methamphetamine-related traits were rescued, converting them to levels found in methamphetamine-avoiding animals. We used a family of recombinant inbred mouse strains for interval mapping and to examine independent and epistatic effects of Taar1 and Oprm1. Both methamphetamine intake and the thermal response mapped to Taar1 and the independent effect of Taar1 was dependent on genotype at Oprm1. Our findings encourage investigation of the contribution of Taar1 and Oprm1 variants to human methamphetamine addiction.

Regional Analysis of the Brain Transcriptome in Mice Bred for High and Low Methamphetamine Consumption.

Transcriptome profiling can broadly characterize drug effects and risk for addiction in the absence of drug exposure. Modern large-scale molecular methods, including RNA-sequencing (RNA-Seq), have been extensively applied to alcohol-related disease traits, but rarely to risk for methamphetamine (MA) addiction. We used RNA-Seq data from selectively bred mice with high or low risk for voluntary MA intake to construct coexpression and cosplicing networks for differential risk. Three brain reward circuitry regions were explored, the nucleus accumbens (NAc), prefrontal cortex (PFC), and ventral midbrain (VMB). With respect to differential gene expression and wiring, the VMB was more strongly affected than either the PFC or NAc. Coexpression network connectivity was higher in the low MA drinking line than in the high MA drinking line in the VMB, oppositely affected in the NAc, and little impacted in the PFC. Gene modules protected from the effects of selection may help to eliminate certain mechanisms from significant involvement in risk for MA intake. One such module was enriched in genes with dopamine-associated annotations. Overall, the data suggest that mitochondrial function and glutamate-mediated synaptic plasticity have key roles in the outcomes of selective breeding for high versus low levels of MA intake.

Depression-like symptoms of withdrawal in a genetic mouse model of binge methamphetamine intake.

Binge methamphetamine (MA) users have higher MA consumption, relapse rates and depression-like symptoms during early periods of withdrawal, compared with non-binge users. The impact of varying durations of MA abstinence on depression-like symptoms and on subsequent MA intake was examined in mice genetically prone to binge-level MA consumption. Binge-level MA intake was induced using a multiple-bottle choice procedure in which mice were offered one water drinking tube and three tubes containing increasing concentrations of MA in water, or four water tubes (control group). In two studies, depression-like symptoms were measured using a tail-suspension test and a subsequent forced-swim test, after forced abstinence of 6 and 30 hours from a 28-day course of chronic MA intake. An additional study measured the same depression-like symptoms, as well as MA intake, after prolonged abstinence of 1 and 2 weeks. MA high drinking mice and one of their progenitor strains DBA/2J escalated their MA intake with increasing MA concentration; however, MA high drinking mice consumed almost twice as much MA as DBA/2J mice. Depression-like symptoms were significantly higher early after MA access was withdrawn, compared to levels in drug-naïve controls, with more robust effects of MA withdrawal observed in MA high drinking than DBA/2J mice. When depression-like symptoms were examined after 1 or 2 weeks of forced abstinence in MA high drinking mice, depression-like symptoms dissipated, and subsequent MA intake was high. The MA high drinking genetic mouse model has strong face validity for human binge MA use and behavioral sequelae associated with abstinence.

Verification of a genetic locus for methamphetamine intake and the impact of morphine.

A quantitative trait locus (QTL) on proximal chromosome (Chr) 10 accounts for > 50% of the genetic variance in methamphetamine (MA) intake in mice selectively bred for high (MAHDR) and low (MALDR) voluntary MA drinking. The µ-opioid receptor (MOP-r) gene, Oprm1, resides at the proximal end of Chr 10, and buprenorphine reduces MA intake in MAHDR mice. However, this drug has only partial agonist effects at MOP-r. We investigated the impact of a full MOP-r agonist, morphine, on MA intake and saccharin intake, measured MOP-r density and affinity in several brain regions of the MA drinking lines and their C57BL/6J (B6) and DBA/2J (D2) progenitor strains, and measured MA intake in two congenic strains of mice to verify the QTL and reduce the QTL interval. Morphine reduced MA intake in the MAHDR line, but also reduced saccharin and total fluid intake. MOP-r density was lower in the medial prefrontal cortex of MAHDR, compared to MALDR, mice, but not in the nucleus accumbens or ventral midbrain; there were no MOP-r affinity differences. No significant differences in MOP-r density or affinity were found between the progenitor strains. Finally, Chr 10 congenic results were consistent with previous data suggesting that Oprm1 is not a quantitative trait gene, but is impacted by the gene network underlying MA intake. Stimulation of opioid pathways by a full agonist can reduce MA intake, but may also non-specifically affect consummatory behavior; thus, a partial agonist may be a better pharmacotherapeutic.

Trace amine-associated receptor 1 regulation of methamphetamine-induced neurotoxicity.

Trace amine-associated receptor 1 (TAAR1) is activated by methamphetamine (MA) and modulates dopaminergic (DA) function. Although DA dysregulation is the hallmark of MA-induced neurotoxicity leading to behavioral and cognitive deficits, the intermediary role of TAAR1 has yet to be characterized. To investigate TAAR1 regulation of MA-induced neurotoxicity, Taar1 transgenic knock-out (KO) and wildtype (WT) mice were administered saline or a neurotoxic regimen of 4 i.p. injections, 2h apart, of MA (2.5, 5, or 10mg/kg). Temperature data were recorded during the treatment day. Additionally, striatal tissue was collected 2 or 7days following MA administration for analysis of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and tyrosine hydroxylase (TH) levels, as well as glial fibrillary acidic protein (GFAP) expression. MA elicited an acute hypothermic drop in body temperature in Taar1-WT mice, but not in Taar1-KO mice. Two days following treatment, DA and TH levels were lower in Taar1-KO mice compared to Taar1-WT mice, regardless of treatment, and were dose-dependently decreased by MA. GFAP expression was significantly increased by all doses of MA at both time points and greater in Taar1-KO compared to Taar1-WT mice receiving MA 2.5 or 5mg/kg. Seven days later, DA levels were decreased in a similar pattern: DA was significantly lower in Taar1-KO compared to Taar1-WT mice receiving MA 2.5 or 5mg/kg. TH levels were uniformly decreased by MA, regardless of genotype. These results indicate that activation of TAAR1 potentiates MA-induced hypothermia and TAAR1 confers sustained neuroprotection dependent on its thermoregulatory effects.