All behavioral tests were performed using male mice during the light cycle. For each genotype, at least two independent cohorts of mice were tested. All experiments and data analyses were performed in a blinded manner.

Open-field activity test. The experimental animal was placed in the center of an open-field apparatus (50 × 50 × 40 cm3) illuminated by a light-emitting diode and allowed to move freely for 15 min. Parameters including distance traveled, time spent in the central area of the open field (30% of total area), and rearing time were measured every minute. Data were recorded and analyzed by using TimeOFCR4 (O’Hara & Co. Ltd.).

Resident-intruder social interaction test. The experimental animal was acclimated to its cage before the experiment. A distinct 6-week-old WT male DBA/2CrSlc (Japan SLC Inc.) was introduced to the experimental animal for a 10- or 5-min period for Dlx5-creERT2 and CaMKII-cre mice, respectively. Interaction between the animals was recorded using a digital video camera, with total contact time and number of contacts initiated by the experimental animal quantified.

Three-chamber social interaction test. The animal was first acclimated to the test apparatus made of clear fiberglass and consisted of three chambers with small entrances in between them for 10 min with objects in the opposing chambers. Following acclimatization, an unfamiliar mouse (stranger) and an object were introduced into opposing chambers, with the experimental mouse allowed to explore for an additional 10 min to evaluate the social approach. Social novelty was examined by replacing the object with a second unfamiliar mouse into the opposite chamber, with the experimental mouse allowed to explore for 10 min. The first stranger mouse was considered to be the familiar mouse in the last stage of the test. Time spent in each chamber was quantified and presented. Distinct 6-week-old WT male C57BL/6JJmsSlc mice (Japan SLC Inc.) were used as unfamiliar mice in this experiment. Time spent in the left and right chambers were compared for each genotype separately to examine social approach (object versus mouse) and social novelty (familiar versus stranger mouse), as detailed previously by Silverman and colleagues (48). The social interaction preference index was also calculated to compare across genotypes. This was defined as a percentage of the time in a selected chamber (e.g., chamber with mouse) relative to the total time spent in both chambers (e.g., sum of time in chamber with mouse and time in chamber with object). Therefore, the percentage of time in the selected chamber will be 50 if there is no preference.

Elevated plus maze test. The animal was placed in the center of a plus-shaped maze with two opposing ends lined by clear fiberglass (closed arms) and the two other ends without any barriers (open arms). The animal was allowed to move freely on the elevated maze for a period of 5 min. Parameters such as total distance traveled, distance traveled and time spent in open and closed arms, and time in center were measured. Data were recorded and analyzed by TimeEP2 (O’Hara & Co. Ltd.).

Tail suspension test. The experimental animal was suspended by holding its tail onto a force measurement apparatus for a 10-min period. The duration of immobility was recorded every minute. Data were recorded and analyzed by using TimeFZ2 (O’Hara & Co. Ltd.).

Nesting behavior test. Nesting behavior was examined and quantified according to the scoring system, as described previously (49). Briefly, mice were placed into individual cages with corncob bedding and a nestlet before the dark phase. Photos of the cages were taken every 12 hours for 2 or 3 days, and a score at each time point was assigned according to the rating scale.

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