Chloride intracellular channel 4 (CLIC4) expression profile in the mouse medial prefrontal cortex and its regulation by ethanol.

BACKGROUND: Chloride intracellular channel 4 (CLIC4) is a multifunctional metamorphic protein for which a growing body of evidence supports a major role in the brain's molecular and behavioral responses to ethanol (EtOH). Although key to understanding the functional biology underlying this role, little is known about the cellular and subcellular expression patterns of CLIC4 in brain and how they are affected by EtOH. METHODS: We used qRT-PCR to assess Clic4 mRNA expression in the medial prefrontal cortex (mPFC) of C57BL/6J mice in the absence and presence of acute EtOH exposure. Two complementary immunohistochemical techniques were employed to assess the subcellular localization of the CLIC4 protein and its pattern of expression across brain cell types in the mPFC in the absence and presence of acute EtOH. RESULTS: Through immunohistochemical and stereological techniques, we show that CLIC4 protein is robustly expressed by oligodendrocytes (most abundant), microglia, and astrocytes, with minimal expression in neurons. Following acute EtOH exposure, we observed a rapid increase in Clic4 mRNA expression in female but not male mice and an overall increase in the number of oligodendrocytes and astrocytes expressing the CLIC4 protein. CONCLUSIONS: These findings suggest that Clic4 functions as an early response gene for acute EtOH in brain, which likely underlies its ability to modulate EtOH behavior. Our results also suggest that the role of CLIC4 in the brain's response to EtOH is mediated through oligodendrocytes.

GCSscore: an R package for differential gene expression analysis in Affymetrix/Thermo-Fisher whole transcriptome microarrays.

BACKGROUND: Despite the increasing use of RNAseq for transcriptome analysis, microarrays remain a widely-used methodology for genomic studies. The latest generation of Affymetrix/Thermo-Fisher microarrays, the ClariomD/XTA and ClariomS array, provide a sensitive and facile method for complex transcriptome expression analysis. However, existing methods of analysis for these high-density arrays do not leverage the statistical power contained in having multiple oligonucleotides representing each gene/exon, but rather summarize probes into a single expression value. We previously developed a methodology, the Sscore algorithm, for probe-level identification of differentially expressed genes (DEGs) between treatment and control samples with oligonucleotide microarrays. The Sscore algorithm was validated for sensitive detection of DEGs by comparison with existing methods. However, the prior version of the Sscore algorithm and a R-based implementation software, sscore, do not function with the latest generations of Affymetrix/Fisher microarrays due to changes in microarray design that eliminated probes previously used for estimation of non-specific binding. RESULTS: Here we describe the GCSscore algorithm, which utilizes the GC-content of a given oligonucleotide probe to estimate non-specific binding using antigenomic background probes found on new generations of arrays. We implemented this algorithm in an improved GCSscore R package for analysis of modern oligonucleotide microarrays. GCSscore has multiple methods for grouping individual probes on the ClariomD/XTA chips, providing the user with differential expression analysis at the gene-level and the exon-level. By utilizing the direct probe-level intensities, the GCSscore algorithm was able to detect DEGs under stringent statistical criteria for all Clariom-based arrays. We demonstrate that for older 3'-IVT arrays, GCSscore produced very similar differential gene expression analysis results compared to the original Sscore method. However, GCSscore functioned well for both the ClariomS and ClariomD/XTA newer microarrays and outperformed existing analysis approaches insofar as the number of DEGs and cognate biological functions identified. This was particularly striking for analysis of the highly complex ClariomD/XTA based arrays. CONCLUSIONS: The GCSscore package represents a powerful new application for analysis of the newest generation of oligonucleotide microarrays such as the ClariomS and ClariomD/XTA arrays produced by Affymetrix/Fisher.

Identifying a novel biological mechanism for alcohol addiction associated with circRNA networks acting as potential miRNA sponges.

