Name: Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder (Video) | JoVE Journal
Uploaded: 2023-06-23T13:50:00.000+00:00
Duration: 5 min 12 s
Description: Learn the chronic intermittent ethanol vapor and two-bottle choice protocol to model alcohol dependence in mice. This method enables automated monitoring of drinking behavior and reveals neurobiological changes linked to alcohol use disorder.

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5 minutes

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 experimental design
NOTE: Two-bottle choice (2BC)/chronic intermittent ethanol vapor exposure (CIE) is a preclinical mouse model to explore ethanol dependence51.
<ol>
	<li>During the 2BC weeks, ensure that the mice have access to a bottle containing ethanol (15% w/v) and a bottle containing water for 2 h daily (Monday to Friday), beginning 2 h into the dark cycle.</li>
	<li>At first, allow the mice a minimum of 2 weeks of baseline 2BC drinking until a stable level of ethanol intake has been reached. Depending on the experimental needs, divide the mice into two groups (a CIE group and an Air group) or more if additional treatments are included based on the average baseline ethanol intake.</li>
	<li>After the baseline 2BC weeks, expose the mice from the CIE group to ethanol vapor (CIE) for four consecutive days (Monday to Thursday) with 16 consecutive hours of vapor on and 8 consecutive hours of vapor off. To ensure stable and sustained blood ethanol concentrations throughout the exposure period, pyrazole was co-administered as an alcohol dehydrogenase inhibitor, thereby preventing rapid ethanol metabolism and promoting consistent systemic ethanol levels. 
	NOTE: To control for potential chamber-specific variables such as lighting, vibration, noise, and temperature, mice in the Air group were housed in an identical chamber under the same environmental conditions as the CIE group, but without alcohol vapor exposure.</li>
	<li>During the CIE/Air week, ensure 2BC does not occur. After 1 week of CIE/Air, resume the mice on 2BC for the following Monday to Friday.</li>
	<li>Allow mice alternate weeks of CIE/Air and 2BC for the remainder of the procedure until the escalation of alcohol drinking occurs in the CIE mice. Collect blood from the mice at least once per week of CIE to determine their blood ethanol concentration (BEC)52.</li>
</ol>
2. Experimental preparation
<ol>
	<li>Acquire all the following supplies before the start of the study: mice, cages/metal cage tops, drinking bottles to fit two per cage side by side, bedding, food, a scale, a chronic intermitted vapor chamber, and an Analox machine or another method to analyze the BECs (see Table of Materials).</li>
	<li>Ensure all the required chemicals are ready for the experiment: drinking water, 95% ethanol, pyrazole, heparin and sterile saline.</li>
</ol>
3. Animal habituation
<ol>
	<li>If the mice are not already housed in the same location as the experiments, allow at least 1 week for them to acclimate to the new environment. 
	NOTE: During this period, mice should also be habituated to gentle handling to minimize stress-related confounds and improve animal welfare. Handling sessions were conducted once daily for five consecutive days, each lasting approximately 2&#8211;3 min per animal. Mice were cupped or gently guided into the experimenter&#39;s hands without tail lifting, allowing them to voluntarily explore the handler&#8217;s hand. This technique facilitates routine interaction, reduces stress during subsequent experimental procedures, and ensures more reliable behavioral data. Additionally, animals were transferred to the experimental area for 30 min daily to further acclimate them to the testing environment.</li>
	<li>Ensure that the mice are given 1 week of two-bottle choices with both bottles filled with water to allow them to adjust to the different cage setup and being placed into an individual cage for drinking.</li>
</ol>
4. Two-bottle choice with 15% w/v ethanol and water
<ol>
	<li>Two-bottle choice habituation
	<ol>
		<li>The two-bottle choice testing paradigm is the same throughout the week of two-bottle choice with water and the later testing with water and ethanol. Move the group-housed mice to their individual drinking cages 30 min before the lights turn off, and then allow the mice to drink for 2 h.</li>
		<li>Record the volumes of each drinking bottle before and after the 2 h drinking period. Each group undergoes 2BC for 5 days, from Monday to Friday. 
		NOTE: The purpose of the habituation week is to let the animals adjust to two bottles being in the cage to minimize any bias caused by the natural side position of a single bottle. During this week, both bottles are filled with water. If the supplied metal hopper is designed to hold only one bottle, carefully use the space between its bars adjacent to the first bottle to create space for the second bottle. The bottles used for drinking experiments are custom-made using 10 mL and 25 mL pipettes with the ends cut to allow a straight sipper with a ball bearing to be inserted for the mice to drink from. Using binder clips to clip the bottles to the wire lid will ensure that the bottles do not move during the drinking period (Supplementary Figure 1 and Supplementary Figure 2).</li>
	</ol>
	</li>
	<li>Baseline two-bottle choice
	<ol>
		<li>After habituation, allow the mice to undergo at least 2 weeks of baseline 2BC drinking to establish a stable ethanol intake. During the baseline drinking, test the mice for 2 h per day and 5 days per week (Monday to Friday) with one water bottle and a second bottle with 15% w/v ethanol. Record the volumes of each drinking bottle before and after the 2 h drinking period. 
		NOTE: To establish a stable baseline of ethanol intake, repeat the weeks of 2BC until each mouse has approximately less than 15% variation in intake between days.</li>
		<li>After the mice achieve a stable baseline of ethanol intake, evenly divide the mice based on their ethanol intake into ethanol-nondependent (Air treatment) and ethanol-dependent (CIE treatment) groups. Once the baseline ethanol intake has been established, the mice undergo CIE (or Air exposure) the following week. 
