Male and female variability in response to chronic stress and morphine in C57BL/6J, DBA/2J, and their BXD progeny.

Drug addiction is a multifactorial syndrome in which genetic predispositions and exposure to environmental stressors constitute major risk factors for the early onset, escalation, and relapse of addictive behaviors. While it is well known that stress plays a key role in drug addiction, the genetic factors that make certain individuals particularly sensitive to stress and thereby more vulnerable to becoming addicted are unknown. In an effort to test a complex set of gene x environment interactions-specifically gene x chronic stress -here we leveraged a systems genetics resource: BXD recombinant inbred mice (BXD5, BXD8, BXD14, BXD22, BXD29, and BXD32) and their parental mouse lines, C57BL/6J and DBA/2J. Utilizing the chronic social defeat stress (CSDS) and chronic variable stress (CVS) paradigms, we first showed sexual dimorphism in the behavioral stress response between the mouse strains. Further, we observed an interaction between genetic background and vulnerability to prolonged exposure to non-social stressors. Finally, we found that DBA/2J and C57BL/6J mice pre-exposed to stress displayed differences in morphine sensitivity. Our results support the hypothesis that genetic variation in predisposition to stress responses influences morphine sensitivity and is likely to modulate the development of drug addiction.

Limited WKY chromosomal regions confer increases in anxiety and fear memory in a F344 congenic rat strain.

This study investigated the interaction between genetic differences in stress reactivity/coping and environmental challenges, such as acute stress during adolescence on adult contextual fear memory and anxiety-like behaviors. Fischer 344 (F344) and the inbred F344;WKY-Stresp3/Eer congenic strain (congenic), in which chromosomal regions from the Wistar-Kyoto (WKY) strain were introgressed into the F344 background, were exposed to a modified forced swim test during adolescence, while controls were undisturbed. In adulthood, fear learning and memory, assessed by contextual fear conditioning, were significantly greater in congenic animals compared with F344 animals, and stress during adolescence increased them even further in males of both strains. Anxiety-like behavior, measured by the open field test, was also greater in congenic than F344 animals, and stress during adolescence increased it further in both strains of adult males. Whole genome sequencing of the F344;WKY-Stresp3/Eer strain revealed an enrichment of WKY genotypes in chromosomes 9, 14, and 15. An example of functional WKY sequence variations in the congenic strain, cannabinoid receptor interacting protein 1 (Cnrip1) had a Cnrip1 transcript isoform that lacked two exons. Although the original hypothesis that the genetic predisposition to increased anxiety of the WKY donor strain would exaggerate fear memory relative to the background strain was confirmed, the consequences of adolescent stress were strain independent but sex dependent in adulthood. Molecular genomic approaches combined with genetic mapping of WKY sequence variations in chromosomes 9, 14, and 15 could aid in finding quantitative trait genes contributing to the variation in fear memory.NEW & NOTEWORTHY This study found that 1) whole genome sequencing of congenic strains should be a criterion for their recognition; 2) sequence variations between Wistar-Kyoto and Fischer 344 strains at regions of chromosomes 9, 14, and 15 contribute to differences in contextual fear memory and anxiety-like behaviors; and 3) stress during adolescence affects these behaviors in males, but not females, and is independent of strain.

Effects of genetics and sex on adolescent behaviors following neonatal ethanol exposure in BXD recombinant inbred strains.

