Ultrasound microphone

An ultrasound microphone (Avisoft) and Avisoft Recorder software were used to record mice ultrasonic vocalizations (USVs). For recording adult USVs, subject male mice were placed in a home cage with an age-matched unfamiliar C57BL/6J female counterpart, and USVs were recorded for 5 min. For pup USVs, pups at the age of postnatal day 4, 6, 8, and 10 were separated from dams and placed in a glass container, and USVs were recorded for 3 min. Recorded USVs were analyzed as previously described (Kim et al., 2018 (link)). Briefly, Avisoft SASLab Pro software (RRID:SCR_014438) was used to analyzed USVs. Signals were filtered from 1 Hz to 100 kHz and digitized with a sampling frequency of 250 kHz, 16 bits per sample (Avisoft UltraSoundGate 116H). To generate spectrograms, the following parameters were used: FFT length, 256; frame size, 100; window, FlatTop; overlap, 75%, which resulted in a frequency resolution of 977 Hz and a temporal resolution of 0.256 ms. Frequencies lower than 45 kHz were filtered out to reduce background white noises.

On PND 9, the pups were individually placed into a Plexiglas arena (30 (length) × 30 (width) × 30 (height) cm), located inside a sound-attenuating and temperature-controlled chamber, with a camera positioned above the arena. The ultrasonic vocalizations (USVs) emitted by the pup were detected for 3 min by an ultrasound microphone (Avisoft Bioacoustics, Glienicke, Germany) sensitive to frequencies between 10 and 200 kHz. Pup axillary temperature was measured before and after the test by a digital thermometer. The USVs were analyzed, both quantitatively and qualitatively, using Avisoft Recorder software (Version 5.1), and classified in six different groups according to number of syllables, frequency modulation and duration:^{44 (link), 45 (link), 46 (link)} flat calls (calls with constant frequency with a maximum variation of ±5 kHz); up/downward (single syllable calls emitted at ±5 kHz with a single frequency modulation); flat high frequency calls (calls with similar constant frequency, but emitted at >75 kHz); syllable calls (calls composed by two or more syllables); complex calls (calls displaying complex frequency modulation); short calls (calls with durations shorter than 5 ms).

Isolation-induced ultrasonic vocalizations (USVs) are emitted by rodent pups when they are removed from the nest. These vocalizations play an important communicative role in mother–offspring interactions. The isolation-induced USVs emitted by SAL- and LPS-exposed pups were recorded as previously described [37 (link),45 (link)] at PNDs 5 and 9. Briefly, pups were individually removed from the nest and placed into a black Plexiglass arena (30 cm × 30 cm), located inside a sound-attenuating and temperature-controlled chamber. Pup USVs were detected for 3 min by an ultrasound microphone (Avisoft Bioacoustics, Berlin, Germany) sensitive to frequencies between 10 and 200 kHz and fixed at 15 cm above the arena. They were analyzed quantitatively (number of calls/3 min).