Our lab and others have shown that chronic alcohol use leads to gene and miRNA expression changes across the mesocorticolimbic (MCL) system. Circular RNAs (circRNAs) are noncoding RNAs that form closed-loop structures and are reported to alter gene expression through miRNA sequestration, thus providing a potentially novel neurobiological mechanism for the development of alcohol dependence (AD). Genome-wide expression of circRNA was assessed in the nucleus accumbens (NAc) from 32 AD-matched cases/controls. Significant circRNAs (unadj. p ≤ 0.05) were identified via regression and clustered in circRNA networks via weighted gene co-expression network analysis (WGCNA). CircRNA interactions with previously generated mRNA and miRNA were detected via correlation and bioinformatic analyses. Significant circRNAs (N = 542) clustered in nine significant AD modules (FWER p ≤ 0.05), within which we identified 137 circRNA hubs. We detected 23 significant circRNA-miRNA-mRNA interactions (FDR ≤ 0.10). Among these, circRNA-406742 and miR-1200 significantly interact with the highest number of mRNA, including genes associated with neuronal functioning and alcohol addiction (HRAS, PRKCB, HOMER1, and PCLO). Finally, we integrate genotypic information that revealed 96 significant circRNA expression quantitative trait loci (eQTLs) (unadj. p ≤ 0.002) that showed significant enrichment within recent alcohol use disorder (AUD) and smoking genome-wide association study (GWAS). To our knowledge, this is the first study to examine the role of circRNA in the neuropathology of AD. We show that circRNAs impact mRNA expression by interacting with miRNA in the NAc of AD subjects. More importantly, we provide indirect evidence for the clinical importance of circRNA in the development of AUD by detecting a significant enrichment of our circRNA eQTLs among GWAS of substance abuse.

Assessing the Role of Long Noncoding RNA in Nucleus Accumbens in Subjects With Alcohol Dependence.

BACKGROUND: Long noncoding RNA (lncRNA) have been implicated in the etiology of alcohol use. Since lncRNA provide another layer of complexity to the transcriptome, assessing their expression in the brain is the first critical step toward understanding lncRNA functions in alcohol use and addiction. Thus, we sought to profile lncRNA expression in the nucleus accumbens (NAc) in a large postmortem alcohol brain sample. METHODS: LncRNA and protein-coding gene (PCG) expressions in the NAc from 41 subjects with alcohol dependence (AD) and 41 controls were assessed via a regression model. Weighted gene coexpression network analysis was used to identify lncRNA and PCG networks (i.e., modules) significantly correlated with AD. Within the significant modules, key network genes (i.e., hubs) were also identified. The lncRNA and PCG hubs were correlated via Pearson correlations to elucidate the potential biological functions of lncRNA. The lncRNA and PCG hubs were further integrated with GWAS data to identify expression quantitative trait loci (eQTL). RESULTS: At Bonferroni adj. p-value ≤ 0.05, we identified 19 lncRNA and 5 PCG significant modules, which were enriched for neuronal and immune-related processes. In these modules, we further identified 86 and 315 PCG and lncRNA hubs, respectively. At false discovery rate (FDR) of 10%, the correlation analyses between the lncRNA and PCG hubs revealed 3,125 positive and 1,860 negative correlations. Integration of hubs with genotype data identified 243 eQTLs affecting the expression of 39 and 204 PCG and lncRNA hubs, respectively. CONCLUSIONS: Our study identified lncRNA and gene networks significantly associated with AD in the NAc, coordinated lncRNA and mRNA coexpression changes, highlighting potentially regulatory functions for the lncRNA, and our genetic (cis-eQTL) analysis provides novel insights into the etiological mechanisms of AD.

Genome-Wide Meta-Analyses of FTND and TTFC Phenotypes.

