Introduction

● Alcohol is among the most used drugs in the United States. While most alcohol users can be characterized as casual drinkers, 1 in 10 people ages 12 and older suffered from an Alcohol Use Disorder (AUD) in the year 2024, underscoring their severe impact on public health(1).

● Understanding sex differences is critical in the context of addressing issues associated with AUDs. Historically men have been diagnosed with AUDs more than women; however, that gap is starting to close as drinking behaviors in women have started to converge(2).

● Drinking context is thought to be a critical aspect of these differences, as various models used to assess this likely evaluate different aspects of drinking behaviors. Operant self-administration models of alcohol more effectively assess the reinforcing efficacy of ethanol and provide insight into how motivation to obtain it varies across environmental and contextual conditions(3,4).

● The present study was used to evaluate sex differences in ethanol drinking behavior using an operant self-administration paradigm. For the latter part of the study, an aversive stimulus–foot shock–was employed to assess whether the aversion was sufficient to alter these behaviors.

Experimental Timeline

Reward Conditioning

Aversive Control

Methods

Subjects

C57BL/6J male (n=12) and female (n=12)mice were generated from breeding pairs purchased from The Jackson Laboratory, Bar Harbor, ME. One week prior to the study, male and female mice were food-restricted to motivate drug self-administration. Subjects remained on food-restriction throughout the 21-day testing period and were fed after each 30-minute session

Apparatus

Operant chambers with lickometers were used in this study to evaluate ethanol self-administration drinking behaviors. Correct responses were evaluated by the number of nose-pokes initiated by the animal. Following a correct response, a light in the chamber would illuminate, serving as a critical discriminative stimuli, that signals a correct response will produce reinforcement. Reinforcement was delivered via the lickometer lowering, providing 120 seconds of sipper access.

Behavioral Testing

Subjects began self-administration habituation in the operant chamber with 10% sucrose on day 1. This habituation period was necessary to reduce novelty-induced arousal and ensure that ethanol self-administration behaviors were driven by the drug, rather than the environment. Following 3 days of habituation, subjects began a fixed-ratio 1 (FR1) schedule of reinforcement for 3 days. Under a FR1 schedule, mice were rewarded with sipper access after each correct response. After these sessions, subjects transitioned to a fixed ratio 3 (FR3) schedule for 3 days. Under FR3, mice were rewarded with sipper access after three correct responses. The FR1 and FR3 schedules were used to facilitate the acquisition and strengthening of self-administration behavior using the lickometer. Once responding was established by acquisition testing, subjects were trained to initiate voluntary ethanol (EtOH) consumption using a sucrose-fade procedure, through which they were given 10% EtOH with 5% sucrose for 5 days. This training was necessary, as alcohol is initially aversive to the mice due to its bitter taste. Following this, subjects transitioned to a 5-day period where they were given 10% EtOH. This period was used to evaluate EtOH self-administration drinking behaviors. We measured correct responses, sip count, volume consumption, and number of rewards per session. Subsequently, subjects initiated a 2-day period in which correct responses alternated between being rewarded (10% EtOH) and being shocked (50% randomization) to evaluate the effects of aversive stimuli on drinking behavior, using the same measures. On day 1, shock was administered at 0.25 mA, serving as the baseline output, as mice typically exhibit consistent responding at this intensity. On day 2, shock was administered at 0.35 mA, to evaluate whether a higher intensity would further reduce response rates.

Data Analysis

All analyses were conducted in GraphPadPrism (v.10.) using repeated measures Two-Way-ANOVA with Sex and Session as factors.