Modeling Alcohol Consumption in Rodents Using Two-Bottle Choice Home Cage Drinking and Microstructural Analysis

This protocol describes a standard intermittent-access two-bottle choice home cage drinking paradigm to model voluntary alcohol consumption in rats. In addition, it provides step-by-step instructions to augment the standard protocol with a DIY lick detection system that enables microstructural analysis of drinking behavior. Two-bottle choice home cage drinking is a widely used preclinical paradigm to study alcohol consumption in rodents.

Animals are giving access to two drinking bottles, one with water and one with ethanol. It can be easily implemented to measure voluntary alcohol consumption in preference without specialized equipment or analysis. The resolution of drinking data acquired using this approach is limited by how often the researcher measures bottle weight.

Consequently, the standard approach is unable to capture drinking patterns, which are known to impact both the risk for and severity of alcohol abuse disorder. Our protocol addresses the need to increase data resolution in preclinical models by augmenting the standard two-bottle choice paradigm with a low-cost, homemade lickometer system. Researchers will be able to easily capture various measures of drinking microstructure and obtain precise estimates of ethanol intake with high temporal resolution.

This protocol affords researchers the flexibility to use either standard bottles or a lickometer-equipped apparatus to measure drinking depending on their experimental needs. Importantly, measurement of drinking microstructure allows researchers to investigate individual differences in drinking patterns and how drinking patterns change in response to experimental intervention. To begin, remove the filter tops from the cages.

On Mondays, Wednesdays, and Fridays, remove the Tuesday, Thursday, and Saturday bottles from the cage tops. Weigh and record the initial weight of the ethanol and water bottles for the day's drinking session. To start the session, place the Monday, Wednesday, and Friday bottles on each rat's cage top with tape facing up to prevent the rat from chewing the bottle labels.

On Tuesdays, Thursdays, and Saturdays, remove the Monday, Wednesday, and Friday bottles from each cage. Weigh each bottle and record the end weight for the previous drinking session. Then, measure the body weight of each rat.

Place the Tuesday, Thursday, and Saturday water bottles on each cage top with tapes facing up to prevent the rat from chewing the bottle labels. To begin building sensor unit A, cover a two-pin female connector cable with red heat-shrink tubing. Insert the red and black wire ends of the cable into pin 0 and pin 1 on the sensor board and solder them in place.

Similarly, solder five more cables with distinctly colored heat-shrink tubing. To assemble sensor unit A connect sensor board A to a communication breakout endpoint with a four-pin cable. Then, connect the endpoint to the 3-volt output of the level shifter.

House sensor unit A into the 3D-printed case and gently close the top. After building sensor units B and C in a similar fashion, daisy-chain all three sensor units using ethernet cables to complete the sensor setup. To set up the microcontroller interface, stack the modified data logger onto the microcontroller.

Then, stack the modified touchscreen onto the data logger. Insert the microSD card and adapter into the data logger. Plug the four-pin jumper wires and an extra grounding wire into the microcontroller.

House the fully assembled microcontroller interface inside the 3D-printed case with the four-pin jumper wires and grounding wire threaded through the opening at the top. To install the LIQ HDR system in cage components, attach the lickometer and the laser-cut acrylic panel to the cage top using two sets of screws and nuts. Next, install the assembled sipper blocker onto the cage top using a screw set, ensuring both sipper blockers align properly with the sipper openings.

To use the LIQ HDR system in the vivarium, mount each sensor unit onto the vivarium shelving. Arrange and secure all female connector cables to prevent overlap or excessive bending. Mount the microcontroller interface next to sensor unit A and plug its connector into the level shifter connected to sensor unit A.Use electrical tape to secure the extra ground wire to the metal shelving.

Securely connect all male lickometer connector cables to the matching female sensor connector cables. Plug in the power supply to turn on the microcontroller interface. To start the lickometer-equipped two-bottle choice procedure, use the sipper to cover the sipper openings on all cages.

Before recording, select the correct experimental side on the main GUI page corresponding to the side with the ethanol bottle for that day. After installing all Monday, Wednesday, and Friday bottles, tap Start on the main page to begin sensor calibration. Once the calibration is completed, the system will display the cumulative lick counts for each bottle on the recording page.

Raise all sipper blockers above the cage tops to allow rats access to the sippers. Gently return all filter tops to the cages. Tap Refresh on the screen to update the lick counts.

On Tuesdays, Thursdays, and Saturdays, tap Save Quit to stop recording and return to the main GUI page. To remove the SD card for data transfer, tap Eject SD.Insert the SD card into a computer. Then, make a copy of the CSV data file with the date of the recording session onto the computer.

To prepare for the next recording session, reinsert the SD card into the interface and tap Mount SD.Ethanol intake in rats showed a pattern of escalation during the initial days of two-bottle choice access, followed by a plateau. Many rats displayed binge-like ethanol consumption shortly after the onset of the drinking session. Ethanol preference was significantly higher than water during binge-like drinking, but often remained below 50%when measured over 24 hours.

The cumulative number of licks detected by the LIQ HDR system strongly correlated with the change in bottle weights for both ethanol and water bottles over 24 hours. Rats consumed more ethanol and water during the dark cycle compared to the light cycle, as depicted in drinking patterns binned into 30-minute intervals. Binge-like drinking behavior was concentrated in the first five minutes of the drinking session as indicated by the higher-resolution data analysis.

Blood ethanol concentrations taken 15 minutes after session onset correlated well with the number of licks detected, even at low ethanol intake levels. Drinking microstructural analysis revealed a similar number of licks and lick bouts between male and female rats associated with a similar level of ethanol intake.