INTRODUCTION: FTND (FagerstrÓ§m test for nicotine dependence) and TTFC (time to smoke first cigarette in the morning) are common measures of nicotine dependence (ND). However, genome-wide meta-analysis for these phenotypes has not been reported. METHODS: Genome-wide meta-analyses for FTND (N = 19,431) and TTFC (N = 18,567) phenotypes were conducted for adult smokers of European ancestry from 14 independent cohorts. RESULTS: We found that SORBS2 on 4q35 (p = 4.05 × 10-8), BG182718 on 11q22 (p = 1.02 × 10-8), and AA333164 on 14q21 (p = 4.11 × 10-9) were associated with TTFC phenotype. We attempted replication of leading candidates with independent samples (FTND, N = 7010 and TTFC, N = 10 061), however, due to limited power of the replication samples, the replication of these new loci did not reach significance. In gene-based analyses, COPB2 was found associated with FTND phenotype, and TFCP2L1, RELN, and INO80C were associated with TTFC phenotype. In pathway and network analyses, we found that the interconnected interactions among the endocytosis, regulation of actin cytoskeleton, axon guidance, MAPK signaling, and chemokine signaling pathways were involved in ND. CONCLUSIONS: Our analyses identified several promising candidates for both FTND and TTFC phenotypes, and further verification of these candidates was necessary. Candidates supported by both FTND and TTFC (CHRNA4, THSD7B, RBFOX1, and ZNF804A) were associated with addiction to alcohol, cocaine, and heroin, and were associated with autism and schizophrenia. We also identified novel pathways involved in cigarette smoking. The pathway interactions highlighted the importance of receptor recycling and internalization in ND. IMPLICATIONS: Understanding the genetic architecture of cigarette smoking and ND is critical to develop effective prevention and treatment. Our study identified novel candidates and biological pathways involved in FTND and TTFC phenotypes, and this will facilitate further investigation of these candidates and pathways.

Beyond genome-wide significance: integrative approaches to the interpretation and extension of GWAS findings for alcohol use disorder.

Alcohol use disorder (AUD) is a heritable complex behavior. Due to the highly polygenic nature of AUD, identifying genetic variants that comprise this heritable variation has proved to be challenging. With the exception of functional variants in alcohol metabolizing genes (e.g. ADH1B and ALDH2), few other candidate loci have been confidently linked to AUD. Genome-wide association studies (GWAS) of AUD and other alcohol-related phenotypes have either produced few hits with genome-wide significance or have failed to replicate on further study. These issues reinforce the complex nature of the genetic underpinnings for AUD and suggest that both GWAS studies with larger samples and additional analysis approaches that better harness the nominally significant loci in existing GWAS are needed. Here, we review approaches of interest in the post-GWAS era, including in silico functional analyses; functional partitioning of single nucleotide polymorphism heritability; aggregation of signal into genes and gene networks; and validation of identified loci, genes and gene networks in postmortem brain tissue and across species. These integrative approaches hold promise to illuminate our understanding of the biological basis of AUD; however, we recognize that the main challenge continues to be the extremely polygenic nature of AUD, which necessitates large samples to identify multiple loci associated with AUD liability.

A Whole Methylome Study of Ethanol Exposure in Brain and Blood: An Exploration of the Utility of Peripheral Blood as Proxy Tissue for Brain in Alcohol Methylation Studies.

BACKGROUND: Recent reviews have highlighted the potential use of blood-based methylation biomarkers as diagnostic and prognostic tools of current and future alcohol use and addiction. Due to the substantial overlap that often exists between methylation patterns across different tissues, including blood and brain, blood-based methylation may track methylation changes in brain; however, little work has explored the overlap in alcohol-related methylation in these tissues. METHODS: To study the effects of alcohol on the brain methylome and identify possible biomarkers of these changes in blood, we performed a methylome-wide association study in brain and blood from 40 male DBA/2J mice that received either an acute ethanol (EtOH) or saline intraperitoneal injection. To investigate all 22 million CpGs in the mouse genome, we enriched for the methylated genomic fraction using methyl-CpG binding domain (MBD) protein capture followed by next-generation sequencing (MBD-seq). We performed association tests in blood and brain separately followed by enrichment testing to determine whether there was overlapping alcohol-related methylation in the 2 tissues. RESULTS: The top result for brain was a CpG located in an intron of Ttc39b (p = 5.65 × 10-08 ), and for blood, the top result was located in Espnl (p = 5.11 × 10-08 ). Analyses implicated pathways involved in inflammation and neuronal differentiation, such as CXCR4, IL-7, and Wnt signaling. Enrichment tests indicated significant overlap among the top results in brain and blood. Pathway analyses of the overlapping genes converge on MAPKinase signaling (p = 5.6 × 10-05 ) which plays a central role in acute and chronic responses to alcohol and glutamate receptor pathways, which can regulate neuroplastic changes underlying addictive behavior. CONCLUSIONS: Overall, we have shown some methylation changes in brain and blood after acute EtOH administration and that the changes in blood partly mirror the changes in brain suggesting the potential for DNA methylation in blood to be biomarkers of alcohol use.

