BioProject

Lasting behavioral and physiological changes such as abusive consumption, dependence, and withdrawal are characteristic features of alcohol use disorders (AUD). More...

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. Overall design: Adult male C57BL/6J mice (n=48) received either ethanol vapor exposure (CIE) in plexiglass inhalation chambers, or only air exposure in the inhalation chambers (Ctrl). Ethanol vapor concentrations were monitored daily and air flow was adjusted to maintain ethanol concentrations within a range (10-13 mg/l air). Before each chronic ethanol exposure cycle, intoxication was initiated in the CIE group by administration of ethanol (1.6 g/kg), and blood ethanol concentration was stabilized by injection of the alcohol dehydrogenase inhibitor pyrazole (1 mmol/kg). Both ethanol and pyrazole were administered intraperitoneally (i.p.) in a volume of 0.02 ml/g body weight. Ctrl mice were handled similarly, but administered saline and pyrazole (i.p.) prior to being placed in air only chambers. Mice spent 4 days in the inhalation chamber for 16hr/day. Following 4 days in the inhalation chamber mice underwent 7 days of complete abstinence from ethanol. At the end of the abstinence period, mice were returned to the inhalation chamber to begin the next cycle of CIE. This pattern of 4 days CIE (or air) exposure followed by 7 days abstinence was repeated for four complete cycles. Immediately following the last cycle of air or ethanol exposure as above, mice were removed from the inhalation chambers and euthanized at the appropriate time point by decapitation. Time points collected were 0, 8, and 72 hours (h) and 7 days (d), with n=6 for each treatment/time group. Gene expression was quantified with Affymetrix GeneChip Mouse Genome 430 2.0 arrays. Scanning data was stored in CEL file format using Affymetrix Expression Console software. Microarray data was analyzed using RMA normalization; ComBat to minimize batch effects; linear models for microarrays (LIMMA) to determine differences in gene expression between CIE and Ctrl groups; and Weighted Gene Correlated Network Analysis (WGCNA) to identify modules of co-expressed genes representing specific biological pathways. Modules were overlapped with genes differentially expressed between CIE and Ctrl mice at each time-point to identify modules regulated at specific times after CIE. Open source bioinformatics resources were further used to determine biological pathways represented by WGCNA modules. Less...