Introduction: Fetal alcohol spectrum disorders (FASD) are the leading preventable neurodevelopmental disorders and two hallmark symptoms of FASD are abnormal behavior, and cognitive and learning deficits. The severity of alcohol's teratogenic effects on the developing brain is influenced by genetics and sex. We previously identified recombinant inbred BXD mouse strains that show differential vulnerability to ethanol-induced cell death in the developing hippocampus, a brain region important in learning and memory. The present study aimed to test the hypothesis that strains with increased vulnerability to ethanol-induced cell death in the hippocampus have concomitant deficits in multiple hippocampal-related behaviors during adolescence. Methods: The current study evaluated the effects of developmental ethanol exposure on adolescent behavior in two BXD strains that show high cell death (BXD48a, BXD100), two that show low cell death (BXD60, BXD71), and the two parental strains (C57BL/6 J (B6), DBA/2 J (D2)). On postnatal day 7, male and female neonatal pups were treated with ethanol (5.0 g/kg) or saline given in two equal doses 2 h apart. Adolescent behavior was assessed across multiple behavioral paradigms including the elevated plus maze, open field, Y-maze, and T-maze. Results: Our results demonstrate that the effects of developmental ethanol exposure on adolescent behavioral responses are highly dependent on strain. The low cell death strains, BXD60 and BXD71, showed minimal effect of ethanol exposure on all behavioral measures but did present sex differences. The parental -B6 and D2-strains and high cell death strains, BXD48a and BXD100, showed ethanol-induced effects on activity-related or anxiety-like behaviors. Interestingly, the high cell death strains were the only strains that showed a significant effect of postnatal ethanol exposure on hippocampal-dependent spatial learning and memory behaviors. Discussion: Overall, we identified effects of ethanol exposure, strain, and/or sex on multiple behavioral measures. Interestingly, the strains that showed the most effects of postnatal ethanol exposure on adolescent behavior were the BXD strains that show high ethanol-induced cell death in the neonatal hippocampus, consistent with our hypothesis. Additionally, we found evidence for interactions among strain and sex, demonstrating that these factors have a complex effect on alcohol responses and that both are important considerations.

Divergent risky decision-making and impulsivity behaviors in Lewis rat substrains with low genetic difference.

Substance use disorder (SUD) is associated with a cluster of cognitive disturbances that engender vulnerability to ongoing drug seeking and relapse. Two of these endophenotypes-risky decision-making and impulsivity-are amplified in individuals with SUD and are augmented by repeated exposure to illicit drugs. Identifying genetic factors underlying variability in these behavioral patterns is critical for early identification, prevention, and treatment of SUD-vulnerable individuals. Here, we compared risky decision-making and different facets of impulsivity between two fully inbred substrains of Lewis rats-LEW/NCrl and LEW/NHsd. We performed whole genome sequencing of both substrains to identify almost all relevant variants. We observed substantial differences in risky decision-making and impulsive behaviors. Relative to LEW/NHsd, the LEW/NCrl substrain accepts higher risk options in a decision-making task and higher rates of premature responses in the differential reinforcement of low rates of responding task. These phenotypic differences were more pronounced in females than males. We defined a total of ∼9,000 polymorphisms between these substrains at 40× whole genome short-read coverage. Roughly half of variants are located within a single 1.5 Mb region of Chromosome 8, but none impact protein-coding regions. In contrast, other variants are widely distributed, and of these, 38 are predicted to cause protein-coding variants. In conclusion, Lewis rat substrains differ significantly in risk-taking and impulsivity and only a small number of easily mapped variants are likely to be causal. Sequencing combined with a reduced complexity cross should enable identification of one or more variants underlying multiple complex addiction-relevant behaviors. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Influence of prenatal cannabinoid exposure on early development and beyond.

Public perception surrounding whether cannabis use is harmful during pregnancy often diverges greatly from the recommendations of doctors and healthcare providers. In contrast to the medical guidance of abstinence before, during, and after pregnancy, many women of reproductive age believe cannabis use during pregnancy is associated with little potential harm. Legalization and social cues support public perceptions that cannabis use during pregnancy is safe. Moreover, pregnant women may consider cannabis to be a safe alternative for treating pregnancy related ailments, including morning sickness. Compounding the problem is a lack of medical and federal guidance on safe, low, or high-risk levels of cannabis use. These issues mirror the continuing debate surrounding alcohol use and health, in particular, whether there are safe or lower risk levels of alcohol consumption during pregnancy. Clinical studies to date suffer from several limitations. First, most human studies are correlative in nature, meaning that causal associations cannot be made between in utero cannabis exposure and health and behavioral outcomes later in life. Due to obvious ethical constraints, it is not possible to randomly assign pregnant mothers to cannabis or other drug exposure conditions-a requirement needed to establish causality. In addition, clinical studies often lack quantitative information on maternal exposure (i.e., dose, frequency, and duration), include a small number of individuals, lack replication of outcome measures across cohorts, rely on self-report to establish maternal drug use, and suffer from unmeasured or residual confounding factors. Causal associations between maternal cannabis exposure and offspring outcomes are possible in preclinical cohorts but there is a large amount of heterogeneity across study designs and developmental differences between rodents and humans may limit translatability. In this review, we summarize research from human and preclinical models to provide insight into potential risks associated with prenatal cannabinoid exposure (PCE). Finally, we highlight gaps in knowledge likely to contribute to the growing divide between medical guidance and public attitudes regarding cannabis use during pregnancy.