Nicotine Enhances the Hypnotic and Hypothermic Effects of Alcohol in the Mouse.

BACKGROUND: Ethanol (EtOH) and nicotine abuse are 2 leading causes of preventable mortality in the world, but little is known about the pharmacological mechanisms mediating co-abuse. Few studies have examined the interaction of the acute effects of EtOH and nicotine. Here, we examine the effects of nicotine administration on the duration of EtOH-induced loss of righting reflex (LORR) and characterize the nature of their pharmacological interactions in C57BL/6J mice. METHODS: We assessed the effects of EtOH and nicotine and the nature of their interaction in the LORR test using isobolographic analysis after acute injection in C57BL/6J male mice. Next, we examined the importance of receptor efficacy using nicotinic partial agonists varenicline and sazetidine. We evaluated the involvement of major nicotinic acetylcholine receptor (nAChR) subtypes using nicotinic antagonist mecamylamine and nicotinic α4- and α7-knockout mice. The selectivity of nicotine's actions on EtOH-induced LORR was examined by testing nicotine's effects on the hypnotic properties of ketamine and pentobarbital. We also assessed the development of tolerance after repeated nicotine exposure. Last, we assessed whether the effects of nicotine on EtOH-induced LORR extend to hypothermia and EtOH intake in the drinking in the dark (DID) paradigm. RESULTS: We found that acute nicotine injection enhances EtOH's hypnotic effects in a synergistic manner and that receptor efficacy plays an important role in this interaction. Furthermore, tolerance developed to the enhancement of EtOH's hypnotic effects by nicotine after repeated exposure of the drug. α4* and α7 nAChRs seem to play an important role in nicotine-EtOH interaction in the LORR test. In addition, the magnitude of EtOH-induced LORR enhancement by nicotine was more pronounced in C57BL/6J than DBA/2J mice. Furthermore, acute nicotine enhanced ketamine and pentobarbital hypnotic effects in the mouse. Finally, nicotine enhanced EtOH-induced hypothermia but decreased EtOH intake in the DID test. CONCLUSIONS: Our results demonstrate that nicotine synergistically enhances EtOH-induced LORR in the mouse.

Analysis of global changes in gene expression induced by human polynucleotide phosphorylase (hPNPase(old-35)).

As a strategy to identify gene expression changes affected by human polynucleotide phosphorylase (hPNPase(old-35)), we performed gene expression analysis of HeLa cells in which hPNPase(old-35) was overexpressed. The observed changes were then compared to those of HO-1 melanoma cells in which hPNPase(old-35) was stably knocked down. Through this analysis, 90 transcripts, which positively or negatively correlated with hPNPase(old-35) expression, were identified. The majority of these genes were associated with cell communication, cell cycle, and chromosomal organization gene ontology categories. For a number of these genes, the positive or negative correlations with hPNPase(old-35) expression were consistent with transcriptional data extracted from the TCGA (The Cancer Genome Atlas) expression datasets for colon adenocarcinoma (COAD), skin cutaneous melanoma (SKCM), ovarian serous cyst adenocarcinoma (OV), and prostate adenocarcinoma (PRAD). Further analysis comparing the gene expression changes between Ad.hPNPase(old-35) infected HO-1 melanoma cells and HeLa cells overexpressing hPNPase(old-35) under the control of a doxycycline-inducible promoter, revealed global changes in genes involved in cell cycle and mitosis. Overall, this study provides further evidence that hPNPase(old-35) is associated with global changes in cell cycle-associated genes and identifies potential gene targets for future investigation.

Identification of ethanol analgesia quantitative trait loci and candidate genes in BXD recombinant inbred mouse lines.

