Anticipatory Food-Reward Elicitation of Appetitive Ultrasonic Vocalizations in Laboratory Rats and Mice

Ultrasonic vocalizations (USVs) in laboratory rats and mice provide a valuable model for investigating vocal function, affective behavior, and the underlying neural mechanisms that drive vocalization. Many commonly used USV elicitation paradigms rely on mating or social interaction, which can introduce hormonal and behavioral confounds that complicate interpretation in studies of aging, neurodegeneration, and endocrine modulation. To address these limitations, this article describes a food-reward USV elicitation paradigm that reliably evokes appetitive 50 kHz vocalizations in anticipation of palatable food. It includes standardized procedures for animal handling, acclimation, and behavioral conditioning to establish consistent anticipatory responses to a palatable reward. Animals undergo repeated exposure to the recording environment followed by reward delivery to promote stable vocal output across sessions. USVs are recorded in sound-attenuated chambers under controlled environmental conditions to ensure a high signal-to-noise ratio and reproducibility. A structured workflow for automated detection and classification is provided, which enables the extraction of key acoustic parameters, including the total number of USVs, rate, frequency range, duration, maximum power, and complexity. The protocol includes quality control steps to verify detection accuracy and minimize artifacts. By eliminating mating-related variables and standardizing recording conditions, this method provides a controlled approach for eliciting and quantifying appetitive 50 kHz vocalizations across repeated sessions. The paradigm is well-suited for investigating affective-motivational processes and cranial sensorimotor function and can be adapted for diverse experimental contexts, including studies of neurological disease, neurodegeneration, and hormone manipulation, with appropriate study-specific validation.