Ultrasonic Vocalization Analysis in Mice

The Ultrasonic vocalization (USV) test^{18 (link)} was performed in mice at P7 because the number, duration, and average peak frequency of USVs had a peak around P7^{82 (link)}. Both male and female pups at P7 were isolated from their mothers at random and gently placed into a container (plastic cylinder, 5 cm high, 5 cm in diameter). USV was recorded for 5 min from the time of isolation. USV emission was monitored by an ultrasonic microphone (UltraSoundGate CM 16; Avisoft Bioacoustics, Berlin, Germany). The isolation container and microphone were placed in a sound attenuating box (63.5 × 42 × 37 cm). The microphone (sensitive to frequencies of 15–250 kHz) was connected to a personal computer with a sampling rate of 250 kHz in 16-bit format by Avisoft RECORDER (version 4.2.16; Avisoft Bioacoustics) via a USB recording interface (UltraSoundGate 116Hb; Avisoft Bioacoustics, Berlin, Germany).