Animal behavioral tests

The EPM test was performed as previously described [17]. Mice were acclimatized to the room for 30 min before behavioral observation. The EPM (Med Associates, St. Albans, Vermont) consisted of two open arms (250 lux) and two closed arms (35 lux) situated opposite to each other. For each test, individual animals were placed in the center square and allowed to move freely for 5 min. An observer, sitting quietly 1.5 m from the maze, measured the number of entries, and time spent in each arm was recorded. The maze was cleaned with ethanol 70 % and Virox® after each mouse was tested. For the purpose of analysis: a) open-arm activity was quantified as a percentage of the ratio between the time spent in the open arms and the total time spent in the elevated plus-maze (time open arms/total time × 100), b) the number of entries into open arms as the sum of them, and c) the total number of entries as the sum of entries into the closed and open arms (entries open + entries closed = total entries). The entry in the arm was considered as such when a mouse crossed the same with all four paws. Mice, having undergone five sessions of the repeated EPM, were tested for 5 min every 30 min. After the first session of EPM, all mice were individually housed until the beginning of the second session. This group of mice was retested after 24 h from the last (fifth) session. A different group of mice was exposed to a single EPM and retested after 24 h.

The mice were exposed to an open field box, a Plexiglas® square white box (43.2 × 43.2 × 30.5 cm3; Med Associates, St. Albans, Vermont) for 30 min and their locomotor activity was monitored by Activity Monitor system from Med Associates (St. Albans, VT). Briefly, this system used paired sets of photo beams to detect movement in the open field and movement was recorded as beam breaks (number of photo beams: 16; space between the beams: 2.5 cm; number of zones: X: 17, Y: 17). The open field was placed inside an isolation chamber with dim illumination. Each subject was placed in the corner of the open field. The central zone was defined as an area: start (X = 4; Y = 4), end (X = 12; Y = 12). Different groups of mice C57Bl/6, either naïve or undergone five sessions of the repeated EPM, were tested on open field 1 h after the last session.