Chapter Nine - Genes and Alcohol Consumption: Studies with Mutant Mice
Introduction
Alcohol use disorder (AUD) is a multifactorial disease, and its risk factors are determined by the interplay of genetic and environmental factors, combined with neuroadaptations following acute and repeated alcohol exposure. Alcohol targets ion channels and signaling cascades, producing intoxication, anxiolysis, and a sense of reward. After prolonged, repeated exposure, alcohol-induced changes in gene expression and synaptic function are thought to contribute to the development tolerance, sensitization, and compulsive consumption and drug seeking.
More than 100 genes have been shown to affect alcohol consumption and other alcohol-related behaviors in mouse models (Crabbe, Phillips, Harris, Arends, & Koob, 2006). Excessive alcohol consumption is a common model of addictive behavior, and animal models of voluntary self-administration are valuable for profiling genetic determinants of AUD (Green & Grahame, 2008). The preference to drink alcohol is a reliable measure that depends upon mouse genotype and has been consistent across laboratories despite variations of the drinking protocol used. Preclinical models, in conjunction with human genetic studies, may expose overlapping target genes and identify the most relevant drinking models and biological systems associated with AUD.
In this review, we focus on a single phenotype, voluntary alcohol self-administration, and summarize the global genetic manipulations in mice published to date on this behavior. Most of the studies used two-bottle choice (2BC) tests, where mice had a choice between water and ethanol and access was usually measured in continuous 24-h periods. In some cases, 2BC access to ethanol was intermittent (eg, every other day), which typically (Hwa et al., 2011, Melendez, 2011, Rosenwasser et al., 2013), but not always (Crabbe, Harkness, Spence, Huang, & Metten, 2012), results in higher ethanol intake compared to continuous access. A few studies used four-bottle choice (4BC) access, where mice have simultaneous access to water and three different concentrations of ethanol. Because rodents distribute their drinking across the circadian cycle and because limited access to ethanol tends to increase intake, restricted access during the dark cycle is often used to study periods of high consumption and to model binge-like drinking in humans (Thiele & Navarro, 2014). In the classic mouse drinking in the dark (DID) test, drinking session times begin a few hours after the start of the dark cycle and usually last 2–4 h over a few days. High levels of ethanol drinking and pharmacologically relevant blood ethanol concentrations (BECs) are achieved using this model (Thiele & Navarro, 2014). The scheduled high access consumption (SHAC) test uses fluid restriction to promote drinking of a low ethanol concentration (Finn et al., 2005). This chronic drinking model can also produce high BECs. Fluid access is first restricted and then gradually relaxed until the effects of fluid limitation are minimized. In operant self-administration tests, mice are trained to self-administer quantities of ethanol that produce moderate to high BECs. Removal of access to alcohol followed by restored access transiently increases consumption in dependent mice, and the effects of mutant genes on this alcohol deprivation effect (ADE; Rodd et al., 2004, Vengeliene et al., 2014), a model of relapse drinking, are also presented. As shown in the tables throughout the chapter, the effects of some mutants can depend on the drinking test used as well as the ethanol concentration, time of access, genetic background, and sex of the mice (Vanderlinden, Saba, Bennett, Hoffman, & Tabakoff, 2015).
There are important considerations regarding genetic engineering methods, including the potential alteration of genes other than the mutated gene and the influence of the background strain carrying the genotype. C57BL/6J (B6) mice are a high alcohol-drinking strain and, as the tables in this chapter demonstrate, occupy a central role in voluntary drinking studies. This review focuses on global homozygous knockouts, although a few studies used hypofunctional or overexpressing transgenic lines. Strategies to reduce confounding and compensatory effects of null mutations include the use of knockin mice and brain regional or cell-specific knockouts, and a few of these studies are noted. In this review, we do not debate the genetic engineering methods used; instead, our aim is to provide a summary of homozygous null or overexpressing mutants and their role (or lack thereof) in alcohol consumption in mice. Current mouse gene and protein names from Uniprot (http://www.uniprot.org) are listed in the tables, which in some cases differ from the nomenclature used in the published studies. We first review the effects of mutant neurotransmitter receptor subunits on alcohol drinking in mice and then examine mutations in other ion channel receptors, cannabinoid and opioid receptors, neuropeptides, kinases/enzymes, and immune-related genes.
