Behavioral tests

The apparatus consist of two custom-built black Plexiglas platforms (6 cm wide × 15 cm long × 20 cm high) connected by two identical 2-cm diameter pipes that together form a runway with a manually adjustable gap distance. Two 5 × 5 cm walls were attached to both sides of the platform upper surfaces at the facing ends for the mice to easily recognize the gap distance. Gap-crossing procedures were conducted as follows. The mice were food deprived for 24 h before testing. To prevent the use of visual information, the tests were conducted in a darkened room. The experimenter kept a red-filtered flashlight on hand, but it was not shined on the mice, so the ambient light exposure was less than 1 Lux. The mice were initially trained to find a reward (a small food pellet) at the opposite end of the connected runway. After this training phase, a gap was inserted in the runway and was widened in 0.5-cm intervals. If the mice crossed the gap to obtain the food reward within 2 min, it was considered a successful trial. The gap was widened until the mice would no longer step across it within the 2-min period. This protocol was repeated two times in succession to obtain an average maximum gap width that the individual mouse would cross.

The testing apparatus consisted of a 30 cm wide × 60 cm long × 20 cm high Plexiglas box divided into three chambers as described previously [27, 28]. The mice could move between the chambers through a small opening (6 × 6 cm) in each chamber divider. Plexiglas restraining cylinders were placed in each of the two side chambers, one of which contained a probe mouse. Numerous holes in the cylinders enabled contact between the test and probe mice. Mice to be tested were placed in the center chamber and allowed 5 min to explore the entire box, after which an unfamiliar, same-sex probe was placed in one of the two restraining cylinders. Test mouse movements were recorded using a video camera positioned above the Plexiglas box. The time spent in the social and opposite (empty) chamber was measured.

Social dominance between control and BWT10 mice was measured by the tube test as previously described [29]. Briefly, the apparatus was a transparent Plexiglas tube 30 cm in length with a 3-cm inner diameter. The tube could be separated into sections by two removal gates 13 cm from each end. The diameter was sufficient to permit only one mouse to walk through without reversing direction. Prior to the test trial, each mouse was released at either end of the tube without the gates down for 2 min. After this 2-min habituation period, two unfamiliar mice of approximately the same age (P8W–P9W) and matched as closely as possible for body size and weight (37–40 g), one control and one BWT10, were simultaneously released at the opposite ends of the tube. The dominant mouse was considered the one that advanced across the midline or the one that pushed the other mouse out of the other end within 2 min. Each mouse was matched with two different opponents with an at least 5-min interval between trials.

The custom-built apparatus consisted of a center platform with eight radiating arms, each 5 cm wide × 25 cm long × 15 cm high, numbered 1 to 8. The arm floors were made of black Plexiglas and surrounded by clear 6-mm thick Plexiglas walls. First, a food deprivation schedule was administered to reduce body weight to 85% of baseline. For this purpose, feeding was restricted to 2 h per day for 2 consecutive days. One day before the actual training began, groups of four mice were habituated to the apparatus by placing them at the center and allowing free exploration for 5 min both with and without a bait placed at the arm ends. In the baited condition, the mice were allowed to retrieve the bait, a single 3-g food pellet placed in a food cup. Following habituation and shaping, each animal was individually placed in the center of the maze and trained once a day for 12 consecutive days. The inner wall surfaces of four arms (numbers 1, 3, 5, and 7) were covered with a wire net (1.4 cm mesh; 21 cm long × 7 cm high), and the food cups in these four arms were baited with a single 10-mg food pellet per cup for each daily training trial, while an empty food cup was placed at the ends of the other four arms without wire nets (numbers 2, 4, 6 and 8). Each mouse was allowed to freely explore until it had taken all the pellets or 5 min had elapsed. Measures were made of the ratio of entries into the net-covered/baited arms to total arm entries (ratio of net arm choice) and number of arm revisits. At 2 h after the task, the brains of the mice were processed for immunocytochemistry.