Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder
June 23rd, 2023
Presented here is a protocol for the 2BC/CIE model of alcohol dependence in mice to study alcohol use disorder.
Our research pertained the field of the alcohol using disorder. This study highlights the methodology commonly using in the established the Alco dependence mouse model. This model is a chronic intermittent ethanol vapor paired with the two bottle choice paradigm.
The CIE two bottle choice paradigm. Automated two bottle choice equipment allows for the monitoring of mouse drinking behavior by recording the frequency of super touches. This enables us to acquire additional information beyond the volume consumed, such as timing of drinking bouts throughout a drinking session.
In preclinical studies of the alcohol using disorder, the approaches such as the whole brain imaging, chemogenetics, and optogenetics with various of the viral transgenetic mice are often used to advance the research in this field. Other approaches such as the computational modeling are also be used. The two bottle choice CIE model for alcohol dependence has been established for many years.
However, we are hoping to make the adoption of the approach more readily accessible to the broader research community. Compare with the other techniques using to study the alcohol using disorder. The two bottle choice and the CIE paradigm has a several advantages that are increased the translational relevance, such as induction of the significant withdrawal symptoms and the development of the escalation of the alcohol intake.
This approach is ideal for its use to study the relevant neuro biological changes caused by the alcohol dependence. To begin habitation, move the group housed mice into their individual drinking cages 30 minutes before the lights turn off. Record the volume of each drinking bottle and then allow the mice to drink for two hours.
Record the volumes of the bottles after the drinking period. Now expose the mice to at least two weeks of baseline two bottle choice drinking to establish a stable ethanol intake. Test the mice by giving them a water bottle and a second bottle containing 15%ethanol.
Record the volumes of each bottle before and after the two hour drinking period. Once the mice have achieved a stable baseline of ethanol intake, divide them into two groups based on their intake and put them back in their home cage. Intraperitoneally inject the mice with an ethanol-pyrazole solution.
Inject all the air controls and the other groups with the same pyrazol dose mixed with saline. After injections, place the air control mice in their home cages and place the CIE cages inside the vapor chamber. Lock the doors and set the pump to the appropriate vapor level.
Next, set the runtime of the experiment and then press start on the CIE vapor chamber control screen 30 minutes before the vapor system turns off. Determine the blood ethanol concentration of the mice. After collecting the blood, return the mice to the two bottle choice testing the week after the CIE.
Centrifuge the blood samples at 208 G for 1.0 minutes at four degrees Celsius to separate the plasma. Use an oxygen rate ethanol analyzer to determine the blood ethanol concentration. The initial baseline ethanol intake during the three week period stabilized at 2.00 plus or minus 0.21 grams per kilogram before the CIE.
A Bonferroni post hoc test showed that the CIE mice increased their ethanol intake significantly at post vapor week six compared to their baseline drinking volume. The CIE mice consumed significantly more ethanol than the non-dependent mice at PV6. The Bonferroni post hoc test also showed that at PV6, the ethanol dependent mice had significantly higher ethanol intake compared to their own baseline and non-dependent mice.
The analyzed blood ethanol concentrations of the CIE group showed a weekly average of 173.12 milligrams per deciliter.
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Overview
This study presents a protocol for the two-bottle choice/chronic intermittent ethanol (CIE) model of alcohol dependence in mice to investigate alcohol use disorder. The model leverages automated equipment to monitor mouse drinking behavior, providing insights into the neurobiological changes linked to alcohol dependence.
Key Study Components
Area of Science
- Neuroscience
- Behavioral Pharmacology
- Alcohol Dependence Studies
Background
- The two-bottle choice and CIE paradigm has been used to study alcohol dependence for many years.
- Automated monitoring enables detailed analysis of drinking patterns beyond just volume.
- This model can induce significant withdrawal symptoms and escalate alcohol intake.
- Research in alcohol use disorder often employs techniques like imaging and genetic manipulation.
Purpose of Study
- To provide an accessible protocol for studying alcohol dependence in mice.
- To highlight the relevance of the two-bottle choice/CIE model in preclinical studies.
- To encourage broader adoption of this methodology in alcohol research.
Methods Used
- The main platform used is the two-bottle choice paradigm paired with CIE exposure.
- Mice are individually housed and monitored for their alcohol intake over a specified period.
- Baseline drinking is established before administering ethanol and CIE treatment.
- Methods include intraperitoneal injections and blood ethanol concentration measurements.
Main Results
- CIE exposure significantly increased ethanol intake in dependent mice over their baseline measurements.
- Blood ethanol levels demonstrated that the CIE group maintained higher average concentrations compared to non-dependent mice.
- Behavioral changes following CIE reflect crucial neurobiological alterations linked to alcohol dependence.
Conclusions
- This study establishes the two-bottle choice/CIE model as a reliable technique for investigating alcohol dependence.
- The methodology facilitates an in-depth examination of withdrawal symptoms and alcohol intake escalation.
- Insights derived from this model can enhance our understanding of the neurobiological underpinnings of alcohol use disorders.
Frequently Asked Questions
Executive Industry Relevance
Chronic intermittent ethanol vapor exposure paired with two-bottle choice (2BC/CIE) provides a translationally relevant preclinical model for alcohol use disorder (AUD) research. This model enables quantitative assessment of voluntary alcohol intake escalation and withdrawal, supporting mechanistic de-risking and predictive confidence in target validation. Its adoption strengthens the discovery pipeline for neurobiological targets implicated in AUD.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of neurobiological pathways underlying alcohol dependence and withdrawal.
- Supports functional target validation by modeling escalation of voluntary alcohol intake.
- Facilitates mechanistic de-risking through controlled induction of dependence and withdrawal symptoms.
Screening & Assay Development
- Provides a validated behavioral system for evaluating candidate compounds affecting alcohol consumption.
- Automated monitoring yields reproducible, quantitative drinking behavior data for assay standardization.
- Enables scalable screening of interventions in a disease-relevant context.
Translational & Preclinical Research
- Aligns with translational biomarker strategies by modeling escalation and withdrawal phenotypes observed in human AUD.
- Supports continuity from discovery through preclinical validation of neurobiological mechanisms.
- Informs risk-adjusted advancement decisions for CNS-targeted portfolios.
Pipeline & Workflow Integration
The 2BC/CIE model integrates into the discovery continuum from early hypothesis testing to preclinical lead evaluation in AUD research.
- Discovery Biology: Enables hypothesis-driven testing of neurobiological mechanisms driving alcohol dependence.
- Screening: Provides reproducible, quantitative outputs for compound evaluation in a validated behavioral paradigm.
- Analytics: Supports statistical comparison of intake escalation and withdrawal metrics across experimental groups.
- Translational Research: Bridges preclinical findings to human-relevant phenotypes of alcohol use disorder.
- Enterprise Reuse: Offers a standardized, reusable platform for cross-program evaluation of AUD interventions.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in AUD target validation.
- Operational Value: Delivers standardized, scalable, and reproducible behavioral data for cross-study comparison.
- Strategic Value: Improves go/no-go decision-making and capital efficiency in CNS disorder portfolios.
- Portfolio Impact: Enables risk-adjusted prioritization of neurobiological targets and candidate interventions.
Implementation Considerations
- Requires expertise in behavioral neuroscience and animal model management.
- Needs access to automated two-bottle choice equipment and ethanol vapor chambers.
- Demands rigorous cross-team standardization of intake measurement and withdrawal assessment.
- Adaptation across mouse strains or genetic backgrounds may require protocol optimization.
- Individual behavioral variability can impact reproducibility and must be statistically addressed.
