Materials and Reagents

1. Paper towels for cleaning the maze with ethanol
   
2. Rats: Our experiments were conducted using 10-week old male Wistar rats but other rodents can be used. If using females, estrous cycle stage should be taken into consideration since spatial reference memory is attenuated during the pro-estrous phase of the cycle (Bowman et al., 2001; Pompili et al., 2010). If using young rats or adult mice, a mouse RAM of smaller dimensions should be used, see below.
   Notes:
   1. The rats are housed under normal controlled laboratory conditions, in weight-matched pairs, under a 12 h dark/light cycle, with ad libitum access to food and tap water prior to the task. The testing protocol should be undertaken during the light phase of the 12 h light cycle between 0700 and 1900 h. Circadian rhythm does not need to be accounted for throughout the experiment as the rodents undergo bi-daily sessions across a large part of the 12 h light phase. To ensure there are no time biases between the groups, spread the testing of control and treated rodents throughout the day. 
      
   2. During the testing protocol, access to food should be restricted to 80% of the rat’s usual food intake to encourage food-seeking behaviour in the maze. Adult (10-week old) male Wistar rats have an approximate daily food intake between 15-20 g during the 12 h dark phase and around 7 g during the 12 h light phase. Adult female Wistar rats have an approximate daily food intake between 10-15 g during the 12 h dark phase and around 5 g during the 12 h light phase (Stefanidis and Spencer, 2012).
      
   3. Prior to commencement and throughout the experiment, normal controlled laboratory light settings should be in place. The maximum allowable light intensity is equivalent to 300 lux at one meter height. There is no additional light source during the experimental protocol to alter the locomotor activity of the rodent.  
      
3. Ethanol, 70% (v/v), diluted in distilled water
   
4. Standard chow grain pellets (45 mg) (Bio-Serv, USA)
   

Equipment

1. RAM
   The task is carried out in an eight-arm radial maze (Lafayette Instrument Company, USA), consisting of an octagonal central platform (34 cm diameter) and eight equally-spaced radial arms (87 cm long, 10 cm wide). At the end of each arm is a food well (2 cm in diameter and 0.5 cm deep; Figure 1). At the entrance to each arm is a clear Perspex door that controlled access in and out of the central area. Each door is controlled by a computerized control system (Lafayette) enabling the experimenter to regulate access to the arms. Salient visual cues of different geometric shapes and contrasting colours are placed around the maze on the walls of the room.
   Note: If using young rats or adult mice, a mouse RAM of smaller dimensions should be used. The central platform should be approximately 22 cm in diameter with arms 25 cm long, 6 cm wide and 6 cm high that is transparent to enable the mice to see extra-maze visual cues (Crusio and Schwegler, 2005). The mouse RAM, should be placed on the floor to avoid elevation-induced anxiety (Crusio and Schwegler, 2005).
   
2. Digital video camcorder. In this experiment a Canon Legria FS200 was used. However, any camcorder can be used.
   
3. Tripod. The video camcorder is attached to a tripod or to the ceiling to allow recording of the entire maze.
   
   
   Figure 1. Radial Arm Maze (Lafayette Instrument Company) with automated Perspex doors and spatial cues placed around the maze. The camcorder is placed on the tripod facing downwards (approximately 45° to the maze). The camcorder can also be attached to the ceiling directly above the maze.
   

Procedure