Ultrasound recording was performed using a condenser microphone (UltraSoundGate CM16/CMPA, Avisoft Bioacoustics, Berlin, Germany) as previously reported [5] (link). When recording songs from adult males, the microphone was placed beside a cage (12×20×11 cm) with 6-cm diameter holes and covered with a 0.5-cm wide mesh. Ultrasonic sounds were recorded for 3 min when a male mouse encountered a devocalized female. Females were devocalized by sectioning of the inferior laryngeal nerve according to previous report [24] (link). Playback sounds consisted of 20 s of ultrasounds representing the strain character from 2 individuals each of B6 (number of syllables, 133 and 162) and BALB (number of syllables, 108 and 166). Similarly, playback sounds of ICR (number of syllables, 106) and KJR (number of syllables, 133; kindly gifted by Dr. Koide of the National Institute of Genetics, Mishima, Japan) were prepared. Figure S5 shows the principal components in 6 playback songs used in each behavior test. These sounds were repeatedly generated during the tests. Before each playback experiment, we played the song and simultaneously observed the sound pressure levels in the experimental box by using the same microphone kept away from the speaker, and adjusted the pressure levels.
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