Project description:Ethanol is the most common substance of abuse in the US, and abuse can lead to physical dependence, addiction, brain damage and premature death. The cycle of alcohol addiction has been described as a composite consisting of three stages: intoxication, withdrawal and craving/abstinence. As a complex brain disorder, there is evidence for both a genetic contribution to risk and sexually-dimorphic responses in alcoholism, but an overall understanding of the biological contributions and the neuroadaptive underpinnings of alcohol addiction is limited. Utilizing novel genetic animal models with highly divergent withdrawal severity, Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) selected lines of mice and by examining both sexes, the distinct or common contributions of response to alcohol genotype/phenotype and of sex to addiction stages over time were characterized. Transcriptional profiling was performed to identify neuroadaptive changes as a consequence of chronic intoxication in the medial prefrontal cortex (mPFC). Significant expression differences were identified for each line and tracked over a behaviorally-relevant time course that covered each stage of alcohol addiction; i.e., after chronic intoxication, during peak withdrawal, and after a defined period of abstinence. Females were more responsive to ethanol with higher fold expression differences. Data structure was analyzed by bioinformatics, which showed a strong effect of sex with high similarity of male vs. female expression profiles during chronic intoxication and at peak withdrawal irrespective of genetic background. However, during abstinence, striking differences were observed instead between the lines/phenotypes irrespective of sex. Because sex was the strongest influence on neuroadaptive changes overall, confirmation analysis compared males vs. females. Notably, results revealed distinct inflammatory signaling between males and females at peak withdrawal, with a pro-inflammatory inflammotoxic phenotype in females but in contrast overall suppression of immune signaling in males. Thus, the early response to chronic intoxication is strongly influenced by sex while pathways that are altered during a period of abstinence are dependent on genotype. Combined, these results suggest that each stage of the addiction cycle is influenced differentially by sex vs. genetic background and support the development of distinct translational targets for stage- and sex-specific therapies for the treatment of alcohol withdrawal and the maintenance of sobriety.

Project description:Ethanol is the most common substance of abuse in the US, and abuse can lead to physical dependence, addiction, brain damage and premature death. The cycle of alcohol addiction has been described as a composite consisting of three stages: intoxication, withdrawal and craving/abstinence. As a complex brain disorder, there is evidence for both a genetic contribution to risk and sexually-dimorphic responses in alcoholism, but an overall understanding of the biological contributions and the neuroadaptive underpinnings of alcohol addiction is limited. Utilizing novel genetic animal models with highly divergent withdrawal severity, Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) selected lines of mice and by examining both sexes, the distinct or common contributions of response to alcohol genotype/phenotype and of sex to addiction stages over time were characterized. Transcriptional profiling was performed to identify neuroadaptive changes as a consequence of chronic intoxication in the medial prefrontal cortex (mPFC). Significant expression differences were identified for each line and tracked over a behaviorally-relevant time course that covered each stage of alcohol addiction; i.e., after chronic intoxication, during peak withdrawal, and after a defined period of abstinence. Females were more responsive to ethanol with higher fold expression differences. Data structure was analyzed by bioinformatics, which showed a strong effect of sex with high similarity of male vs. female expression profiles during chronic intoxication and at peak withdrawal irrespective of genetic background. However, during abstinence, striking differences were observed instead between the lines/phenotypes irrespective of sex. Because sex was the strongest influence on neuroadaptive changes overall, confirmation analysis compared males vs. females. Notably, results revealed distinct inflammatory signaling between males and females at peak withdrawal, with a pro-inflammatory inflammotoxic phenotype in females but in contrast overall suppression of immune signaling in males. Thus, the early response to chronic intoxication is strongly influenced by sex while pathways that are altered during a period of abstinence are dependent on genotype. Combined, these results suggest that each stage of the addiction cycle is influenced differentially by sex vs. genetic background and support the development of distinct translational targets for stage- and sex-specific therapies for the treatment of alcohol withdrawal and the maintenance of sobriety. A total of 32 microarrays were run with 4 biological replicates per treatment, line, and sex. Selection replicates (i.e. WSP-1 and WSP-2) for each treatment, line, and sex were collapsed to improve statistical power (n=4) and to facilitate in the identification of phenotype related effects and exclude selection artifacts. For comparisons, EtOH regulation was determined by comparing 4 arrays from (for example) Male WSR EtOH treated versus 4 arrays from Male WSR Air treated arrays.

