Materials and Reagents

1. Paper towels (Renown, catalog number: REN06116-WB )
   
2. Red paper towels (KCWW, Kimberly-Clark, catalog number: 05930 )
   
3. C57BL/6J male adult mice (3-18 months of age)
   Note: C57BL/6J male adult mice (3-18 months of age) housed in a room with reversed light cycle for at least two weeks prior to test. Behavior experiment is conducted during the dark cycle (7 AM-7 PM, see Note 1).
   
4. 70% ethanol (v/v in ddH₂O) in a spray bottle
   

Equipment

1. A well-lit (1,200 lux) behavior room isolated from noises and a close-by holding room (Figure 1A)
   
2. White shower curtains (Figures 1B and 1C)
   
3. Two large visual clues (Figures 1B and 1C)
   
4. A Polystyrene circular DMP dry maze platform (Diameter = 122 cm, thickness = 1.2 cm) with 40 escape holes (D = 5 cm)
   Note: 16 holes on the outer ring, 16 on the middle ring and 8 holes on the inner ring with distance of 50, 35 and 20 cm to the center of platform, respectively (Figure 1B). ABS tubes (Inner diameter = 52 mm, outer diameter = 60 mm) are attached to each escape hole which allows easy attach and detach of the escape tube (Figure 1D). It is important to use black or dark colored escape tube so mice would prefer to enter. In addition, dark color minimizes the chance of leaving visual clues around escape holes over time.
   
5. A 3” ABS plug (NIBCO, catalog number: 5818 )
   
6. Escape tube assembled using black ABS pipes (2”, NIBCO, catalog numbers: C5806-2 and C5807-V Figure 1E) with a removable plug (to be attached to escape holes) at the other end (NIBCO, catalog number: 5818 , Figure 1F). When connected to the escape hole, the resulting depth from the top pf maze to the floor of escape tube is about 8 cm
   
7. A metal stand to support the platform to 90 cm above the floor (Figure 1 D)
   
8. A small non-transparent transfer box
   Note: We use a pipette tip box without the lid (Figure 1I).
   
9. Fish net with extended handle or a similar item (Figure 1I)
   
10. Timer
    
11. Speakers capable of playing at 85 dB or louder (Figure 1B)
    
12. Noise-cancelling headphones or similar items (Figure 1I)
    
13. Overhead camera (GigE, catalog number: XCFS-BC6o# )
    
14. A computer (with Windows 7 64bit Professional) connected to the camera
    
    
    Figure 1. Platform and room setup. A. Sketch of the behavior room and holding room layout; B. A picture showing the details of platform layout and visual clues on two sides of the platform; C. A picture of visual clues on other two sides; D. A picture showing the bottom of platform; E-H. Pictures to show the escape tube, an escape hole and how they are connected; I. Other items needed for the task.
    

Software

1. Recording and tracking software (Noldus, Ethovision XT v 11.5.1026)
   
2. Audio file of a recorded tone at 960 Hz with > 90 sec length (audio file 1)
   

Procedure