Effects of Genetics and Sex on Acute Gene Expression Changes in the Hippocampus Following Neonatal Ethanol Exposure in BXD Recombinant Inbred Mouse Strains.

Fetal alcohol spectrum disorders (FASD) are prevalent neurodevelopmental disorders. Genetics have been shown to have a role in the severity of alcohol's teratogenic effects on the developing brain. We previously identified recombinant inbred BXD mouse strains that show high (HCD) or low cell death (LCD) in the hippocampus following ethanol exposure. The present study aimed to identify gene networks that influence this susceptibility. On postnatal day 7 (3rd-trimester-equivalent), male and female neonates were treated with ethanol (5.0 g/kg) or saline, and hippocampi were collected 7hrs later. Using the Affymetrix microarray platform, ethanol-induced gene expression changes were identified in all strains with divergent expression sets found between sexes. Genes, such as Bcl2l11, Jun, and Tgfb3, showed significant strain-by-treatment interactions and were involved in many apoptosis pathways. Comparison of HCD versus LCD showed twice as many ethanol-induced genes changes in the HCD. Interestingly, these changes were regulated in the same direction suggesting (1) more perturbed effects in HCD compared to LCD and (2) limited gene expression changes that confer resistance to ethanol-induced cell death in LCD. These results demonstrate that genetic background and sex are important factors that affect differential cell death pathways after alcohol exposure during development that could have long-term consequences.

GeneCup: mining PubMed and GWAS catalog for gene-keyword relationships.

Interpreting and integrating results from omics studies typically requires a comprehensive and time consuming survey of extant literature. GeneCup is a literature mining web service that retrieves sentences containing user-provided gene symbols and keywords from PubMed abstracts. The keywords are organized into an ontology and can be extended to include results from human genome-wide association studies. We provide a drug addiction keyword ontology that contains over 300 keywords as an example. The literature search is conducted by querying the PubMed server using a programming interface, which is followed by retrieving abstracts from a local copy of the PubMed archive. The main results presented to the user are sentences where gene symbol and keywords co-occur. These sentences are presented through an interactive graphical interface or as tables. All results are linked to the original abstract in PubMed. In addition, a convolutional neural network is employed to distinguish sentences describing systemic stress from those describing cellular stress. The automated and comprehensive search strategy provided by GeneCup facilitates the integration of new discoveries from omic studies with existing literature. GeneCup is free and open source software. The source code of GeneCup and the link to a running instance is available at https://github.com/hakangunturkun/GeneCup.

Correction of the hypomorphic Gabra2 splice site variant in mouse strain C57BL/6J modifies the severity of Scn8a encephalopathy.

De novo gain-of-function mutations of SCN8A are a significant cause of developmental and epileptic encephalopathy (DEE) (MIM: 614558). The severely affected individuals exhibit refractory seizures, developmental delay, and cognitive disabilities, often accompanied by impaired movement. Individuals with the identical SCN8A variant often differ in clinical course, suggesting a role for modifier genes in disease severity. In a previous study we demonstrated genetic linkage between a hypomorphic mutation in the Gabra2 gene and seizure severity in a mouse model of the human SCN8A pathogenic variant p.Arg1872Trp. Homozygosity for the hypomorphic Gabra2 mutation was associated with early seizure onset and shortened lifespan. We have now confirmed Gabra2 as the modifier gene using a knock-in allele that corrects the splice site variant in strain C57BL/6J. Correction of the Gabra2 variant restores transcript abundance, increases the age of seizure onset, and extends survival of the Scn8a mutant mice. GABRA2 encodes the α2 subunit of the GABAA receptor that provides inhibitory input to dendrites and the the axon initial segment of excitatory neurons. Quantitative variation in human GABAA receptor expression could contribute to variation in the severity of genetic epilepsies and suggests a potential therapeutic intervention.

Identification of cyclin D1 as a major modulator of 3-nitropropionic acid-induced striatal neurodegeneration.

