Rats (Rattus norvegicus) exhibit defensive behaviors such as hiding, freezing, and fleeing when confronted with predators. They often emit long-duration (~22-kHz) ultrasonic vocalizations (USVs) in response to predator detection, unlike shorter (~50-kHz) USVs associated with positive states. These vocalizations can also trigger defensive behaviors in other rats. We studied the impact of a predator-associated stimulus (cat fur) and the context in which it was experienced on ~22-kHz and ~50-kHz USVs in laboratory rats, along with how the presence of conspecifics (none, one, or two familiar rats) influenced these vocalizations. Group-housed rats were habituated to a testing arena with a hide box alone or in pairs or trios. The next day, they were exposed to cat fur (1 gram) in the arena, and defensive behaviors and USVs were recorded. Subsequently, the rats returned to the arena without cat fur to assess contextual fear. Cat fur prompted significant hiding and ~22-kHz USVs, especially in social groups, but not in the context alone. Cat fur also suppressed ~50-kHz USVs, with recovery faster in trios, indicating a social buffering effect. These findings suggest that predator cues induce ~22-kHz USVs influenced by social context, highlighting an altruistic communicative function.

These files comprise data from  90 experimentally naïve male Wistar rats (6-8 weeks).

Experimental Apparatus

The behavioral responses of these rats were tested in a cat odor avoidance apparatus consisting of a rectangular chamber (80 cm x 44 cm x 49 cm) with a smaller red Perspex “hide box” (23 cm x 15 cm x 49 cm) placed at one end. A glass Petri dish either containing the odor cue (cat fur) or empty (control) is fixed at the opposite end of the chamber using a Velcro sticker (Velcro brand, 2022 Velcro IP Holdings LLC). An ultrasonic microphone (UltraSoundGate CM16/CMPA, 2022 Avisoft Bioacoustics) and recording hardware (UltraSoundGate 416H, 2022 Avisoft Bioacoustics) were fitted to the top of the chamber on the opposite side of the hide box to record any vocalizations the animals emitted. This was accomplished using Avisoft RECORDER software (2022 Avisoft Bioacoustics). An infrared CCTV camera (Panasonic WV-CP300 Series 650 TVL Day/Night IR Dual Voltage Fixed Camera, fitted with a computer CS-Mount 2.9–8.2 mm Varifocal Lens) was located above the chamber for video recording.

Cat fur sourcing and storage

Cat fur was obtained from veterinary clinics and animal shelters across Sydney, Australia. Sampling was opportunistic, either as part of routine grooming or through shaving areas for medical procedures, and stored at -20^oC until use.

Experimental procedure

The experimental procedure occurred over three consecutive days, with rats placed in the testing apparatus for 20 minutes each day according to social grouping allocation (i.e., singles (n=17 rats), in 14 pairs (n=28 rats) or 15 trios (n=45 rats)). Social grouping allocation was done with animals housed together, with pairs and trios housed in the same home cage; thus, all social groupings were composed of familiar individuals throughout the experiment. The experimental apparatus contained no cat fur on the first Habituation day. On the second Exposure day, 1 g of cat fur was placed in a petri dish, warmed to 36 ^oC, and then located opposite the hide box. The fur was heated (36 ^oC) before Exposure to mimic the natural body temperature of a cat and increase the potential release of volatiles. Finally, on the third Conditioned Fear day, an empty heated petri dish was placed opposite the hide box. The testing apparatus was thoroughly cleaned with 70% ethanol at the end of each test.

Data acquisition and analysis

Video recordings of behavior were manually scored to determine the time at least one rat remained in the hide box and the time all rats present spent in the hide box. Avisoft SASLab Pro (2022 Avisoft Bioacoustics) software was used to record and analyze USVs. Spectrograms from each recording were obtained with the following parameters: Temporal resolution overlap of 50%, frequency resolution of 977 Hz, Bandwith 1699 Hz, frame size 100%, and Kaiser–Bessel window. Denoising was achieved by using a -80 dB threshold and a reduction by 90 dB. Spectrograms were matched for start and end times to the video recordings of behavior. The Automated Parameter measurements function, with Element separation set to “whistles”, allowed filtering of the data from the spectrogram into two frequency ranges: 18-32kHz frequency and longer than 100 ms in duration, i.e., ~22-kHz USVs and 35-72-kHz frequency, i.e., ~50-kHz.