An alcohol withdrawal test battery measuring multiple behavioral symptoms in mice

Abstract

Despite acceptance that risk for alcohol-use disorder (AUD) has a large genetic component, the identification of genes underlying various components of risk for AUD has been hampered in humans, in part by the heterogeneity of expression of the phenotype. One aspect of AUD is physical dependence. Alcohol withdrawal is a serious consequence of alcohol dependence with multiple symptoms, many of which are seen in multiple species, and can be experienced over a wide-ranging time course. In the present three studies, we developed a battery of withdrawal tests in mice, examining behavioral symptoms from multiple domains that could be measured over time. To permit eventual use of the battery in different strains of mice, we used male and female mice of a genetically heterogeneous stock developed from intercrossing eight inbred strains. Withdrawal symptoms were assessed using commonly used tests after administration of ethanol in vapor for 72 continuous hours. We found significant effects of ethanol withdrawal versus air-breathing controls on nearly all symptoms, spanning 4 days following ethanol vapor inhalation. Withdrawal produced hypothermia, greater neurohyperexcitability (seizures and tremor), anxiety-like behaviors using an apparatus (such as reduced transitions between light and dark compartments), anhedonia (reduced sucrose preference), Straub tail, backward walking, and reductions in activity; however, there were no changes in thermal pain sensitivity, hyper-reactivity to handling, or anxiety-like emergence behaviors in other apparatus. Using these data, we constructed a refined battery of withdrawal tests. Individual differences in severity of withdrawal among different tests were weakly correlated at best. This battery should be useful for identifying genetic influences on particular withdrawal behaviors, which should reflect the influences of different constellations of genes.

Affective Behavior in Withdrawal Seizure–Prone and Withdrawal Seizure–Resistant Mice during Long‐Term Alcohol Abstinence

“…In the LDT, EtOH‐exposed mice showed longer latencies to enter the light compartment and fewer total transitions between compartments than air‐controls across the entire duration of post‐Tx testing, suggesting that CIE induced a rapid and long‐lasting increase in behavioral anxiety. These observations are consistent with previous work showing that forced abstinence from EtOH vapor can elicit anxiety‐like behaviors in diverse mouse populations (Finn et al., ; Kliethermes et al., ; McCool and Chappell, ; Metten et al., ; Sidhu et al., ).…”

Affective Disruption During Forced Ethanol Abstinence in C57BL/6J and C57BL/6NJ Mice

“…Interestingly, however, anxiety‐ and depressive‐like behaviors in mice may follow distinct temporal trajectories during prolonged abstinence. Across diverse EtOH administration protocols and behavioral test procedures, mice usually display significant anxiety‐like behavior during the first 72 hours of abstinence, a phase referred to as “acute withdrawal” (Finn et al, 2000; Gong et al, 2017; Kash et al, 2009; Kliethermes et al, 2004; Perez and De Biasi, 2015; Pleil et al, 2015; Rose et al, 2016); however, several negative findings have also been reported (Cox et al, 2013; Daut et al, 2015; Lee et al, 2015; Metten et al, 2018). At 4 days to 3 weeks into abstinence, generally referred to as “early abstinence” (Heilig et al, 2010), anxiety‐like behavior becomes markedly less prominent in mice (Becker et al, 2017; Fukushiro et al, 2012; Holleran et al, 2016; Lee et al, 2015; Pang et al, 2013).…”

“…Unlike with studies of anxiety‐like behavior, inconsistent findings regarding depressive‐like behavior during acute withdrawal have been reported, and results appear to be significantly dependent on the method of EtOH administration and to some extent, the behavioral assay utilized. In mice, forced methods of EtOH administration (Arora and Vohora, 2016; Karadayian et al, 2013; Metten et al, 2018), as opposed to long‐term voluntary consumption paradigms (Holleran et al, 2016; Lee et al, 2015; Lee et al, 2016; Lee et al, 2017; Stevenson et al, 2009), seem to more reliably produce depressive‐like behavior during acute withdrawal. Nonetheless, there is abundant evidence for the presentation of depressive‐like behavior in mice during early abstinence, across various methods of EtOH administration and behavioral assays (Gong et al, 2017; Holleran et al, 2016; Kim et al, 2017; Pang et al, 2013; Roni and Rahman, 2017; Stevenson et al, 2009).…”

RiboTag-Seq Reveals the Activation of the Unfolded Protein Response in Striatal Microglia Induced by Ethanol Withdrawal

“…GSEA is a method that used previously curated sets of functionally defined genes and examines whether there is a statistical over-representation of genes associated with these gene sets in any of the experimentally manipulated treatment groups. Using this method, we found a pattern of changes that suggested the induction of inflammatory and cytokine signaling in microglia after repeated cycles of ethanol exposure (EtOH-0h), as well as an upregulation in the TNFα cytokine gene set exclusively at 8 hours of withdrawal from ethanol vapor, a timepoint that has previously been associated with intense biological and behavioral signs of ethanol withdrawal 40 . Although GSEA is a very sensitive strategy for detecting meaningful patterns of gene changes, it is based on the observations that were used to curate the gene sets in the first place.…”