This work was supported by National Institutes of Health (NIH) grants AA027301 and AA029985.

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...

Modeling Alcohol Use Disorder Using Chronic Intermittent Ethanol Vapor Exposure

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/CIE20,34,39,40,47,53,54,55. Here, this established m...

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 consequences1,2,3,4,5,6,7. 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&#39;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 hallucinations14,15. 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 vapor4,19,20,21,22,23,24,25,26,27,28,29,30. 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 drinking22,32,33,34,35, 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 intake36,37,38, whereas the mouse model involves CIE and two-bottle choice (2BC) drinking39,40. Preclinical models of alcohol dependence have consistently shown that animals increase their ethanol intake after chronic ethanol vapor exposure23,41,42,43. In mice specifically, repeated cycles of CIE have been shown to escalate voluntary ethanol intake3,21,44,45,46. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>500 Eppendorf Tubes</td>
			<td>Eppendorf</td>
			<td>L203896J</td>
			<td></td>
		</tr>
		<tr>
			<td>95% ethanol</td>
			<td>Decon laboratories</td>
			<td>2816</td>
			<td></td>
		</tr>
		<tr>
			<td>Analox machine</td>
			<td>Analox Instruments</td>
			<td>Analox-AM1</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Weighing Scale</td>
			<td>Kent Scientific</td>
			<td>SCL-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Binder Clips</td>
			<td>Office Depot</td>
			<td>560394</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5418R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chronic intermitted vapor chamber</td>
			<td>La Jolla Alcohol Research Inc</td>
			<td></td>
			<td>Custom made materials</td>
		</tr>
		<tr>
			<td>Heparin/EDTA</td>
			<td>Sagent Pharmaceuticals</td>
			<td>TS/DRUGS/2/2015</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse bedding</td>
			<td>Bed-o&#8217;Cobs</td>
			<td>8B</td>
			<td>fill with 1/8&#34; deep</td>
		</tr>
		<tr>
			<td>Mouse drinking bottle</td>
			<td></td>
			<td></td>
			<td>Custom made materials</td>
		</tr>
		<tr>
			<td>Pyrazole</td>
			<td>Sigma-Aldrich</td>
			<td>bccc6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Teklad global 18% protein (mouse food)</td>
			<td>Teklad global</td>
			<td>Envigo 2018</td>
			<td></td>
		</tr>
	</tbody>
</table>

chronic intermittent ethanol vapor exposure paired with two-bottle choice to model alcohol use disorder

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.

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

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

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 &#177; 0.21 g/kg (n = 15) before...

Watch this Scientific Journal Video about Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder at JoVE.com

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

Alcohol use disorder (AUD) is a chronic alcohol-related disorder that typically presents as uncontrolled drinking and preoccupation with alcohol. A key ...