DOI: 10.5061/dryad.t76hdr890
title: Conspecific presence facilitates 22-kHz ultrasonic calls in male rats exposed to cat odor

description: 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.
description: These files comprise data from  90 experimentally naïve male
 Wistar rats (6-8 weeks). <em>Experimental
 Apparatus</em> 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. <em>Cat
 fur sourcing and storage</em> 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<sup>o</sup>C until use.
 <em>Experimental procedure</em>
 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 <sup>o</sup>C, and then located opposite the hide box. The
 fur was heated (36 <sup>o</sup>C) 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.
 <em>Data acquisition and analysis</em>
 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.
description: # Conspecific presence facilitates 22-kHz ultrasonic calls in male rats
 exposed to cat odor
 [https://doi.org/10.5061/dryad.t76hdr890](https://doi.org/10.5061/dryad.t76hdr890) ## Description of the data and file structure The two data files correspond to the vocalization and behavioral data over 20-minute tests (Cumulative data) and 5-minute bins (Timecourse data). ### Files and variables #### File: Cumulative\_data.xlsx **Description:** This file contains total vocalization and behavioral data over 20-minute tests. ##### Variables * Sample: Categorical variable representing the experimental unit ID, i.e., Unique identifier of the Single, Pair, or Trio of rats. * Treatment: Categorical variable representing the social "Treatment", i.e., 1.Singles, 2.Pairs, 3. Trios. * Animals: Integer values from 1 to 3 that represent the number of animals composing the experimental unit. To be used as an offset to normalize the data. * Stage: Categorical variable representing the assay day or "Stage", i.e., 1. Habituation, 2. Exposure, 3. Conditioned Fear * HideTime1Animal: Continuous value from 0 to 1200, representing the time in seconds at least one animal in the social group remained in the Hide Box. * HideTimeAllAnimal: Continuous value from 0 to 1200, representing the time in seconds all animals in the social group remained in the Hide Box. * Duration22kHz: Continuous value from 0 to 1200, representing the time in seconds during which vocalization 18-32kHz frequency and longer than 100 ms in duration, i.e., ~22-kHz were recorded. * Count22kHz: Integer value representing the number of individual 18-32kHz frequency and longer than 100 ms in duration, i.e., ~22-kHz vocalization recorded. * Duration50kHz: Continuous value from 0 to 1200, representing the time in seconds during which vocalization 35-72-kHz frequency, i.e., ~50-kHz were recorded. * Count50kHz: Integer value representing the number of individual 35-72-kHz frequency, i.e., ~50-kHz vocalization recorded. #### File: Timecourse\_data.xlsx **Description:** This file contains vocalization data in 5-minute bins. ##### Variables * Bin: Integer value from 1 to 4, representing each 5 minute bin within a 20 minute test, where 1 = 5 minutes, 2 = 10 minutes, 3 = 15 minutes, and 4 = 20 minutes. * Sample: Categorical variable representing the experimental unit ID, i.e., Unique identifier of the Single, Pair, or Trio of rats. * Treatment: Categorical variable representing the social "Treatment", i.e., 1.Singles, 2.Pairs, 3. Trios. * Animals: Integer values from 1 to 3 that represent the number of animals composing the experimental unit. To be used as an offset to normalize the data. * Stage: Categorical variable representing the assay day or "Stage", i.e., 1. Habituation, 2. Exposure, 3. Conditioned Fear * Duration22kHz: Continuous value representing the time in seconds during which vocalization 18-32kHz frequency and longer than 100 ms in duration, i.e., ~22-kHz were recorded. * Count22kHz: Integer value representing the number of individual 18-32kHz frequency and longer than 100 ms in duration, i.e., ~22-kHz vocalization recorded. * Duration50kHz: Continuous value representing the time in seconds during which vocalization 35-72-kHz frequency, i.e., ~50-kHz were recorded. * Count50kHz: Integer value representing the number of individual 35-72-kHz frequency, i.e., ~50-kHz vocalization recorded. ## Code/software These files are in excel. ## Access information Other publicly accessible locations of the data: * NA Data was derived from the following sources: * NA