Alcohol consumption produces acute analgesic effects, and people experiencing pain conditions may drink alcohol to alleviate discomfort. However, tolerance to the analgesic properties of alcohol could prompt escalating consumption and dependence. Both nociception and alcohol-induced analgesia are under significant genetic control. Understanding the genetic architecture of these processes could inform better treatment options for people with pain conditions. This study aims to identify quantitative trait loci (QTL) driving variation in ethanol-induced analgesia across BXD recombinant inbred mouse lines. Male and female mice from 62 BXD strains received ethanol or saline oral gavage for five days and were tested for hot plate (HP) latency at baseline, Day 1, and Day 5. QTL mapping of HP phenotypes identified a significant provisional QTL on chromosome 17 for Day 1 HP latency in mice receiving ethanol. An additional highly suggestive QTL was present on chromosome 9 for the difference in pre- and post-ethanol thermal nociception. Candidate genes within QTL support intervals were provisionally identified using HP phenotypic correlations to transcriptomic database, expression QTL analysis, and other bioinformatics inquiries. The combined behavioral and bioinformatic analyses yielded strong ethanol analgesia candidate genes, specifically Myo6 . Thus, the results of this genetic study of ethanol-induced analgesia in BXD mouse strains may contribute significantly to our understanding of the molecular basis for individual variation in the analgesic response to acute ethanol.

Identification of novel genetic loci and candidate genes for progressive ethanol consumption in diversity outbred mice.

Mouse behavioral genetic mapping studies can identify genomic intervals modulating complex traits under well-controlled environmental conditions and have been used to study ethanol behaviors to aid in understanding genetic risk and the neurobiology of alcohol use disorder (AUD). However, historically such studies have produced large confidence intervals, thus complicating identification of potential causal candidate genes. Diversity Outbred (DO) mice offer the ability to perform high-resolution quantitative trait loci (QTL) mapping on a very genetically diverse background, thus facilitating identification of candidate genes. Here, we studied a population of 636 male DO mice with four weeks of intermittent ethanol access via a three-bottle choice procedure, producing a progressive ethanol consumption phenotype. QTL analysis identified 3 significant (Chrs 3, 4, and 12) and 13 suggestive loci for ethanol-drinking behaviors with narrow confidence intervals (1-4 Mbp for significant QTLs). Results suggested that genetic influences on initial versus progressive ethanol consumption were localized to different genomic intervals. A defined set of positional candidate genes were prioritized using haplotype analysis, identified coding polymorphisms, prefrontal cortex transcriptomics data, human GWAS data and prior rodent gene set data for ethanol or other misused substances. These candidates included Car8, the lone gene with a significant cis-eQTL within a Chr 4 QTL for week four ethanol consumption. These results represent the highest-resolution genetic mapping of ethanol consumption behaviors in mice to date, providing identification of novel loci and candidate genes for study in relation to the neurobiology of AUD.

Surgical incision pain induced an increase in alcohol consumption in mice.

INTRODUCTION: Large population-based studies have suggested a link between increased alcohol use and reduced pain. In addition, these studies suggest that higher levels of pain intensity are associated with an increase in alcohol consumption and rates of hazardous drinking which potentiates the risk of developing alcohol use disorders (AUD). The mechanisms and determinants of the alcohol-pain interaction can be studied in preclinical studies. METHODS: The overall goal of this study is to use animal models to explore the impact of acute postoperative pain on alcohol intake. To achieve this, we characterized the timeline and levels of alcohol intake and preference in mice after laparotomy in the 2-bottle choice paradigm. RESULTS: Our results show that laparotomy surgery increased alcohol intake and preference in male mice but not females in the 2-bottle choice and 3-bottle choice assays. In addition, ketoprofen administration blocked the increase in alcohol consumption in male mice after laparotomy. We also found that changes in alcohol initial sensitivity and acute functional tolerance, using loss of righting reflex (LORR) response, occur after surgery in mice. CONCLUSION: Taken together, these findings suggests that sex, pain and alcohol sensitivity-related factors may modulate the relationship between alcohol consumption and pain.

A selective GSK3ß inhibitor, tideglusib, decreases intermittent access and binge ethanol self-administration in C57BL/6J mice.

Over 10% of the US population over 12 years old meets criteria for Alcohol Use Disorder (AUD), yet few effective, long-term treatments are currently available. Glycogen synthase kinase 3 beta (GSK3ß) has been implicated in ethanol behaviors and poses as a potential therapeutic target in the treatment of AUD. Here we investigate the role of tideglusib, a selective GSK3ß inhibitor, in ethanol consumption and other behaviors. We have shown tideglusib decreases ethanol consumption in both a model of daily, progressive ethanol intake (two-bottle choice, intermittent ethanol access) and binge-like drinking behavior (drinking-in-the-dark) without effecting water intake. Further, we have shown tideglusib to have no effect on ethanol pharmacokinetics, taste preference, or anxiety-like behavior, though there was a transient increase in total locomotion following treatment. Additionally, we assessed liver health following treatment via serum levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase and showed no effect on aminotransferase levels though there was a decrease in alkaline phosphatase. RNA sequencing studies revealed a role of GSK3ß inhibition via tideglusib on the canonical Wnt signaling pathway, suggesting tideglusib may carry out its effects on ethanol consumption through effects on ß-catenin binding to the transcription factors TCF3 and LEF1. The data presented here further implicate GSK3ß in alcohol consumption and support the use of tideglusib as a potential therapeutic in the treatment of AUD.