Project description:Alcoholism is a relapsing disorder associated with excessive consumption after periods of abstinence. Neuroadaptations in brain structure, plasticity and gene expression occur with chronic intoxication but are poorly characterized. Here we report identification of pathways altered during abstinence in prefrontal cortex, a brain region associated with cognitive dysfunction and damage in alcoholics. To determine the influence of genetic differences, an animal model was employed with widely divergent responses to alcohol withdrawal, the Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) lines. Mice were chronically exposed to highly intoxicating concentrations of ethanol and withdrawn, then left abstinent for 21 days. Transcriptional profiling by microarray analyses identified a total of 562 genes as significantly altered during abstinence. Hierarchical cluster analysis revealed that the transcriptional response correlated with genotype/withdrawal phenotype rather than sex. Gene Ontology category overrepresentation analysis identified thyroid hormone metabolism, glutathione metabolism, axon guidance and DNA damage response as targeted classes of genes in low response WSR mice, with acetylation and histone deacetylase complex as highly dimorphic between WSR and WSP mice. Confirmation studies in WSR mice revealed both increased neurotoxicity by histopathologic examination and elevated triidothyronine (T3) levels. Most importantly, relapse drinking was reduced by inhibition of thyroid hormone synthesis in dependent WSR mice compared to controls. These findings provide in vivo physiological and behavioral validation of the pathways identified. Combined, these results indicate a fundamentally distinct neuroadaptive response during abstinence in mice genetically selected for divergent withdrawal severity. Identification of pathways altered in abstinence may aid development of novel therapeutics for targeted treatment of relapse in abstinent alcoholics. A total of 32 microarrays were run with 4 biological replicates per treatment, line, and sex. Selection replicates (i.e. WSP-1 and WSP-2) for each treatment, line, and sex were collapsed to improve statistical power (n=4) and to facilitate in the identification of phenotype related effects and exclude selection artifacts. For comparisons, EtOH regulation was determined by comparing 4 arrays from (for example) Male WSR EtOH treated versus 4 arrays from Male WSR Air treated arrays.

Project description:Alcoholism is a relapsing disorder associated with excessive consumption after periods of abstinence. Neuroadaptations in brain structure, plasticity and gene expression occur with chronic intoxication but are poorly characterized. Here we report identification of pathways altered during abstinence in prefrontal cortex, a brain region associated with cognitive dysfunction and damage in alcoholics. To determine the influence of genetic differences, an animal model was employed with widely divergent responses to alcohol withdrawal, the Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) lines. Mice were chronically exposed to highly intoxicating concentrations of ethanol and withdrawn, then left abstinent for 21 days. Transcriptional profiling by microarray analyses identified a total of 562 genes as significantly altered during abstinence. Hierarchical cluster analysis revealed that the transcriptional response correlated with genotype/withdrawal phenotype rather than sex. Gene Ontology category overrepresentation analysis identified thyroid hormone metabolism, glutathione metabolism, axon guidance and DNA damage response as targeted classes of genes in low response WSR mice, with acetylation and histone deacetylase complex as highly dimorphic between WSR and WSP mice. Confirmation studies in WSR mice revealed both increased neurotoxicity by histopathologic examination and elevated triidothyronine (T3) levels. Most importantly, relapse drinking was reduced by inhibition of thyroid hormone synthesis in dependent WSR mice compared to controls. These findings provide in vivo physiological and behavioral validation of the pathways identified. Combined, these results indicate a fundamentally distinct neuroadaptive response during abstinence in mice genetically selected for divergent withdrawal severity. Identification of pathways altered in abstinence may aid development of novel therapeutics for targeted treatment of relapse in abstinent alcoholics.

Project description:Alcohol use disorder (AUD) is a chronic mental illness in which patients often achieve protracted periods of abstinence prior to relapse. Epigenetic mechanisms may provide an explanation for the persisting gene expression changes that can be observed even after long periods of abstinence and may contribute to relapse. In this study we examined two stable histone modifications, histone 3 lysine 4 tri-methylation (H3K4me3) and histone 3 lysine 27 tri-methylation (H3K27me3), in the prefrontal cortex of Withdrawal Seizure Resistant (WSR) mice 21 days after 72 hours of ethanol vapor exposure. These histone modifications were selected because they are associated with active promoters (H3K4me3) and repressed gene expression in a euchromatic environment (H3K27me3). We performed a genome-wide analysis to identify differences in H3K4me3 and H3K27me3 levels in post-ethanol exposure vs. control mice by ChIP-seq. We detected a global reduction in H3K4me3 peaks and increase in H3K27me3 peaks in post-ethanol exposure mice compared to controls, these changes are consistent with persistent reductions in gene expression and suggest differential epigenetic regulation of genes during this post-exposure period. We also examined the correspondence between genes that displayed changes in H3K4me3 and/or H3K27me3 and were differentially expressed. The expression of 52% of the genes with altered H3K4me3 binding and 40% of genes with H3K27me3 differences found here is altered by ethanol exposure based on analysis of prior studies. Finally, pathway analysis of genes displaying changes in H3K4me3 and H3K27me3 revealed enrichment for genes involved in proteoglycan and calcium signaling pathways, respectively. The chromatin changes associated with the 21-day post-exposure period suggest that this period is a unique state in the addiction cycle that differs from ethanol intoxication and acute withdrawal. These results provide insights into the enduring effects of ethanol on proteoglycan and calcium signaling genes in the brain.

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