Detailed spectrographic analysis of rat ultrasonic vocalizations emitted during the acoustic startle response test

•Rats emit ultrasonic vocalizations during the acoustic startle response test.•Enhanced startle responses are associated with a predominant 22-kHz call profile.•Emission of 22-kHz ultrasonic vocalizations reflect a negative affective state. Rats emit ultrasonic vocalizations (USV). During aversive situations, rats emit 22-kHz USV, which are considered "alarm calls" and supposed to reflect a negative affective state of the sender. During appetitive situations, rats emit 50-kHz USV, which are believed to reflect a positive affective state. Here, we recorded USV emission in adult male rats during the acoustic startle response test. Our results indicate varied USV emission in both the 22- and 50-kHz USV ranges. Enhanced startle responses were observed in rats with a predominant 22-kHz call profile, supporting the notion that 22-kHz USV emission is associated with a negative affective state.