Molecular Profiles of Drinking Alcohol to Intoxication in C57BL/6J Mice

University of Texas at Austin, Waggoner Center for Alcohol and Addiction Research, Austin, Texas, USA.
Alcoholism Clinical and Experimental Research (Impact Factor: 3.21). 04/2011; 35(4):659-70. DOI: 10.1111/j.1530-0277.2010.01384.x
Source: PubMed


Alcohol addiction develops through a series of stages, and mechanistic studies are needed to understand the transition from initial drug use to sustained controlled alcohol consumption followed by abuse and physical dependence. The focus of this study was to examine the effects of voluntary alcohol consumption on brain gene expression profiles using a mouse model of binge drinking. The main goal was to identify alcohol-responsive genes and functional categories after a single episode of drinking to intoxication.
We used a modification of a "Drinking In the Dark" (DID) procedure (Rhodes et al., 2005) that allows mice to experience physiologically relevant amounts of alcohol in a non-stressful environment and also allows for detection of alcohol-sensitive molecular changes in a dose-dependent manner. C57BL/6J male mice were exposed to either 20% ethanol solution or water (single bottle) starting 3 hours after lights off for 4 hours and brains were harvested immediately after the drinking session. cDNA microarrays were used to assess the effects of voluntary drinking on global gene expression in 6 brain regions. We employed three statistical approaches to analyze microarray data.
A commonly used approach that applies a strict statistical threshold identified the eight top statistically significant genes whose expression was significantly correlated with blood ethanol concentration (BEC) in one of the brain regions. We then used a systems network approach to examine brain region-specific transcriptomes and identify modules of co-expressed (correlated) genes. In each brain region, we identified alcohol-responsive modules, i.e., modules significantly enriched for genes whose expression was correlated with BEC. A functional over-representation analysis was then applied to examine the organizing principles of alcohol-responsive modules. Genes were clustered into modules according to their roles in different physiological processes, functional groups, and cell types, including blood circulation, signal transduction, cell-cell communication, and striatal neurons. Finally, a meta-analysis across all brain regions suggested a global role of increasing alcohol dose in coordination of brain blood circulation and reaction of astrocytes.
This study showed that acute drinking resulted in small but consistent changes in brain gene expression which occurred in a dose-dependent manner. We identified both general and region-specific changes, some of which represent adaptive changes in response to increasing alcohol dose, which may play a role in alcohol-related behaviours, such as tolerance and consumption. Our systems approach allowed us to estimate the functional values of individual genes in the context of their genetic networks and formulate new refined hypotheses. An integrative analysis including other alcohol studies suggested several top candidates for functional validation, including Mt2, Gstm1, Scn4b, Prkcz, and Park7.

