Key words: Alcohol-Preferring Rats, Drinking-In-The-Dark, Emotional Status, Negative Affect, Excessive Alcohol Intake Abbreviations: AUDs Alcohol Use Disorders DID Drinking in the Dark EtOH Ethanol FM Frequency-modulated P rat Selectively-bred alcohol-preferring rat USVs ultrasonic vocalizations

Background: Emotional states are often thought to drive excessive alcohol intake and influence the development of alcohol use disorders (AUDs). To gain insight into affective properties associated with excessive alcohol intake, we utilized ultrasonic vocalization (USV) detection and analyses to characterize the emotional phenotype of selectively bred alcoholpreferring “P rats”; an established animal model of excessive alcohol intake. USVs emitted by rodents have been convincingly associated with positive (50-55 kHz) and negative (22-28 kHz) affective states. Therefore, we hypothesized that 50-55 and 22-28 kHz USV emission patterns in alcohol-preferring P rats would reveal a unique emotional phenotype sensitive to alcohol experience. Methods: 50-55 kHz frequency-modulated (FM) and 22-28 kHz USVs elicited from male alcohol-preferring P rats were assessed during access to water, 15% and 30% EtOH (v/v). (EtOH; n=12) or water only (Control; n=4) across 8 weeks of daily drinking-in-the-dark (DID) sessions. Results: Spontaneous 22-28 kHz USVs are emitted by alcohol-naïve P rats and are enhanced by alcohol experience. During DID sessions when alcohol was not available (e.g., “EtOH OFF” intervals), significantly more 22-28 kHz than 50-55 kHz USVs were elicited, while significantly more 50-55 kHz than 22-28 kHz USVs were emitted when alcohol was available (e.g., “EtOH ON” intervals). In addition, USV acoustic property analyses revealed chronic effects of alcohol experience on 22-28 kHz USV mean frequency, indicative of lasting alcohol-mediated alterations to neural substrates underlying emotional response. Conclusions: Our findings demonstrate that acute and chronic effects of alcohol exposure are reflected in changes in 22-28 and 50-55 kHz USV counts and acoustic patterns. These data support the notion that initiation and maintenance of alcohol intake in P rats may be due to a unique, alcohol-responsive emotional phenotype and further suggest that spontaneous 22-28 kHz USVs serve as behavioral markers for excessive drinking vulnerability.