		NOTE: Not all experiments require a non-dependent group to be included, and this must be determined based on the focus of the study. For example, ethanol-dependent mice with and without specific treatment may be a more relevant comparison.</li>
	</ol>
	</li>
	<li>Two-bottle choice (2BC)
	<ol>
		<li>Resume the 2BC procedure as performed during baseline drinking after the first CIE week. 
		&#8203;NOTE: The two-bottle choice mouse drinking formula is a 15% w/v ethanol mixture (1 L): 95% ethanol (195 mL) and mouse drinking water (805 mL).</li>
	</ol>
	</li>
</ol>
5. Chronic intermittent ethanol vapor (CIE) exposure
<ol>
	<li>Arrange the following materials: custom-made CIE vapor chambers (Supplementary Figure 3), 95% ethanol, an ethanol-pyrazole solution (68.1 mg/kg pyrazole mixed in 20% ethanol), a saline-pyrazole solution (68.1 mg/kg pyrazole mixed in saline), syringes (1 mL), and needles.</li>
	<li>Chronic intermittent ethanol vapor (CIE) procedure
	<ol>
		<li>Prior to being put into the vapor chambers for CIE, intraperitoneally inject the mice with an ethanol-pyrazole solution (0.0075 mL/g), which is used to create an initial ethanol intoxication, inhibit ethanol dehydrogenase, and block the metabolism of ethanol53.</li>
		<li>Inject all the Air controls and other groups with the same pyrazole dose mixed with saline (0.0075 mL/g) instead of ethanol. It is recommended to give a half dose (0.00375 mL/g) of the pyrazole solutions on the first day and last day of CIE each week.</li>
		<li>After the injections, return the Air/control mice to their home cages, and place the cages for the CIE group inside the vapor chamber. 
		NOTE: Given the potential health risks associated with chronic ethanol vapor exposure, animals were closely monitored throughout the protocol to ensure their well-being and to detect any adverse effects. Mice were weighed at least twice per week, and health assessments included observation for clinical signs of distress. Animals exhibiting a body weight loss exceeding 20% from baseline or displaying signs such as hunched posture, lethargy, piloerection, or unkempt fur were considered to have reached humane endpoints and were promptly removed from the study in accordance with approved institutional animal care and use guidelines. During CIE, mice are most susceptible to poor health outcomes toward the end of the week, when they enter an extended withdrawal period following the cessation of ethanol exposure. To minimize the severity of withdrawal, a half dose of pyrazole is administered on the final day of vapor exposure each week. Additionally, a half dose is given at the start of each new CIE cycle to ease the animals back into ethanol exposure. Pyrazole, an alcohol dehydrogenase inhibitor, is used to stabilize and prolong blood ethanol concentrations; doses typically range from 0.00375 mL/g to 0.0075 mL/g and may be adjusted to achieve the desired blood ethanol concentration.</li>
		<li>Once the mice are put into the vapor chambers, lock the doors, and set the pump to the appropriate vapor level (e.g., 1-3). Set the run time of the experiment. For the present study, the experiment started at 17:00 PM and finished at 09:00 AM, and a pump setting of 1-2 was used (used throughout).</li>
		<li>Then, press the Start button on the CIE vapor chamber control screen to start the experiment. 
		NOTE: This step could vary depending on the chamber being used and differences in the setup. Here, once all the mice are injected and placed into the ethanol chamber, the chamber should begin pumping 95% ethanol into a glass flask and heat the liquid ethanol into vapor. The vapor flows through the chamber where the mice are held for 16 h/day for 4 days (Monday evening through Friday morning). Each day, the mice experience 8 h of the vapor being off. The mice are then removed from the chamber at the end of the fourth day (Friday morning) and begin 2BC the following Monday. Depending on the specific vapor chamber system and setup, there may be variations in the BECs caused by the different settings/vapor levels. Various factors, such as slight differences in the system components, air flow, exhaust, etc., can cause this. Adjusting the ethanol vapor level instead of the pyrazole dose is suitable to maintain a BEC in the target range. As tolerance develops, the BECs may be too low, and the vapor level may need to be adjusted. If the gradient between the vapor levels is too steep for increasing the BECs, then adjusting the pyrazole dose accordingly is recommended to alter the BECs.</li>
		<li>Measure the BECs (see step 5.3) of the mice at least once per week during CIE. Perform blood collection 30 min before the vapor stops to ensure the BECs have not started to drop. 
		NOTE: The vapor level may need to be adjusted during the experiment as ethanol tolerance builds over the course of several weeks. BECs should be tested before and after adjusting the vapor settings.</li>
		<li>Return the mice to 2BC testing on the week following the CIE. Repeat step 4 and step 5. Keep the mice alternating between CIE and 2BC for four to eight cycles (8-16 weeks) until they have significant ethanol consumption escalation compared with their baseline drinking volume.</li>
	</ol>
	</li>
	<li>Blood measurements
	<ol>
		<li>Arrange the following materials: equipment to measure BECs, such as an Analox system, a blade, a tube (0.2 mL), heparin/EDTA, and a centrifuge (see Table of Materials).</li>
		<li>Tail-snip the CIE mice once during the CIE week, 30 min before the vapor system turns off, to collect blood for determining the BEC. The blood collection can be performed using several methods depending on the preferences of the lab54. 
		NOTE: The blood is collected into heparinized or EDTA-coated 0.5 mL tubes (see Table of Materials).</li>
		<li>Maintain the collected blood samples at 4 &#176;C until processed.</li>
		<li>To process the samples, first centrifuge at 208 x g for 10 min at 4 &#176;C to separate the plasma.</li>
		<li>Determine the blood ethanol concentrations using an oxygen-rate ethanol analyzer (see Table of Materials) or other systems capable of determining BECs.</li>
	</ol>
	</li>
</ol>