Mitochondria dysfunction occurs in the aging brain as well as in several neurodegenerative disorders and predisposes neuronal cells to enhanced sensitivity to neurotoxins. 3-nitropropionic acid (3-NP) is a naturally occurring plant and fungal neurotoxin that causes neurodegeneration predominantly in the striatum by irreversibly inhibiting the tricarboxylic acid respiratory chain enzyme, succinate dehydrogenase (SDH), the main constituent of the mitochondria respiratory chain complex II. Significantly, although 3-NP-induced inhibition of SDH occurs in all brain regions, neurodegeneration occurs primarily and almost exclusively in the striatum for reasons still not understood. In rodents, 3-NP-induced striatal neurodegeneration depends on the strain background suggesting that genetic differences among genotypes modulate toxicant variability and mechanisms that underlie 3-NP-induced neuronal cell death. Using the large BXD family of recombinant inbred (RI) strains we demonstrate that variants in Ccnd1 - the gene encoding cyclin D1 - of the DBA/2 J parent underlie the resistance to 3-NP-induced striatal neurodegeneration. In contrast, the Ccnd1 variant inherited from the widely used C57BL/6 J parental strain confers sensitivity. Given that cellular stress triggers induction of cyclin D1 expression followed by cell-cycle re-entry and consequent neuronal cell death, we sought to determine if the C57BL/6 J and DBA/2 J Ccnd1 variants are differentially modulated in response to 3-NP. We confirm that 3-NP induces cyclin D1 expression in striatal neuronal cells of C57BL/6 J, but this response is blunted in the DBA/2 J. We further show that striatal-specific alternative processing of a highly conserved 3'UTR negative regulatory region of Ccnd1 co-segregates with the C57BL/6 J parental Ccnd1 allele in BXD strains and that its differential processing accounts for sensitivity or resistance to 3-NP. Our results indicate that naturally occurring Ccnd1 variants may play a role in the variability observed in neurodegenerative disorders involving mitochondria complex II dysfunction and point to cyclin D1 as a possible therapeutic target.

A quantitative trait variant in Gabra2 underlies increased methamphetamine stimulant sensitivity.

Psychostimulant (methamphetamine, cocaine) use disorders have a genetic component that remains mostly unknown. We conducted genome-wide quantitative trait locus (QTL) analysis of methamphetamine stimulant sensitivity. To facilitate gene identification, we employed a Reduced Complexity Cross between closely related C57BL/6 mouse substrains and examined maximum speed and distance traveled over 30 min following methamphetamine (2 mg/kg, i.p.). For maximum methamphetamine-induced speed following the second and third administration, we identified a single genome-wide significant QTL on chromosome 11 that peaked near the Cyfip2 locus (LOD = 3.5, 4.2; peak = 21 cM [36 Mb]). For methamphetamine-induced distance traveled following the first and second administration, we identified a genome-wide significant QTL on chromosome 5 that peaked near a functional intronic indel in Gabra2 coding for the alpha-2 subunit of the GABA-A receptor (LOD = 3.6-5.2; peak = 34-35 cM [66-67 Mb]). Striatal cis-expression QTL mapping corroborated Gabra2 as a functional candidate gene underlying methamphetamine-induced distance traveled. CRISPR/Cas9-mediated correction of the mutant intronic deletion on the C57BL/6J background to the wild-type C57BL/6NJ allele was sufficient to reduce methamphetamine-induced locomotor activity toward the wild-type C57BL/6NJ-like level, thus validating the quantitative trait variant (QTV). These studies show the power and efficiency of Reduced Complexity Crosses in identifying causal variants underlying complex traits. Functionally restoring Gabra2 expression decreased methamphetamine stimulant sensitivity and supports preclinical and human genetic studies implicating the GABA-A receptor in psychostimulant addiction-relevant traits. Importantly, our findings have major implications for studying psychostimulants in the C57BL/6J strain-the gold standard strain in biomedical research.

Gene-by-environment modulation of lifespan and weight gain in the murine BXD family.

How lifespan and body weight vary as a function of diet and genetic differences is not well understood. Here we quantify the impact of differences in diet on lifespan in a genetically diverse family of female mice, split into matched isogenic cohorts fed a low-fat chow diet (CD, n = 663) or a high-fat diet (HFD, n = 685). We further generate key metabolic data in a parallel cohort euthanized at four time points. HFD feeding shortens lifespan by 12%: equivalent to a decade in humans. Initial body weight and early weight gains account for longevity differences of roughly 4-6 days per gram. At 500 days, animals on a HFD typically gain four times as much weight as control, but variation in weight gain does not correlate with lifespan. Classic serum metabolites, often regarded as health biomarkers, are not necessarily strong predictors of longevity. Our data indicate that responses to a HFD are substantially modulated by gene-by-environment interactions, highlighting the importance of genetic variation in making accurate individualized dietary recommendations.

