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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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'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.

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 experimental design

NOTE: Two-bottle choice (2BC)/chronic intermittent ethanol vapor exposure (CIE) is a preclinical mouse model to explore ethanol dependence47.

  1. 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.
  2. 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.
  3. 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.
    NOTE: Mice from the Air group remain in their regular housing setting so they are exposed to Air with no ethanol vapor.
  4. 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.
  5. 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)48.

2. Experimental preparation

  1. 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).
  2. Ensure all the required chemicals are ready for the experiment: drinking water, 95% ethanol, pyrazole, heparin and sterile saline.

3. Animal habituation

  1. 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. During this time, handle the mice regularly, and transfer them to any areas where experimentation will occur for 30 min daily.
  2. 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.

4. Two-bottle choice with 15% w/v ethanol and water

  1. Two-bottle choice habituation
    1. 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.
    2. 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).
  2. Baseline two-bottle choice
    1. 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.
    2. 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.
  3. Two-bottle choice (2BC)
    1. Resume the 2BC procedure as performed during baseline drinking after the first CIE week.
      ​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).

5. Chronic intermittent ethanol vapor (CIE) exposure

  1. 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.
  2. Chronic intermittent ethanol vapor (CIE) procedure
    1. 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 ethanol49.
    2. 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.
    3. 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: During CIE, the mice are most susceptible to poor health outcomes from vapor exposure at the end of the week as they enter an extended withdrawal period; thus, a half dose of pyrazole is given on the last day of CIE to minimize the withdrawal occurring after the CIE is paused for the weekend and the following week. A half dose of pyrazole is also given each week on the first day of CIE to ease the mice back into the weekly vapor exposure. The dose of pyrazole may need to be adjusted in order to calibrate the BEC to be higher or lower, as desired. It is recommended to use a pyrazole dose ranging from 0.00375 mL/g to 0.0075 mL/g.
    4. 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).
    5. 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.
    6. 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.
    7. 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.
  3. Blood measurements
    1. 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).
    2. 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 lab50.
      NOTE: The blood is collected into heparinized or EDTA-coated 0.5 mL tubes (see Table of Materials).
    3. Maintain the collected blood samples at 4 °C until processed.
    4. To process the samples, first centrifuge at 208 x g for 10 min at 4 °C to separate the plasma.
    5. Determine the blood ethanol concentrations using an oxygen-rate ethanol analyzer (see Table of Materials) or other systems capable of determining BECs.

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

Disclosures

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

Acknowledgements

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

Materials

NameCompanyCatalog NumberComments
500 Eppendorf TubesEppendorfL203896J
95% ethanolDecon laboratories2816
Analox machineAnalox InstrumentsAnalox-AM1
Animal Weighing ScaleKent ScientificSCL-4000
Binder ClipsOffice Depot560394
C57BL/6J miceThe Jackson Laboratory000664
CentrifugeEppendorf5418R
Chronic intermitted vapor chamberLa Jolla Alcohol Research IncCustom made materials
Heparin/EDTASagent PharmaceuticalsTS/DRUGS/2/2015
Mouse beddingBed-o’Cobs8Bfill with 1/8" deep
Mouse drinking bottleCustom made materials
PyrazoleSigma-Aldrichbccc6397
Teklad global 18% protein (mouse food)Teklad globalEnvigo 2018

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