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Understanding the pharmacogenomics of opioid metabolism and behavior is vital to therapeutic success as mutations can dramatically alter therapeutic efficacy and addiction liability. We found robust, sex-dependent BALB/c substrain differences in oxycodone behaviors and whole brain concentration of oxycodone metabolites. BALB/cJ females showed robust state-dependent oxycodone reward learning as measured via conditioned place preference when compared to the closely related BALB/cByJ substrain. Accordingly, BALB/cJ females also showed a robust increase in brain concentration of the inactive metabolite noroxycodone and the active metabolite oxymorphone compared to BALB/cByJ mice. Oxymorphone is a highly potent full agonist at the mu opioid receptor that could enhance drug-induced interoception and state-dependent oxycodone reward learning. Quantitative trait locus (QTL) mapping in a BALB/c F2 reduced complexity cross revealed one major QTL on chromosome 15 underlying brain oxymorphone concentration that explained 32% of the female variance. BALB/cJ and BALB/cByJ differ by fewer than 10,000 variants which can greatly facilitate candidate gene/variant identification. Hippocampal and striatal cis-expression QTL (eQTL) and exon-level eQTL analysis identified Zhx2, a candidate gene coding for a transcriptional repressor with a private BALB/cJ retroviral insertion that reduces Zhx2 expression and sex-dependent dysregulation of CYP enzymes. Whole brain proteomics corroborated the Zhx2 eQTL and identified upregulated CYP2D11 that could increase brain oxymorphone in BALB/cJ females. To summarize, Zhx2 is a highly promising candidate gene underlying brain oxycodone metabolite levels. Future studies will validate Zhx2 and its site of action using reciprocal gene editing and tissue-specific viral manipulations in BALB/c substrains.Significance StatementOur findings show genetic variation can result in sex-specific alterations in whole brain concentrations of bioactive opioid metabolites following oxycodone administration, and reinforces the need for sex as a biological factor in pharmacogenomic studies. The co-occurrence of female-specific increased oxymorphone and state-dependent reward learning suggests that this minor yet potent and efficacious metabolite of oxycodone could increase opioid interoception and drug-cue associative learning of opioid reward which has implications for cue-induced relapse of drug-seeking behavior.

Methamphetamine addiction remains a major public health concern in the United States that has paralleled the opioid epidemic. Psychostimulant use disorders have a heritable genetic component that remains unexplained. Methamphetamine targets membrane and vesicular transporters to increase synaptic dopamine, norepinephrine, and serotonin. We previously identifiedHnrnph1(heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying methamphetamine behavioral sensitivity.Hnrnph1encodes the RNA-binding protein hnRNP H1 that is ubiquitously expressed in neurons throughout the brain. Gene-edited mice with a heterozygous frameshift deletion in the first coding exon ofHnrnph1showed reduced methamphetamine-induced dopamine release and behaviors. To inform the mechanism linking hnRNP H dysfunction with reduced methamphetamine neurobehavioral effects, we surveyed the mRNA targetome of hnRNP H via cross-linking immunoprecipitation coupled with RNA-sequencing in striatal tissue at baseline and at 30 min post-methamphetamine. Methamphetamine induced opposite changes in RNA-binding targets of hnRNP H inHnrnph1mutants versus wild-types, including 3’UTR targets in mRNAs enriched for synaptic proteins involved in dopamine release and excitatory synaptic plasticity. Targetome, transcriptome, and spliceome analyses triangulated on a methamphetamine-induced upregulation of the calcium channel subunit transcriptCacna2d2and decreased its 3’UTR usage in hyposensitiveHnrnph1mutants. Pretreatment with pregabalin, an inhibitor of α2δ2 and α2δ1 voltage-gated calcium channel subunits attenuated methamphetamine-induced locomotor activity in wild-type females but not inHnrnph1mutants, supporting Cacna2d2 as a hnRNP H target. Our study identifies a dynamic hnRNP H RNA targetome that can rapidly and adaptively respond to methamphetamine to regulate gene expression and likely synaptic plasticity and behavior.SIGNIFICANCE STATEMENTThe genetic risks mediating psychostimulant addiction are unknown and there are no FDA-approved treatments. We identifiedHnrnph1in modulating methamphetamine behavioral sensitivity in mice.Hnrnph1codes for hnRNP H1, an RNA-binding protein. Here, we asked whether anHnrnph1mutation and methamphetamine treatment would change the hnRNP H RNA targets and whether these targets could tell us howHnrnph1is linked to behavior. We identified a calcium channel subunit that is a primary target of the FDA-approved drug pregabalin (a.k.a. Lyrica®). Like theHnrnph1mutation, pregabalin reduced methamphetamine behaviors in wild-type mice. We propose hnRNP H regulates calcium channels in response to methamphetamine-induced perturbations in neurotransmitter release. Accordingly, pregabalin could represent a novel treatment to restore synaptic function following methamphetamine administration.