Sex and heredity are determinants of drug intake in a novel model of rat oral oxycodone self-administration.

The steady rise in prescription opioids such as oxycodone has led to a virulent epidemic of widespread abuse and deaths in the United States; approximately 80% of affected individuals initiate the habitual use of oxycodone by using prescription oral oxycodone. Given the importance of drug pharmacokinetics in determining abuse potential, we designed an oral operant oxycodone self-administration (SA) procedure in rats to model drug intake by most human users/abusers of oxycodone. Key aspects of the model include limited initial drug intake followed by increasing drug concentrations during extended 4-h sessions on alternating days. Sex and genetic predisposition are major determinants of human opiate abuse. Therefore, we studied females in seven inbred strains (WLI, WMI, LEW, DSS, F344, BN and SHR) and both sexes in three of these strains. All strains increased intake across serially increasing doses (0.025-0.2 mg/ml; p < 0.001): the range of intakes at the final concentration of oxycodone was 0.72 ± 0.17-4.84 ± 1.42 mg/kg (mean ± SEM) - a 6.7-fold difference across strains. In LEW, WLI and WMI strains, oxycodone intake increased significantly across all sessions in both sexes. However, in LEW and WMI male rats but not WLI, daily oxycodone intake was significantly lower across all 4-h sessions than females (p < 0.005). The estimated heritability in oxycodone intake was in the range of 0.21-0.41. In summary, our novel operant oral oxycodone SA model captures the strong abuse potential of oral oxycodone and shows dose, sex and strain-specific drug intake that is significantly dependent on heredity.

Genetic Modulation of Initial Sensitivity to Δ9-Tetrahydrocannabinol (THC) Among the BXD Family of Mice.

Cannabinoid receptor 1 activation by the major psychoactive component in cannabis, Δ9-tetrahydrocannabinol (THC), produces motor impairments, hypothermia, and analgesia upon acute exposure. In previous work, we demonstrated significant sex and strain differences in acute responses to THC following administration of a single dose (10 mg/kg, i.p.) in C57BL/6J (B6) and DBA/2J (D2) inbred mice. To determine the extent to which these differences are heritable, we quantified acute responses to a single dose of THC (10 mg/kg, i.p.) in males and females from 20 members of the BXD family of inbred strains derived by crossing and inbreeding B6 and D2 mice. Acute THC responses (initial sensitivity) were quantified as changes from baseline for: 1. spontaneous activity in the open field (mobility), 2. body temperature (hypothermia), and 3. tail withdrawal latency to a thermal stimulus (antinociception). Initial sensitivity to the immobilizing, hypothermic, and antinociceptive effects of THC varied substantially across the BXD family. Heritability was highest for mobility and hypothermia traits, indicating that segregating genetic variants modulate initial sensitivity to THC. We identified genomic loci and candidate genes, including Ndufs2, Scp2, Rps6kb1 or P70S6K, Pde4d, and Pten, that may control variation in THC initial sensitivity. We also detected strong correlations between initial responses to THC and legacy phenotypes related to intake or response to other drugs of abuse (cocaine, ethanol, and morphine). Our study demonstrates the feasibility of mapping genes and variants modulating THC responses in the BXDs to systematically define biological processes and liabilities associated with drug use and abuse.

TailTimer: A device for automating data collection in the rodent tail immersion assay.

The tail immersion assay is a widely used method for measuring acute thermal pain in a way which is quantifiable and reproducible. It is non-invasive and measures response to a stimulus that may be encountered by an animal in its natural environment. However, quantification of tail withdrawal latency relies on manual timing of tail flick using a stopwatch, and precise temperatures of the water at the time of measurement are most often not recorded. These two factors greatly reduce the reproducibility of tail immersion assay data and likely contribute to some of the discrepancies present among relevant literature. We designed a device, TailTimer, which uses a Raspberry Pi single-board computer, a digital temperature sensor, and two electrical wires, to automatically record tail withdrawal latency and water temperature. We programmed TailTimer to continuously display and record water temperature and to only permit the assay to be conducted when the water is within ± 0.25°C of the target temperature. Our software also records the identification of the animals using a radio frequency identification (RFID) system. We further adapted the RFID system to recognize several specific keys as user interface commands, allowing TailTimer to be operated via RFID fobs for increased usability. Data recorded using the TailTimer device showed a negative linear relationship between tail withdrawal latency and water temperature when tested between 47-50°C. We also observed a previously unreported, yet profound, effect of water mixing speed on latency. In one experiment using TailTimer, we observed significantly longer latencies following administration of oral oxycodone versus a distilled water control when measured after 15 mins or 1 h, but not after 4 h. TailTimer also detected significant strain differences in baseline latency. These findings valorize TailTimer in its sensitivity and reliability for measuring thermal pain thresholds.

