Alcohol-naïve USVs distinguish male HAD-1 from LAD-1 rat strains.

Ultrasonic vocalizations (USVs) are mediated through specific dopaminergic and cholinergic neural pathways and serve as real-time measures of positive and negative emotional status in rodents. Although most USV studies focus primarily on USV counts, each USV possesses a number of characteristics shown to reflect activity in the associated neurotransmitter system. In the present study, we recorded spontaneously emitted USVs from alcohol-naïve high alcohol drinking (HAD-1) and low alcohol drinking (LAD-1) rats. Using our recently developed WAAVES algorithm, we quantified four acoustic characteristics (mean frequency, duration, power, and bandwidth) from each 22-28 kHz and 50-55 kHz frequency-modulated (FM) USV. This rich USV representation allowed us to apply advanced statistical techniques to identify the USV acoustic characteristics that distinguished HAD-1 from LAD-1 rats. Linear mixed models (LMM) examined the predictability of each USV characteristic in isolation and linear discriminant analysis (LDA), and binomial logistic regression examined the predictability of linear combinations of the USV characteristics as a group. Results revealed significant differences in acoustic characteristics between HAD-1 and LAD-1 rats in both 22-28 kHz and 50-55 kHz FM USVs. In other words, these rats selectively bred for high- and low-alcohol consumption can be identified as HAD-1 or LAD-1 rats with high classification accuracy (approximately 92-100%) exclusively based on their emitted 22-28 kHz and 50-55 kHz FM USV acoustic characteristics. In addition, acoustic characteristics of 22-28 kHz and 50-55 kHz FM USVs emitted by alcohol-naïve HAD-1 and LAD-1 rats significantly correlate with their future alcohol consumption. Our current findings provide novel evidence that USV acoustic characteristics can be used to discriminate between alcohol-naïve HAD-1 and LAD-1 rats, and may serve as biomarkers in rodents with a predisposition for, or against, excessive alcohol intake.

Negative Affect-Associated USV Acoustic Characteristics Predict Future Excessive Alcohol Drinking and Alcohol Avoidance in Male P and NP Rats.

BACKGROUND: Negative emotional status and adverse emotional events increase vulnerability to alcohol abuse. Ultrasonic vocalizations (USVs) emitted by rats are a well-established model of emotional status that can reflect positive or negative affective responses in real time. Most USV studies assess counts, yet each USV is a multidimensional data point characterized by several acoustic characteristics that may provide insight into the neurocircuitry underlying emotional response. METHODS: USVs emitted from selectively bred alcohol-naïve and alcohol-experienced alcohol-preferring and nonpreferring rats (P and NP rats) were recorded during 4-hour sessions on alternating days over 4 weeks. Linear mixed modeling (LMM) and linear discriminant analysis (LDA) were applied to USV acoustic characteristics (e.g., frequency, duration, power, and bandwidth) of negative affect (22 to 28 kilohertz [kHz])- and positive (50 to 55 kHz) affect-related USVs. RESULTS: Hundred percent separation between alcohol-naïve P and NP rats was achieved through a linear combination (produced by LDA) of USV acoustic characteristics of 22- to 28-kHz USVs, whereas poor separation (36.5%) was observed for 50- to 55-kHz USVs. 22- to 28-kHz LDA separation was high (87%) between alcohol-experienced P and NP rats, but was poor for 50- to 55-kHz USVs (57.3%). USV mean frequency and duration were the highest weighted characteristics in both the naïve and experienced 22- to 28-kHz LDA representations suggesting that alcohol experience does not alter the representations. LMM analyses of 22- to 28-kHz USV acoustic characteristics matched the LDA results. Poor LDA separation was observed between alcohol-naïve and alcohol-experienced P rats for both 22- to 28-kHz and 50- to 55-kHz USVs. CONCLUSIONS: Advanced statistical analysis of negative affect-associated USV data predicts future behaviors of excessive alcohol drinking and alcohol avoidance in selectively bred rats. USV characteristics across rat lines reveal affect-related motivation to consume alcohol and may predict neural pathways mediating emotional response. Further characterization of these differences could delineate particular neurocircuitry and methods to ameliorate dysregulated emotional states often observed in human alcohol abusers.