Role of adrenal glucocorticoid signaling in prefrontal cortex gene expression and acute behavioral responses to ethanol.

BACKGROUND: Glucocorticoid hormones modulate acute and chronic behavioral and molecular responses to drugs of abuse including psychostimulants and opioids. There is growing evidence that glucocorticoids might also modulate behavioral responses to ethanol ( EtOH ). Acute EtOH activates the hypothalamic-pituitary-adrenal axis, causing the release of adrenal glucocorticoid hormones. Our prior genomic studies suggest that glucocorticoids play a role in regulating gene expression in the prefrontal cortex (PFC) of DBA2/J (D2) mice following acute EtOH administration. However, few studies have analyzed the role of glucocorticoid signaling in behavioral responses to acute EtOH . Such work could be significant, given the predictive value for the level of response to acute EtOH in the risk for alcoholism. METHODS: We studied whether the glucocorticoid receptor (GR) antagonist, RU-486, or adrenalectomy (ADX) altered male D2 mouse behavioral responses to acute (locomotor activation, anxiolysis, or loss-of-righting reflex [LORR]) or repeated (sensitization) EtOH treatment. Whole-genome microarray analysis and bioinformatics approaches were used to identify PFC candidate genes possibly responsible for altered behavioral responses to EtOH following ADX. RESULTS: ADX and RU-486 both impaired acute EtOH (2 g/kg)-induced locomotor activation in D2 mice without affecting basal locomotor activity. However, neither ADX nor RU-486 altered the initiation of EtOH sensitization (locomotor activation or jump counts), EtOH -induced anxiolysis, or LORR. ADX mice showed microarray gene expression changes in PFC that significantly overlapped with acute EtOH -responsive gene sets derived by our prior microarray studies. Q-rtPCR analysis verified that ADX decreased PFC expression of Fkbp5 while significantly increasing Gpr6 expression. In addition, high-dose RU-486 pretreatment blunted EtOH -induced Fkbp5 expression. CONCLUSIONS: Our studies suggest that EtOH 's activation of adrenal glucocorticoid release and subsequent GR activation may partially modulate EtOH 's acute locomotor activation in male D2 mice. Furthermore, because adrenal glucocorticoid basal tone regulated PFC gene expression, including a significant set of acute EtOH -responsive genes, this suggests that glucocorticoid-regulated PFC gene expression may be an important factor modulating acute behavioral responses to EtOH .

Thermal antinociceptive responses to alcohol in DBA/2J and C57BL/6J inbred male and female mouse strains.

BACKGROUND: The phenomenon of alcohol analgesia and tolerance can facilitate misuse and lead to the development of alcohol use disorder (AUD). Numerous alcohol-induced behaviors are genetically influenced; however, it is unknown if alcohol analgesia has a genetic contribution. Rodent studies have shown that alcohol responses differ vastly between two widely studied inbred strains of mice, C57BL/6 J (B6) and DBA/2 J (D2). Here, we used B6 and D2 mice as an initial behavioral genetic analysis of acute alcohol-induced antinociception. METHODS: The antinociceptive effect of orally-administered alcohol was characterized using the hot plate test in B6 and D2 mice of both sexes. Using the opioid receptor antagonist naloxone, the involvement of the opioid system was assessed. Locomotor activity and blood alcohol concentrations were also measured. Ovariectomized mice were used to evaluate the influence of ovarian sex hormones on alcohol-induced antinociception. RESULTS: Alcohol induced an antinociceptive effect in B6 and D2 male mice in a time- and dose-dependent manner. In addition, D2 male mice were more sensitive to the antinociceptive effect of alcohol than B6 male mice. However, locomotion is not impeded by the tested doses of alcohol in B6 mice. Female D2 and B6 mice failed to show significant antinociceptive effects in alcohol dose-response studies. In addition, alcohol-induced antinociception was still not evident in ovariectomized female mice. Male mice of both strains developed tolerance to this effect after repeated administration of alcohol. Strain differences were found in blood alcohol concentration. Finally, no difference was found in the blockade of alcohol antinociception by 2 mg/kg naloxone. CONCLUSION: Our results indicate that the antinociceptive effects of alcohol in the hot plate test are influenced by strain and sex. These findings support further genetic analysis of alcohol-induced antinociception to identify operative mechanisms and better assess the contribution of this phenotype to AUD.

