Five-trial social recognition memory test

This test assesses cognition and sociability, namely the ability to recognize novel versus familiar animals. The test was performed according to the methods outlined in previous reports.^21,25 Experiment mice were habituated in the test arena for 10 minutes/day for 2 days before testing. Age- and size-matched C57BL/6J male stimulus mice were used and habituated to a cylinder holder made of transparent acryl (30-cm high, 10.5 cm-diameter with 16 holes of 0.5-cm diameter) for 30 minutes before testing. In this test, the same stimulus animal was used for both acquisition and recognition phases (intruder 1). Over the course of multiple exposures, experiment mice became habituated to intruders and no longer regarded as interesting as they did for completely novel intruders. During testing, each experiment mouse was given four 5-minutes exposures, with 15 to 20 minutes interval period, in a transparent acrylic box (60 cm × 60 cm × 30 cm). In the fifth trial, the experiment mice encountered an entirely novel intruder (intruder 2). All test trials were recorded with Any-maze tracking system, and total investigating time was subsequently analyzed. The time spent by each mouse (nose-point) in close proximity (~2 cm) to the cylinder holder was measured as interaction time (ie, sniffing time, approach) to evaluate the approach behavior.