Ultrasoundgate 116h

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.

Vocalization of the mouse during the fear conditioning was recorded using an ultrasonic microphone (CM16/CMPA, Avisoft) amplified (UltraSoundGate 116H, Avisoft) and digitized at 250 kHz (by a software, Avisoft-RECORDER USGH, Avisoft). For the visualization of the vocalization, we generated a spectrogram using a multitaper method^{58 (link)}. In this analysis, multiple time tapers were designed using a set of 6 series of discrete prolate spheroidal sequences with the time half bandwidth parameter set to 3⁵⁹ . The length of these tapers was set to 512 data samples (~2 ms at 250 kHz). The stored waveform was multiplied by each taper and transformed into the frequency domain. These multiplied waveforms were averaged in the frequency domain. This procedure could produce a stable spectrotemporal representation of vocal sound with background noise attenuated.

Adult subject male mice were isolated in their home cage for 3 days before the test, whereas age-matched intruder female mice were group-housed (6–7 mice/cage). We did not measure female cycles on the assumption that group housing might synchronize cycles. Basal ultrasonic vocalizations (USVs) of an isolated male mouse in its home cage under light conditions of ∼60 lux in a soundproof chamber were recorded for 5 min in the absence of a female intruder. Next, a randomly chosen stranger C57BL/6J female mouse was introduced into the cage, and female-induced courtship USVs were recorded for 5 min during free interaction between the male and female. Avisoft SASLab Pro software was used to automatically analyze the number of USV calls, latency to first call, and total duration of calls from recorded USV files. Signals were filtered from 1 to 100 kHz and digitized with a sampling frequency of 250 kHz, 16 bits per sample (Avisoft UltraSoundGate 116H). Spectrograms were generated using the following parameters: FFT length, 256; frame size, 100; window, FlatTop; overlap, 75%. These parameters yielded a frequency resolution of 977 Hz and a temporal resolution of 0.256 ms. Frequencies lower than 25 kHz were filtered out to reduce white background noise.