Whole genome sequencing of nearly isogenic WMI and WLI inbred rats identifies genes potentially involved in depression and stress reactivity.

The WMI and WLI inbred rats were generated from the stress-prone, and not yet fully inbred, Wistar Kyoto (WKY) strain. These were selected using bi-directional selection for immobility in the forced swim test and were then sib-mated for over 38 generations. Despite the low level of genetic diversity among WKY progenitors, the WMI substrain is significantly more vulnerable to stress relative to the counter-selected WLI strain. Here we quantify numbers and classes of genomic sequence variants distinguishing these substrains with the long term goal of uncovering functional and behavioral polymorphism that modulate sensitivity to stress and depression-like phenotypes. DNA from WLI and WMI was sequenced using Illumina xTen, IonTorrent, and 10X Chromium linked-read platforms to obtain a combined coverage of ~ 100X for each strain. We identified 4,296 high quality homozygous SNPs and indels between the WMI and WLI. We detected high impact variants in genes previously implicated in depression (e.g. Gnat2), depression-like behavior (e.g. Prlr, Nlrp1a), other psychiatric disease (e.g. Pou6f2, Kdm5a, Reep3, Wdfy3), and responses to psychological stressors (e.g. Pigr). High coverage sequencing data confirm that the two substrains are nearly coisogenic. Nonetheless, the small number of sequence variants contributes to numerous well characterized differences including depression-like behavior, stress reactivity, and addiction related phenotypes. These selected substrains are an ideal resource for forward and reverse genetic studies using a reduced complexity cross.

Genetic differences in ethanol consumption: effects on iron, copper, and zinc regulation in mouse hippocampus.

One common characteristic of neurodegenerative diseases is dysregulation of iron, usually with observed increases in its concentration in various regions. Heavy alcohol consumption is believed to contribute to such iron dysregulation in the brain with accompanying dementia. To examine this effect and related genetic-based individual differences in an animal model, we subjected female mice from 12 BXD recombinant inbred strains to 16 weeks of alcohol consumption using the drinking in the dark (DID) method. Daily consumption was recorded and at the end of 16 weeks hippocampus tissues harvested. Concentrations of iron, copper and zinc were measured using X-ray fluorescence technology. The results showed that, DID increased iron overall across all strains, ranging from 3 to 68%. Copper and Zinc both decreased, ranging from 0.4-42 and 5-35% respectively. Analysis of variance revealed significant strain by treatment interactions for all three metals. Additionally, in the DID group, we observed strain differences in reduction of hippocampus mass. These findings are particularly interesting to us because high alcohol consumption in humans has been associated with neurodegeneration and dementia related to disruption of iron regulation. The findings of alcohol consumption associated decreases in copper and zinc are novel. The role of copper regulation and neurological function related to alcohol consumption is as yet largely unexplored. The role of zinc is better known as a neuromodulator in the hippocampus and appears to be protective against neurological damage. It would seem then, that the alcohol-related decrease in zinc in the hippocampus would be of concern and warrants further study.

Gabra2 is a genetic modifier of Dravet syndrome in mice.

Pathogenic variants in epilepsy genes result in a spectrum of clinical severity. One source of phenotypic heterogeneity is modifier genes that affect expressivity of a primary pathogenic variant. Mouse epilepsy models also display varying degrees of clinical severity on different genetic backgrounds. Mice with heterozygous deletion of Scn1a (Scn1a+/-) model Dravet syndrome, a severe epilepsy most often caused by SCN1A haploinsufficiency. Scn1a+/- mice recapitulate features of Dravet syndrome, including spontaneous seizures, sudden death, and cognitive/behavioral deficits. Scn1a+/- mice maintained on the 129S6/SvEvTac (129) strain have normal lifespan and no spontaneous seizures. In contrast, admixture with C57BL/6J (B6) results in epilepsy and premature lethality. We previously mapped Dravet Survival Modifier loci (Dsm1-Dsm5) responsible for strain-dependent differences in survival. Gabra2, encoding the GABAA α2 subunit, was nominated as a candidate modifier at Dsm1. Direct measurement of GABAA receptors found lower abundance of α2-containing receptors in hippocampal synapses of B6 mice relative to 129. We also identified a B6-specific single nucleotide deletion within Gabra2 that lowers mRNA and protein by nearly 50%. Repair of this deletion reestablished normal levels of Gabra2 expression. In this study, we used B6 mice with a repaired Gabra2 allele to evaluate Gabra2 as a genetic modifier of severity in Scn1a+/- mice. Gabra2 repair restored transcript and protein expression, increased abundance of α2-containing GABAA receptors in hippocampal synapses, and rescued epilepsy phenotypes of Scn1a+/- mice. These findings validate Gabra2 as a genetic modifier of Dravet syndrome, and support enhancing function of α2-containing GABAA receptors as treatment strategy for Dravet syndrome.

