Reciprocal social interaction test. Two mice with identical genotypes housed in different cages were placed in a box (50 cm × 50 cm × 50 cm; O’Hara & Co., Tokyo, Japan) with dim light (10 lux) and allowed to move freely for 10 min. These two mice not only had an identical genotype but also had the same age, sex, and similar body weight. In this test, a pair of mice was used as a sample. Images were captured at a rate of 2 per second by a charge-coupled device camera located above the field. An interaction time was defined as any period of time in which the two mice either contacted each other or remained separated by no more than two body lengths. To avoid observer bias, interaction times were measured automatically using a custom macro for ImageJ.

Measurement of USV. A male mouse was placed in a soundproof box, and then, a female mouse was put in the cage. USVs emitted by the male were recorded by a condenser microphone (Avisoft, Berlin, Germany) for 5 min at a sampling rate of 250 kHz. Before the test day, each mouse was habituated in the field for 5 min without female exposure. Recorded data were analyzed by SASLab Pro (Avisoft). The number of calls was measured manually with single blinding.

Open-field test. The open-field test was performed with modified procedures reported in a previous study (4). Each mouse was permitted to freely move in the open-field apparatus (50 cm × 50 cm × 50 cm). Total distance traveled, vertical activity (rearing measured by counting the number of photobeam interruptions), and time spent in the center area of open-field were recorded for 60 min by using TimeOF (O’Hara & Co.), which allows automated measurements.

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