Elevated plus-maze

The EPM is a classic behavioral test to study anxiety-like behaviors in rodents (Borelli and Brandão, 2008; Handley and Mithani, 1984; Pellow et al., 1985). The apparatus comprised two open (50 × 10 cm) and two closed (50 × 10 × 40 cm) arms extending from a central platform (10 × 10 cm) elevated 50 cm above the floor. All testing was conducted during the light phase of the light/dark cycle, between 10:00 AM and 1:00 PM. The apparatus was located inside a room with constant background noise (50 dB). The animal behaviors were recorded by a video camera (Everfocus, Orange, CA, USA) positioned above the maze, and the signal was relayed to a monitor in another room via a closed-circuit TV camera. Luminosity at the open and closed arms level was 27 lx. The rats were placed individually in the center of the maze facing an open arm and allowed 5-min free exploration. Each rat was tested only once, and the apparatus was cleaned with 20% ethyl alcohol after each session.

The performance of each animal in the maze was scored by an observer blind to the experimental groups. The standard measurements recorded in each section of the maze (closed and open arms) comprised the frequency of open and closed arm entries (an arm entry or exit being defined as all four paws into or out an arm, respectively), total arm entries, and the percentage of time spent on the open and closed arms were calculated as percentage of the total time the rat spent on the maze [arm time % = 100 × (arm time/total time)]. In addition, the frequencies of the following ethological parameters were measured: (1) head-dipping: dipping of the head below the level of the maze floor; (2) end-arm exploration: the number of times the rat reached the end of an open arm; (3) stretched-attend postures: when the animal stretches to its full length with the forepaws (keeping the hind paws in the same place and turns back to the anterior position); and (4) rearing: vertical movements in any direction, including sniffing of maze walls. Most of these categories were defined previously following work with rats and mice (Blanchard et al., 1993; Borelli and Brandão, 2008; Rodgers and Johnson, 1995). In our initial experiment, males and separate groups of female rats at the four different stages of the cycle were tested on the EPM. These experiments revealed significant differences in responding between the P and LD stages. Thus, for subsequent experiments testing the effect of CBD, we compared rats only in P and LD.