Section snippets
γ-Aminobutyric Acid
Alcohol potentiates γ-aminobutyric acid type A (GABAA) receptor-mediated responses and enhances inhibitory neurotransmission, and some of the top candidate genes implicated in alcohol consumption code for specific GABAA receptor subunits (Trudell, Messing, Mayfield, & Harris, 2014). Deletion of the α1 subunit decreased ethanol consumption in operant and 2BC tests (Blednov et al., 2003, June et al., 2007), and knockdown of α5 reduced drinking in male (Boehm, Ponomarev, et al., 2004) but not
Cannabinioids and Opioids
The endocannabinoid system is involved in brain reward signaling and drug-seeking behavior (Panagis, Mackey, & Vlachou, 2014). In several studies of different genetic backgrounds, male and female cannabinoid 1 receptor (CB1R) knockout mice showed reduced ethanol intake and/or preference for ethanol than wild-type mice (Hungund et al., 2003, Lallemand and de Witte, 2005, Naassila et al., 2004, Poncelet et al., 2003, Racz et al., 2003, Thanos, Dimitrakakis, et al., 2005, Vinod, Yalamanchili, et
Immune-Related Genes
The interplay between brain, behavior, and immune responses in the etiology and progression of drug abuse is a current area of interest in addiction research (http://www.arcr.niaaa.nih.gov/arcr/arcr372/toc37_2.htm). The neuroimmune system, encompassing innate immune signaling within the peripheral and central nervous systems, is important in the pathophysiology and potential treatment of alcohol abuse and dependence (Crews and Vetreno, 2015, Mayfield et al., 2013, Robinson et al., 2014).
Ion Channels
Some of the rapid-onset actions of alcohol are likely mediated by direct action on ion channels (Howard et al., 2014, Trudell et al., 2014). In addition to the prominent and well-studied neurotransmitter systems in alcohol dependence discussed previously (eg, GABA, glutamate), other ion channels that have been implicated in alcohol intake and preference are described below.
Protein Kinases
In addition to direct effects on ion channels, ethanol indirectly modulates channel function via phosphorylation and other posttranslational processing mechanisms (Trudell et al., 2014). Mice lacking protein kinase C type ɛ (PKCɛ) drank less ethanol than wild type, and this effect has been observed in different drinking tests across multiple labs (Besheer et al., 2006, Choi et al., 2002, Hodge et al., 1999, Olive et al., 2005, Olive et al., 2000, Wallace et al., 2007; Table 9). Selective
Enzymes
Table 10 shows the effects of other assorted enzymes on ethanol drinking in mice. In particular, aldehyde dehydrogenase (ALDH) is one of the few known genes to affect risk of developing AUD in humans (Chen, Ferreira, Gross, & Mochly-Rosen, 2014). ALDH2 plays a major role in the detoxification of ethanol-derived acetaldehyde, and inhibition of ALDH is the mechanism of action of disulfiram, an FDA-approved drug for AUD. A mutation in ALDH2 produces an enzyme incapable of metabolizing ethanol,
Neuropeptides/Hormones
Table 11 summarizes the effects of deletion or overexpression of classical or putative neuropeptides and hormones and their receptors on voluntary ethanol administration in mice. Of the genes represented here, the corticotropin-releasing factor/urocortin family and other stress-related neuromodulators are promising for future studies of genetic determinants of AUD (Schank, Ryabinin, Giardino, Ciccocioppo, & Heilig, 2012).
Other Gene Targets
Table 12 represents an assortment of genes that did not specifically fit into the previous categories. These genes are associated with synaptic function, development, circadian regulation, and other cellular regulatory functions but are not discussed individually herein. One of the most pronounced phenotypes among mutant mice is the almost complete blockade of alcohol consumption in mice lacking any one of the three taste genes (Gnat3, Tas1r3, and Trpm5) (Blednov et al., 2008).
Concluding Remarks
Mouse models of voluntary ethanol administration have been instrumental for profiling putative behavioral and genetic determinants in human alcoholics, who exhibit excessive consumption as a hallmark of the disease. The impact of more than 150 genes on alcohol consumption has been evaluated by construction of mutant mice. The global knockout strategy has been used extensively in addiction research to link proteins with behavior, and most studies presented in this chapter used this approach.
Medication Development
FDA-approved drugs for AUD have provided only modest benefit and are not routinely prescribed, and so the search continues for more effective drugs. Identifying existing drugs that could be repurposed to treat AUD is a current goal for researchers and, if successful, would fast track therapeutic options for the disease. A strategy for prioritizing relevant genes from a large list of potential targets is to examine the preclinical evidence in combination with genetic association studies in human
Future Directions
While the study of individual genes is informative, combining gene network and systems biology approaches to identify interrelated networks and pathways is critical in the future treatment of AUD. Because complex trait diseases involve coordinated expression changes in multiple gene families, examining gene clusters is an important research direction, as supported by the INIA studies showing that coexpression patterns can distinguish gene modules related to alcohol consumption in animal models (
Acknowledgments
The authors acknowledge funding from NIAAA grants AA006399, AA013520, AA020926, and AA012404. The authors have no conflicts of interest with this material. The views expressed herein are solely those of the authors and do not necessarily represent those of the funding agencies.
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