Project description:While women are more vulnerable than men to many of the medical consequences of alcohol abuse, the role of sex in the response to ethanol is controversial. Neuroadaptive responses that result in the hyperexcitability associated with withdrawal from chronic ethanol likely reflect gene expression changes. We have examined both genders for the effects of withdrawal on brain gene expression using mice with divergent withdrawal severity that have been selectively bred from a genetically heterogeneous population. A total of 295 genes were identified as ethanol regulated from each gender of each selected line by microarray analyses. Hierarchical cluster analysis of the arrays revealed that the transcriptional response correlated with sex rather than with the selected withdrawal phenotype. Consistent with this, gene ontology category over-representation analysis identified cell death and DNA/RNA binding as targeted classes of genes in females, while in males, protein degradation, and calcium ion binding pathways weremore altered by alcohol. Examination of ethanol regulated genes and these distinct signaling pathways suggested enhanced neurotoxicity in females. Histopathological analysis of brain damage following ethanol withdrawal confirmed elevated cell death in female but not male mice. The sexually dimorphic response was observed irrespective of withdrawal phenotype. Combined, these results indicate a fundamentally distinct neuroadaptive response in females compared to males during chronic ethanol withdrawal and are consistent with observations that female alcoholics may be more vulnerable than males to ethanol-induced brain damage associated with alcohol abuse. 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:Lasting behavioral and physiological changes such as abusive consumption, dependence, and withdrawal are characteristic features of alcohol use disorders (AUD). Mechanistically, persistent changes in gene expression are hypothesized to contribute to these brain adaptations leading to ethanol toxicity and abuse. Here we employed repeated chronic intermittent ethanol (CIE) exposure by vapor chamber as a mouse model to simulate the cycles of ethanol exposure and withdrawal commonly seen with AUD. This model has previously been shown to induce progressive ethanol consumption in rodents. Brain regional expression networks contributing to CIE-induced behavioral changes were identified by microarray analysis across five brain regions in the mesolimbic dopamine system and extended amygdala with tissue harvested from 0-120 hours following the last cycle of CIE. Weighted Gene Correlated Network Analysis (WGCNA) was used to identify gene networks over-represented for CIE-induced temporal expression changes across brain regions. Differential gene expression analysis of CIE vs. air-treated controls showed that long-lasting gene regulation occurred 5-days after the final cycle of ethanol exposure only in prefrontal cortex (PFC) and hippocampus. In the majority of brain-regions, however, ethanol regulated gene expression changes occurred only immediately following CIE or within the first 8-hours of removal from ethanol. Bioinformatics analysis of modules identified by WGCNA showed that neuroinflammatory responses were seen across multiple brain regions at early time-points, whereas co-expression modules related to neuroplasticity, chromatin remodeling, and neurodevelopment were seen at later time-points and in specific brain regions (PFC or HPC). In PFC a module containing Bdnf was identified as highly CIE responsive in a biphasic manner, with peak changes at 0 hours and 5 days following CIE, suggesting a possible role in mechanisms underlying long-term molecular and behavioral response to CIE. Strikingly, bioinformatics analysis of this network and several other modules identified Let-7 family microRNAs as potential regulators of gene expression changes induced by CIE. Our results suggest a complex temporal and regional pattern of widespread gene network responses involving neuroinflammatory and neuroplasticity related genes as contributing to physiological and behavioral responses to chronic ethanol. In particular, our identification of a potential role for Let-7 miRNAs and a Bdnf-related expression network in long-lasting expression changes after CIE may lead to future druggable gene target identification for novel intervention in AUD.

Project description:Purpose: Genetic factors significantly affect alcohol consumption and vulnerability to withdrawal. Some genetic models that show predisposition to severe withdrawal are also predisposed to low ethanol consumption and vice versa, even when tested independently in naïve animals. The study was undertaken to understand gene and network differences between mice selectively bred for high withdrawal/low withdrawal or low withdrawal/high consumption. Methods: Beginning with a C57BL/6J×DBA/2J F2 intercross founder population, animals were simultaneously selectively bred for both high alcohol consumption and low acute withdrawal (SOT line), or vice versa (NOT line). Using randomly chosen fourth selected generation (S4) mice (N= 18-22/sex/line), RNA-Seq was employed to assess genome-wide gene expression in the ventral striatum. The Mega-MUGA array was used to detect genome-wide genotypic differences. Differential gene expression and the weighted gene co-expression network analysis (WGCNA) were implemented. Results: The new selection of the SOT and NOT lines was similar to that reported previously (Metten et al. 2014). One thousand eight hundred and fifteen transcripts were detected as being differentially expressed between the lines. For the genes overexpressed in the SOT line there was enrichment in genes associated with cell adhesion, synapse organization and post-synaptic membrane. The genes with a cell adhesion annotation included 23 protocadherins, Mpdz & Dlg2. Genes with a postsynaptic membrane annotation included Gabrb3, Gphn, Grid1, Grin2b, Grin2c & Grm3. The genes overexpressed in the NOT line were enriched in a network module (red) with annotations associated with mitochondrial function. Several of these genes were module hub nodes and these included Nedd8, Guk1, Elof1, Ndufa8, & Atp6v1f. Conclusions: Marked effects of selection on gene expression were detected. The NOT line was characterized by the increased expression of hub nodes associated with mitochondrial function. Genes overexpressed in the SOT aligned with previous findings e.g. Colville et al. (2017) that high ethanol preference and consumption is associated with effects on cell adhesion and glutamate synaptic plasticity.

Project description:Purpose: Fetal alcohol spectrum disorders (FASD) result from ethanol exposure to the developing fetus. FASD occur in up to 1-5% of live births in the United States and there currently is no cure. Ethanol exposure to the developing central nervous system (CNS) has profound effects on learning and memory, impulse control, and motor function, resulting from neuropathology. The purpose of the current study was to perform transcriptome analysis to evaluate the effects of early postnatal ethanol exposure in the hippocampus and cerebellum. Methods: Postnatal C57BL/6 mice were treated with 4g/kg of ethanol from P4-P9, brains were harvested 24h after the final treatment at P10, hippocampi and cerebella were microdissected, RNA was isolated, and RNASeq analysis was performed. We compared differences in gene expression, sex-dependent expression, and global biological pathways associated with disruptions in hippocampal and cerebellar genes between the ethanol and vehicle treated neonates. Results: Ethanol caused both an up and down regulation of genes associated with the hippocampus and cerebellum, which may result in the disruption of normal circuitry and maturation and growth in these brain regions. Ethanol increased the expression of genes associated with the S phase of the cell cycle in both the hippocampus and cerebellum. In the cerebellum, ethanol increased effector gene expression, and in the hippocampus, genes associated with different interneuron lineages were altered. Postnatal ethanol exposure also resulted in altered expression of genes associated with oligodendrocyte lineages and myelination, along with alterations in microglia associated genes. Conclusion: Collectively, these data indicate that ethanol has profound effects on the hippocampus and cerebellum, resulting in alterations of gene expression and biological pathways regulating neurodevelopment. These studies may have important implications concerning alcohol-induced neuropathology and the neurological effects seen across the life span in FASD.

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RNA-sequencing in rat hippocampus following chronic ethanol and withdrawal

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