Alcohol enhances unprovoked 22-28 kHz USVs and suppresses USV mean frequency in High Alcohol Drinking (HAD-1) male rats.

Heightened emotional states increase impulsive behaviors such as excessive ethanol consumption in humans. Though positive and negative affective states in rodents can be monitored in real-time through ultrasonic vocalization (USV) emissions, few animal studies have focused on the role of emotional status as a stimulus for initial ethanol drinking. Our laboratory has recently developed reliable, high-speed analysis techniques to compile USV data during multiple-hour drinking sessions. Since High Alcohol Drinking (HAD-1) rats are selectively bred to voluntarily consume intoxicating levels of alcohol, we hypothesized that USVs emitted by HAD-1 rats would reveal unique emotional phenotypes predictive of alcohol intake and sensitive to alcohol experience. In this study, male HAD-1 rats had access to water, 15% and 30% EtOH or water only (i.e., Controls) during 8 weeks of daily 7-h drinking-in-the-dark (DID) sessions. USVs, associated with both positive (i.e., 50-55 kHz frequency-modulated or FM) and negative (i.e., 22-28 kHz) emotional states, emitted during these daily DID sessions were examined. Findings showed basal 22-28 kHz USVs were emitted by both EtOH-Naïve (Control) and EtOH-experienced rats, alcohol experience enhanced 22-28 kHz USV emissions, and USV acoustic parameters (i.e., mean frequency in kHz) of both positive and negative USVs were significantly suppressed by chronic alcohol experience. These data suggest that negative affective status initiates and maintains excessive alcohol intake in selectively bred HAD-1 rats and support the notion that unprovoked emissions of negative affect-associated USVs (i.e., 22-28 kHz) predict vulnerability to excessive alcohol intake in distinct rodent models.

Alcohol-preferring P rats emit spontaneous 22-28 kHz ultrasonic vocalizations that are altered by acute and chronic alcohol experience.

BACKGROUND: Emotional states are often thought to drive excessive alcohol intake and influence the development of alcohol use disorders. 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 alcohol-preferring (P) rats; an established animal model of excessive alcohol intake. USVs emitted by rodents have been convincingly associated with positive (50-55 kHz frequency-modulated [FM]) and negative (22-28 kHz) affective states. Therefore, we hypothesized that 50-55 and 22-28 kHz USV emission patterns in P rats would reveal a unique emotional phenotype sensitive to alcohol experience. METHODS: 50-55 kHz FM and 22-28 kHz USVs elicited from male P rats were assessed during access to water, 15 and 30% EtOH (v/v). Ethanol (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 FM 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 FM 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.

Automating ultrasonic vocalization analyses: the WAAVES program.

BACKGROUND: Human emotion is a crucial component of drug abuse and addiction. Ultrasonic vocalizations (USVs) elicited by rodents are a highly translational animal model of emotion in drug abuse studies. A major roadblock to comprehensive use of USV data is the overwhelming burden to attain accurate USV assessment in a timely manner. One of the most accurate methods of analyzing USVs, human auditory detection with simultaneous spectrogram inspection, requires USV sound files to be played back 4% normal speed. NEW METHOD: WAAVES (WAV-file Automated Analysis of Vocalizations Environment Specific) is an automated USV assessment program utilizing MATLAB's Signal and Image Processing Toolboxes in conjunction with a series of customized filters to separate USV calls from background noise, and accurately tabulate and categorize USVs as flat or frequency-modulated (FM) calls. In the current report, WAAVES functionality is demonstrated by USV analyses of cocaine self-administration data collected over 10 daily sessions. RESULTS: WAAVES counts are significantly correlated with human auditory counts (r(48)=0.9925; p<0.001). Statistical analyses used WAAVES output to examine individual differences in USV responses to cocaine, cocaine-associated cues and relationships between USVs, cocaine intake and locomotor activity. COMPARISON WITH EXISTING METHOD: WAAVES output is highly accurate and provides tabulated data in approximately 0.3% of the time required when using human auditory detection methods. CONCLUSIONS: The development of a customized USV analysis program, such as WAAVES streamlines USV assessment and enhances the ability to utilize USVs as a tool to advance drug abuse research and ultimately develop effective treatments.