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Available from: Robert Adron Harris, Oct 09, 2015
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    • "Using a similar approach we compiled findings from several transcriptional profiling studies that have identified differentially expressed genes from alcohol-related studies on D. melanogaster (Morozova et al. 2006, 2007, 2009, 2011; Urizar et al. 2007; Awofala 2010; Kong et al. 2010); from alcohol-related expression studies done on mice (Xu et al. 2001; Daniels and Buck 2002; Tabakoff et al. 2003; Hitzemann et al. 2004; Saito et al. 2004; Treadwell and Singh 2004; Kerns et al. 2005; MacLaren et al. 2006; Mulligan et al. 2006, 2011; Saba et al. 2006; Wang et al. 2007; Denmark and Buck 2008; Wolstenholme et al. 2011), and transcriptional profiling data on rats (Rimondini et al. 2002; Edenberg et al. 2005; Worst et al. 2005; Carr et al. 2007; Kimpel et al. 2007; Rodd et al. 2008), and identified human orthologs. In addition, we analyzed six published transcriptional profiling data sets performed on different areas of postmortem human brains and also included candidate genes for alcohol-related phenotypes from the HuGE Navigator database (Lewohl et al. 2000; Mayfield et al. 2002; Sokolov et al. 2003; Iwamoto et al. 2004; Flatscher-Bader et al. 2005; Liu et al. 2006a; Guo et al. 2009). "
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    ABSTRACT: Alcohol abuse and alcoholism incur a heavy socioeconomic cost in many countries. Both genetic and environmental factors contribute to variation in the inebriating effects of alcohol and alcohol addiction among individuals within and across populations. From a genetics perspective, alcohol sensitivity is a quantitative trait determined by the cumulative effects of multiple segregating genes and their interactions with the environment. This review summarizes insights from model organisms as well as human populations that represent our current understanding of the genetic and genomic underpinnings that govern alcohol metabolism and the sedative and addictive effects of alcohol on the nervous system. Electronic supplementary material The online version of this article (doi:10.1007/s00438-013-0808-y) contains supplementary material, which is available to authorized users.
    MGG Molecular & General Genetics 01/2014; 289(3). DOI:10.1007/s00438-013-0808-y · 2.73 Impact Factor
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    • "Many differentially regulated proteins from the present study have been described by previous genomic and proteomic studies in human alcoholic brain [15], [29]–[32] and rat [33]–[37] and mouse [38], [39] brain from animal models of alcohol abuse. Furthermore, some of the differentially expressed proteins reported here (i.e., DYN1, HSP7C, STXB1) were identified in our previous PPI study [40] as interacting partners of the BK channel, a well-established alcohol target which is important in behavioral and molecular tolerance to ethanol [41], [42]. "
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    ABSTRACT: Alcohol abuse causes dramatic neuroadaptations in the brain, which contribute to tolerance, dependence, and behavioral modifications. Previous proteomic studies in human alcoholics and animal models have identified candidate alcoholism-related proteins. However, recent evidences suggest that alcohol dependence is caused by changes in co-regulation that are invisible to single protein-based analysis. Here, we analyze global proteomics data to integrate differential expression, co-expression networks, and gene annotations to unveil key neurobiological rearrangements associated with the transition to alcohol dependence modeled by a Chronic Intermittent Ethanol (CIE), two-bottle choice (2BC) paradigm. We analyzed cerebral cortices (CTX) and midbrains (MB) from male C57BL/6J mice subjected to a CIE, 2BC paradigm, which induces heavy drinking and represents one of the best available animal models for alcohol dependence and relapse drinking. CIE induced significant changes in protein levels in dependent mice compared with their non-dependent controls. Multiple protein isoforms showed region-specific differential regulation as a result of post-translational modifications. Our integrative analysis identified modules of co-expressed proteins that were highly correlated with CIE treatment. We found that modules most related to the effects of CIE treatment coordinate molecular imbalances in endocytic- and energy-related pathways, with specific proteins involved, such as dynamin-1. The qRT-PCR experiments validated both differential and co-expression analyses, and the correspondence among our data and previous genomic and proteomic studies in humans and rodents substantiates our findings. The changes identified above may play a key role in the escalation of ethanol consumption associated with dependence. Our approach to alcohol addiction will advance knowledge of brain remodeling mechanisms and adaptive changes in response to drug abuse, contribute to understanding of organizational principles of CTX and MB proteomes, and define potential new molecular targets for treating alcohol addiction. The integrative analysis employed here highlight the advantages of systems approaches in studying the neurobiology of alcohol addiction.
    PLoS ONE 12/2013; 8(12):e82656. DOI:10.1371/journal.pone.0082656 · 3.23 Impact Factor
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    • "In a 2-or 4-h drinking session, mice typically reach BECs higher than 80 mg/dl and show signs of behavioral intoxication such as motor impairment (Rhodes et al, 2007). Its use is growing and more and more studies are applying this paradigm to investigate neuronal circuits and signaling molecules affected by ethanol drinking (Mulligan et al, 2011; Cozzoli et al, 2012; Sprow and Thiele, 2012). Despite its success in inducing voluntary binge-like ethanol drinking, the behavioral mechanisms underlying the acquisition of this behavior are not understood. "
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    ABSTRACT: Repeated cycles of binge alcohol drinking and abstinence are key components in the development of dependence. However, the precise behavioral mechanisms underlying binge-like drinking and its consequences on striatal synaptic physiology remain unclear. In the present study, ethanol and water drinking patterns were recorded with high temporal resolution over 6 weeks of binge-like ethanol drinking using the 'drinking in the dark' (DID) protocol. The bottle exchange occurring at the beginning of each session prompted a transient increase in the drinking rate that might facilitate the acquisition of ethanol binge-like drinking. Ethanol drinking mice also displayed a 'frontloading' behavior in which the highest rate of drinking was recorded during the first 15 min. This rate increased over weeks and paralleled the mild escalation of blood ethanol concentrations. GABAergic and glutamatergic transmission in the dorsal striatum were examined following DID. Spontaneous glutamatergic transmission and the density of dendritic spines were unchanged after ethanol drinking. However, the frequency of GABAA receptor-mediated inhibitory postsynaptic currents was depressed in medium spiny neurons of ethanol drinking mice. A history of ethanol drinking also increased ethanol preference and altered the acute ethanol effects on GABAergic transmission differentially in dorsolateral and dorsomedial striatum. Together, the study shows that the bottle exchange during DID promotes fast, voluntary ethanol drinking and that this intermittent pattern of ethanol drinking causes a depression of GABAergic transmission in the dorsal striatum.Neuropsychopharmacology accepted article preview online, 2 September 2013. doi:10.1038/npp.2013.230.
    Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 09/2013; 39(3). DOI:10.1038/npp.2013.230 · 7.05 Impact Factor
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