Identification of candidate genes for nicotine withdrawal in C57BL/6J × DBA/2J recombinant inbred mice.

Nicotine is the reinforcing ingredient in tobacco. Following chronic exposure, sudden cessation of nicotine use produces negative symptoms of withdrawal that contribute to dependence. The molecular mechanisms underlying nicotine withdrawal behaviors, however, are poorly understood. Using recombinant inbred mice, chronic nicotine was delivered by minipump and withdrawal induced using mecamylamine. Somatic signs of withdrawal, and anxiety-like behavior using elevated plus maze, were then assessed. Interval mapping was used to identify associations between genetic variation and withdrawal behaviors, and with basal gene expression. Differential gene expression following nicotine exposure and withdrawal was also assessed in progenitor mice using microarrays. Quantitative trait loci mapping identified chromosome intervals with significant genetic associations to somatic signs of withdrawal or withdrawal-induced anxiety-like behavior. Using bioinformatics, and association with basal gene expression in nucleus accumbens, we implicated Rb1, Bnip3l, Pnma2, Itm2b, and Kif13b as candidate genes for somatic signs of withdrawal, and Galr1, which showed trans-regulation from a region of chromosome 14 that was associated with somatic signs of withdrawal. Candidate genes within the chromosome 9 region associated with anxiety-like withdrawal behavior included Dixdc1, Ncam1, and Sorl1. Bioinformatics identified six genes that were also significantly associated with nicotine or alcohol traits in recent human genome-wide association studies. Withdrawal-associated somatic signs and anxiety-like behavior had strong non-overlapping genetic associations, respectively, with regions of chromosome 14 and chromosome 9. Genetic, behavioral and gene expression correlations, and bioinformatics analysis identified several candidate genes that may represent novel molecular targets for modulating nicotine withdrawal symptoms.

Adolescent social housing protects against adult emotional and cognitive deficits and alters the PFC and NAc transcriptome in male and female C57BL/6J mice.

Introduction: Adolescence is a critical period in cognitive and emotional development, characterized by high levels of social interaction and increases in risk-taking behavior including binge drinking. Adolescent exposure to social stress and binge ethanol have individually been associated with the development of social, emotional, and cognitive deficits, as well as increased risk for alcohol use disorder. Disruption of cortical development by early life social stress and/or binge drinking may partly underlie these enduring emotional, cognitive, and behavioral effects. The study goal is to implement a novel neighbor housing environment to identify the effects of adolescent neighbor housing and/or binge ethanol drinking on (1) a battery of emotional and cognitive tasks (2) adult ethanol drinking behavior, and (3) the nucleus accumbens and prefrontal cortex transcriptome. Methods: Adolescent male and female C57BL/6J mice were single or neighbor housed with or without access to intermittent ethanol. One cohort underwent behavioral testing during adulthood to determine social preference, expression of anxiety-like behavior, cognitive performance, and patterns of ethanol intake. The second cohort was sacrificed in late adolescence and brain tissue was used for transcriptomics analysis. Results: As adults, single housed mice displayed decreased social interaction, deficits in the novel object recognition task, and increased anxiety-like behavior, relative to neighbor-housed mice. There was no effect of housing condition on adolescent or adult ethanol consumption. Adolescent ethanol exposure did not alter adult ethanol intake. Transcriptomics analysis revealed that adolescent housing condition and ethanol exposure resulted in differential expression of genes related to synaptic plasticity in the nucleus accumbens and genes related to methylation, the extracellular matrix and inflammation in the prefrontal cortex. Discussion: The behavioral results indicate that social interaction during adolescence via the neighbor housing model may protect against emotional, social, and cognitive deficits. In addition, the transcriptomics results suggest that these behavioral alterations may be mediated in part by dysregulation of transcription in the frontal cortex or the nucleus accumbens.