Systems genetic analysis of binge-like eating in a C57BL/6J x DBA/2J-F2 cross.

Binge eating is a heritable trait associated with eating disorders and refers to the rapid consumption of a large quantity of energy-dense food that is, associated with loss of control and negative affect. Binge eating disorder is the most common eating disorder in the United States; however, the genetic basis is unknown. We previously identified robust mouse inbred strain differences between C57BL/6J and DBA/2J in binge-like eating of sweetened palatable food in an intermittent access, conditioned place preference paradigm. To map the genetic basis of changes in body weight and binge-like eating (BLE) and to identify candidate genes, we conducted quantitative trait locus (QTL) analysis in 128 C57BL/6J x DBA/2J-F2 mice combined with PheQTL and trait covariance analysis in GeneNetwork2 using legacy BXD-RI trait datasets. We identified a QTL on Chromosome 18 influencing changes in body weight across days in females (log of the odds [LOD] = 6.3; 1.5-LOD: 3-12 cM) that contains the candidate gene Zeb1. We also identified a sex-combined QTL influencing initial palatable food intake on Chromosome 5 (LOD = 5.8; 1.5-LOD: 21-28 cM) that contains the candidate gene Lcorl and a second QTL influencing escalated palatable food intake on Chromosome 6 in males (LOD = 5.4; 1.5-LOD: 50-59 cM) that contains the candidate genes Adipor2 and Plxnd1. Finally, we identified a suggestive QTL in females for slope of BLE on distal Chromosome 18 (LOD = 4.1; p = 0.055; 1.5-LOD: 23-35 cM). Future studies will use BXD-RI strains to fine map loci and support candidate gene nomination for gene editing.

A platform for experimental precision medicine: The extended BXD mouse family.

The challenge of precision medicine is to model complex interactions among DNA variants, phenotypes, development, environments, and treatments. We address this challenge by expanding the BXD family of mice to 140 fully isogenic strains, creating a uniquely powerful model for precision medicine. This family segregates for 6 million common DNA variants-a level that exceeds many human populations. Because each member can be replicated, heritable traits can be mapped with high power and precision. Current BXD phenomes are unsurpassed in coverage and include much omics data and thousands of quantitative traits. BXDs can be extended by a single-generation cross to as many as 19,460 isogenic F1 progeny, and this extended BXD family is an effective platform for testing causal modeling and for predictive validation. BXDs are a unique core resource for the field of experimental precision medicine.

Paraquat Toxicogenetics: Strain-Related Reduction of Tyrosine Hydroxylase Staining in Substantia Nigra in Mice.

Paraquat (PQ) is a putative risk factor for the development of sporadic Parkinson's disease. To model a possible genetic basis for individual differences in susceptibility to exposure to PQ, we recently examined the effects of paraquat on tyrosine hydroxylase (TH)-containing neurons in the substantia nigra pars compacta (SNc) of six members of the BXD family of mice (n = 2-6 per strain). We injected males with 5 mg/kg paraquat weekly three times. The density of TH+ neurons counted by immunocytochemistry at 200x in eight or more sections through the SNc is reduced in five of the six strains relative to control (N = 4 ± 2 mice per strain). TH+ loss ranged from 0 to 20% with an SEM of 1%. The heritability was estimated using standard ANOVA and jackknife resampling and is 0.37 ± 0.05 in untreated animals and 0.47 ± 0.04 in treated animals. These results demonstrate genetic modulation and GxE variation in susceptibility to PQ exposure and the loss of TH staining in the substantia nigra.