The opioid epidemic led to an increase in the number of Neonatal Opioid Withdrawal Syndrome (NOWS) cases in infants born to opioid-dependent mothers. Hallmark features of NOWS include weight loss, severe irritability, respiratory problems, and sleep fragmentation. Mouse models provide an opportunity to identify brain mechanisms that contribute to NOWS. Neonatal outbred Swiss Webster Cartworth Farms White (CFW) mice were administered morphine (15mg/kg, s.c.) twice daily for postnatal days (P) 1-14, an approximate of the third trimester of human gestation. Male and female mice underwent behavioral testing on P7 and P14 to determine the impact of opioid exposure on anxiety and pain sensitivity. Ultrasonic vocalizations (USVs) and daily body weights were also recorded. Brainstems containing pons and medulla were collected during morphine withdrawal on P14 for RNA-sequencing. Morphine induced weight loss from P2-14, which persisted during adolescence (P21) and adulthood (P50). USVs markedly increased at P7 in females, emerging earlier than males. On P7 and P14, both morphine exposed female and male mice displayed hyperalgesia on the hot plate and tail flick assays, with females having greater hyperalgesia than males. Morphine-exposed mice exhibited increased anxiety-like behavior in the open-field arena at P21. Transcriptome analysis of the brainstem (medulla plus pons), an area implicated in opioid withdrawal and NOWS, identified pathways enriched for noradrenergic signaling in females and males. We also found sex-specific pathways related to mitochondrial function and neurodevelopment in females and circadian entrainment in males. Sex-specific transcriptomic neuroadaptations implicate unique neurobiological mechanisms underlying NOWS-like behaviors.SIGNIFICANCE STATEMENTNeonatal opioid withdrawal syndrome (NOWS) is a poorly understood condition that has both a genetic and environmental component and is thought to be mechanistically distinct from opioid withdrawal in adults. The development of murine models for measuring neurobehavioral responses is critical for informing the neurobiological adaptations underlying NOWS. Using outbred mice that more closely model human genetic variation, we discovered a surprising degree of sexual dimorphism in behavioral timing and severity of NOWS-model behaviors as well as transcriptomic adaptations in brain tissue that together suggest distinct mechanisms and sex-specific therapeutics for reversing withdrawal symptoms and restoring brain function.

ObjectiveBinge eating is a heritable quantitative trait associated with eating disorders (ED) and refers to the rapid consumption of a large quantity of energy-dense food that is associated with loss of control, anxiety, and depression. Binge Eating Disorder is the most common ED in adults in the US; however, the genetic basis is unknown. We previously identified robust mouse inbred strain differences between C57BL/6J and DBA/2J in binge-like eating (BLE) of sweetened palatable food (PF) in an intermittent access, conditioned place preference paradigm.MethodsTo map the genetic basis of BLE, we phenotyped and genotyped 128 C57BL/6J x DBA/2J-F2 mice.ResultsWe identified a quantitative trait locus (QTL) on chromosome 13 influencing progressive changes in body weight across training days (LOD = 5.5; 26-39 cM). We also identified two sex-combined QTLs influencing PF intake on chromosome 5 (LOD = 5.6; 1.5-LOD interval = 21-28 cM) and 6 (LOD = 5.3; 1.5-LOD interval = 50-59 cM). Furthermore, sex-specific analyses revealed that the chromosome 6 locus was driven by males (1.5-LOD interval: 52-59 cM) and identified a female-selective QTL for BLE on chromosome 18 (LOD = 4.1; 1.5-LOD interval: 23-35 cM). Systems genetic analysis of the chromosome 6 locus for BLE using GeneNetwork legacy trait datasets from BXD recombinant inbred strains identified Adipor2 and Plxnd1 as two positional, functional, biological candidate genes.DiscussionWe identified genetic loci influencing BLE. Future studies will phenotype BXD recombinant inbred strains to fine map loci and support candidate gene nomination and validation.

Binge eating (BE) is a heritable symptom of eating disorders with an unknown genetic etiology. Rodent models for BE of palatable food permit the study of genetic and biological mechanisms. We previously used genetic mapping and transcriptome analysis to map a coding mutation in Cyfip2 associated with increased BE in the BE-prone C57BL/6NJ substrain compared to the BE-resistant C57BL/6J substrain. The increase in BE in C57BL/6NJ mice was associated with a decrease in transcription of genes enriched for myelination in the striatum. Here, we tested the hypothesis that decreasing myelin levels with the demyelinating agent cuprizone would enhance BE. Mice were treated with a 0.3% cuprizone home cage diet for two weeks. Following a three-week recovery period, mice were trained for BE in an intermittent, limited access procedure. Cuprizone induced similar weight loss in both substrains and sexes that recovered within 48 h after removal of the cuprizone diet. Surprisingly, cuprizone reduced BE in male but not female C57BL/6NJ mice while having no effect in C57BL/6J mice. Cuprizone also reduced myelin basic protein (MBP) at seven weeks post-cuprizone removal while having no effect on myelin-associated glycoprotein (MAG) at this time point. C57BL/6N mice also showed less MBP than C57BL/6J mice. There were no statistical interactions of Treatment with Sex on MBP levels, indicating that differences in MBP are unlikely to account for sex differences in BE. To summarize, cuprizone induced an unexpected, male-specific reduction in BE which could indicate sex-specific biological mechanisms that depend on genetic background.