Rats emit aversive 44-kHz ultrasonic vocalizations during prolonged Pavlovian fear conditioning

Rats are believed to communicate their emotional state by emitting two distinct types of ultrasonic vocalizations. The first is long “22-kHz” vocalizations (>300 ms, <32 kHz) with constant frequency, signaling aversive states and the second, is short “50-kHz” calls (<100 ms, >32 kHz), often frequency-modulated, in appetitive situations. Here we are first to describe a new-type of aversive vocalizations emitted at a higher pitch by Wistar and spontaneously hypertensive rats (SHR) in an intensified aversive state – prolonged fear conditioning. These calls, which we named “44-kHz vocalizations”, are long (>150 ms), generally at a constant frequency (usually within 35-50 kHz range) and have an overall spectrographic image similar to 22-kHz calls. Some 44-kHz vocalizations are comprised of both 22-kHz-like and 44-kHz-like elements. Furthermore, two separate clustering methods confirmed that these 44-kHz calls are distinct from other vocalizations. We also show that some of rats’ responses to the playback of 44-kHz calls were more akin to that of aversive calls, e.g. heart rate changes, whereas other responses were at an intermediate level between aversive and appetitive calls. Our results suggest that rats have a wider vocal repertoire than previously believed, and current definitions of major call types may require reevaluation.