Materials and Reagents

1. Laboratory-bred plus maze-naïve mice
   Note: Mice housed in groups of 4-5 per cage, kept in an environment with controlled temperature (around 23 °C) and humidity under a 12-12 h light-dark cycle with food and water ad libitum.
   
2. Paper towels and 70% ethanol for cleaning
   

Equipment

1. Elevated Plus maze (either customized apparatus or commercially available one)
   
2. Video camera (placed directly above the maze)
   
3. Indirect white light
   
4. Digital lux meter (Instrutherm, Mastech, Dr. Meter or similar)
   
5. Digital chronometers for manual analysis, or computer software (Any-maze, Ethovision or others) for automated analysis
   Note: In our laboratory, we use a custom made plus-shaped maze sized for mice (Leo et al., 2014), which is elevated 50 cm above the ground, and consists of two opposite closed arms, two opposite open arms and a central square of 5 cm sides (see image below). The closed arms measure 30 x 5 cm and are constituted of white-painted wood, as are the 15 cm high walls that enclose the arms. Glossy painting should be avoided as to prevent excessive glare, thus a matte finish is recommended. The open arms measure 30 x 5 cm and are made of transparent acrylic; a 0.3 cm high transparent acrylic railing prevents animals from falling while exploring the open arms. The apparatus can be disassembled and moved to and from the experiment room where it is set up for use. Wood-made apparatuses covered with wood lacquer, painted in black or transparent Plexiglas apparatuses are also frequently found. Commercially available apparatuses, such as those from Stoelting Co. or Panlab S.L.U., are recommended as alternatives. The room illumination and apparatus light reflection strongly affect the elevated plus maze test. In our laboratory, a Samsung Flashcam digital camcorder is used, as it is more appropriate for posterior off-line analysis. One should always run pilot experiments to establish reliable baseline parameters and standardize the experimental environment before starting the experimental manipulations (drugs, procedure, transgenic animals, etc.).
   
   
   Figure 1. Plus-shaped maze
   

Software

1. Automated computer software (Any-maze, Ethovision and others)

Procedure