Case-only exome variation analysis of severe alcohol dependence using a multivariate hierarchical gene clustering approach.

BACKGROUND: Variation in genes involved in ethanol metabolism has been shown to influence risk for alcohol dependence (AD) including protective loss of function alleles in ethanol metabolizing genes. We therefore hypothesized that people with severe AD would exhibit different patterns of rare functional variation in genes with strong prior evidence for influencing ethanol metabolism and response when compared to genes not meeting these criteria. OBJECTIVE: Leverage a novel case only design and Whole Exome Sequencing (WES) of severe AD cases from the island of Ireland to quantify differences in functional variation between genes associated with ethanol metabolism and/or response and their matched control genes. METHODS: First, three sets of ethanol related genes were identified including those a) involved in alcohol metabolism in humans b) showing altered expression in mouse brain after alcohol exposure, and altering ethanol behavioral responses in invertebrate models. These genes of interest (GOI) sets were matched to control gene sets using multivariate hierarchical clustering of gene-level summary features from gnomAD. Using WES data from 190 individuals with severe AD, GOI were compared to matched control genes using logistic regression to detect aggregate differences in abundance of loss of function, missense, and synonymous variants, respectively. RESULTS: Three non-independent sets of 10, 117, and 359 genes were queried against control gene sets of 139, 1522, and 3360 matched genes, respectively. Significant differences were not detected in the number of functional variants in the primary set of ethanol-metabolizing genes. In both the mouse expression and invertebrate sets, we observed an increased number of synonymous variants in GOI over matched control genes. Post-hoc simulations showed the estimated effects sizes observed are unlikely to be under-estimated. CONCLUSION: The proposed method demonstrates a computationally viable and statistically appropriate approach for genetic analysis of case-only data for hypothesized gene sets supported by empirical evidence.

Multi-species data integration and gene ranking enrich significant results in an alcoholism genome-wide association study.

BACKGROUND: A variety of species and experimental designs have been used to study genetic influences on alcohol dependence, ethanol response, and related traits. Integration of these heterogeneous data can be used to produce a ranked target gene list for additional investigation. RESULTS: In this study, we performed a unique multi-species evidence-based data integration using three microarray experiments in mice or humans that generated an initial alcohol dependence (AD) related genes list, human linkage and association results, and gene sets implicated in C. elegans and Drosophila. We then used permutation and false discovery rate (FDR) analyses on the genome-wide association studies (GWAS) dataset from the Collaborative Study on the Genetics of Alcoholism (COGA) to evaluate the ranking results and weighting matrices. We found one weighting score matrix could increase FDR based q-values for a list of 47 genes with a score greater than 2. Our follow up functional enrichment tests revealed these genes were primarily involved in brain responses to ethanol and neural adaptations occurring with alcoholism. CONCLUSIONS: These results, along with our experimental validation of specific genes in mice, C. elegans and Drosophila, suggest that a cross-species evidence-based approach is useful to identify candidate genes contributing to alcoholism.

Systems genetic analysis of the effects of iron deficiency in mouse brain.

Iron regulation in the brain is both necessary and highly complex. Too little or too much iron can compromise neurological function, yet we still do not know all of the regulatory processes. In our research, we seek to identify genes and gene networks underlying individual differences in brain iron regulation. To this end, we fed mice from 20+ inbred strains a diet low in iron from weaning to 4 months of age. At sacrifice, we measured iron content in the ventral midbrain (VMB). The VMB contains the substantia nigra, a region particularly vulnerable to iron imbalance. The results showed high, inter-strain variability in dietary iron reduction, from almost no loss to more than 40 % vs. control. When we performed quantitative trait loci (QTL) analysis, we observed a significant area on chromosome 2. Within this QTL, we selected glial high-affinity glutamate transporter 1 (Glt1) as the leading candidate. Expression of this gene is both correlated with VMB iron and is also cis-modulated by local sequence variants that segregate in the BXD family. VMB expression differences of Glt1 in six strains covary with differential susceptibility to VMB iron loss.