Modeling Alcohol Use Disorder Using Chronic Intermittent Ethanol Vapor Exposure

Maury Cole is the owner and operator of La Jolla Alcohol Research Inc.

Alcohol use disorder represents a global public health problem with high prevalence and cost to society56. To study AUD in preclinical animal models, a common method in mice is 2BC/CIE20,34,43,44,51,57,58,59. 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. Moreover, CIE leads to a significant escalation in voluntary alcohol consumption, reflecting the compulsive intake observed in human AUD36. During intoxication phases, mice display behavioral and physiological signs consistent with alcohol intoxication in humans, including ataxia, sedation, and impaired hypothermia37,38,39. These outcomes underscore the utility of the CIE model in capturing both the neurobiological and behavioral aspects of alcohol dependence, making it a valuable tool for elucidating AUD mechanisms and testing potential therapeutic interventions. In preclinical models of AUD, such as chronic intermittent ethanol (CIE) vapor exposure, mice exhibit behavioral and physiological signs that parallel human alcohol intoxication and dependence. During intoxication phases, mice may display signs including ataxia, sedation, decreased exploratory behavior, and impaired righting reflex. Given these risks, animals were closely monitored during and after ethanol vapor exposure to identify signs of severe intoxication or health deterioration.
In rats, the CIE model often involves the operant self-administration of alcohol to measure the escalation of intake40,41,42, whereas the mouse model involves CIE and two-bottle choice (2BC) drinking43,44. Preclinical models of alcohol dependence have consistently shown that animals increase their ethanol intake after chronic ethanol vapor exposure23,45,46,47. In mice specifically, repeated cycles of CIE have been shown to escalate voluntary ethanol intake3,21,48,49,50. 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><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&amp;rsquo;Cobs</td><td>8B</td><td>fill with 1/8&quot; 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

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.

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 the ...

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 ...

chronic-intermittent-ethanol-vapor-exposure-paired-with-two-bottle

Research

JoVE Journal

Neuroscience

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Learn the chronic intermittent ethanol vapor and two-bottle choice protocol to model alcohol dependence in mice. This method enables automated monitoring of drinking behavior and reveals neurobiological changes linked to alcohol use disorder.

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

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