Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder

Summary

Presented here is a protocol for the 2BC/CIE model of alcohol dependence in mice to study alcohol use disorder.

Abstract

Alcohol use disorder (AUD) is a chronic alcohol-related disorder that typically presents as uncontrolled drinking and preoccupation with alcohol. A key component of AUD research is using translationally relevant preclinical models. Over the past several decades, a variety of animal models have been used to study AUD. One prominent model of AUD is the chronic intermittent ethanol vapor exposure (CIE) model, which is a well-established approach for inducing alcohol dependence in rodents through repeated cycles of ethanol exposure via inhalation. To model AUD in mice, the CIE exposure is paired with a voluntary two-bottle choice (2BC) of alcohol drinking and water to measure the escalation of alcohol drinking. The 2BC/CIE procedure involves alternating weeks of 2BC drinking and CIE, which repeat until the escalation of alcohol drinking is achieved. In the present study, we outline the procedures for performing 2BC/CIE, including the daily use of the CIE vapor chamber, and provide an example of escalated alcohol drinking in C57BL/6J mice using this approach.

Introduction

Alcohol use disorder (AUD), which involves chronic excessive alcohol consumption, is one of the most common psychiatric disorders and is a global problem. AUD symptoms involve repeated cycles of intoxication, withdrawal, and cravings and are characterized by the constant consumption of alcohol without regard for the social, occupational, and health consequences^{1,2,3,4,5,6,7}. Alcohol use disorder often occurs in conjunction with other pervasive, persistent, and impairing mental disorders⁸, such as ADHD⁹, anxiety¹⁰, or depression¹¹ and is responsible for approximately 88,000 deaths annually in the United States alone². Excessive or frequent alcohol use can affect a person's work status and social relationships¹² and may lead to increased violence¹³. Physically, acute withdrawal from alcohol can result in anxiety, agitation, tremor, excessive sweating, altered consciousness, and hallucinations^14,15. Furthermore, people may feel withdrawal symptoms when cutting down or stopping drinking and become irritable or cranky¹⁶. Additionally, chronic alcohol consumption can cause memory loss¹⁷ and can result in thiamine deficiency, also known as Wernicke-Korsakoff syndrome (WKS), which contributes significantly to alcohol-induced dementia¹⁸.

To further advance AUD research, it is necessary to have translationally relevant animal models of the disease. The most common model of AUD in rodents is chronic intermittent ethanol vapor exposure (CIE), which is a well-established approach for inducing alcohol dependence through repeated inhalation of alcohol vapor^{4,19,20,21,22,23,24,25,26,27,28,29,30}. Rodent CIE procedures induce withdrawal symptoms such as handling-induced convulsions³¹, hyperexcitability, irritability-like behavior, anxiety-like behavior, and sleep disorders and result in an escalation of alcohol drinking^{22,32,33,34,35}, thus meaning the CIE model has translational validity to human AUD.

In rats, the CIE model often involves the operant self-administration of alcohol to measure the escalation of intake^36,37,38, whereas the mouse model involves CIE and two-bottle choice (2BC) drinking^39,40. Preclinical models of alcohol dependence have consistently shown that animals increase their ethanol intake after chronic ethanol vapor exposure^23,41,42,43. In mice specifically, repeated cycles of CIE have been shown to escalate voluntary ethanol intake^{3,21,44,45,46}. Overall, prior studies demonstrate that the CIE model is sufficient to increase ethanol consumption and model AUD in rodents.

This study aims to highlight the CIE method for studying AUD and, more specifically, focus on the 2BC/CIE mouse model. We go through a detailed process of the steps necessary for performing 2BC/CIE and present an example of the escalation of alcohol drinking after CIE.

Protocol

All procedures were approved by the Purdue University Animal Care and Use Committee. The present study used 8 week old C57BL/6J mice. The CIE group had 5 mice (3 male, 2 female), and the Air group had 10 mice (5 male and 5 female). The animals were obtained from a commercial source (see Table of Materials) and were group-housed on a 12 h light-dark cycle with access to food. The body weights of the mice were measured once per week throughout the experiment. 1. General ex…

Representative Results

In the present representative study, ethanol intake (g/kg) during 2BC is reported during baseline drinking and after weeks of CIE (post vapor). Briefly, as described in the protocol, during 2BC, the mice had access to two bottles: one containing water and the other containing 15% (w/v) ethanol. After the baseline intakes were determined, the subjects were split and evenly assigned to the CIE or Air group. The initial baseline ethanol intake during the 3 week period stabilized at 2.00 g/kg ± 0.21 g/kg (n = 15) before…

Discussion

Alcohol use disorder represents a global public health problem with high prevalence and cost to society⁵². To study AUD in preclinical animal models, a common method in mice is 2BC/CIE^{20,34,39,40,47,53,54,55}. Here, this established m…

Materials

500 Eppendorf Tubes	Eppendorf	L203896J
95% ethanol	Decon laboratories	2816
Analox machine	Analox Instruments	Analox-AM1
Animal Weighing Scale	Kent Scientific	SCL-4000
Binder Clips	Office Depot	560394
C57BL/6J mice	The Jackson Laboratory	000664
Centrifuge	Eppendorf	5418R
Chronic intermitted vapor chamber	La Jolla Alcohol Research Inc		Custom made materials
Heparin/EDTA	Sagent Pharmaceuticals	TS/DRUGS/2/2015
Mouse bedding	Bed-o’Cobs	8B	fill with 1/8" deep
Mouse drinking bottle			Custom made materials
Pyrazole	Sigma-Aldrich	bccc6397
Teklad global 18% protein (mouse food)	Teklad global	Envigo 2018