Materials and Reagents

1. Latex examination gloves (Alliance: LGPF, Safeplus^®)
   
2. Paper towels
   
3. 50-ml polypropylene conical tubes
   
4. C57BL/6 mice
   Note: Animals are described in detailed in Steps A1 and A2.
   
5. 70% ethanol
   Note: The 70% ethanol and paper towels are used to remove mouse excrement from the floor and walls of the U-shaped field and grid cages before the start of each behaviour test sessions.
   

Equipment

1. Two circular grid cages (Figure 1A)
   Two circular grid cages (12 cm in diameter x 33 cm in height) made of tungsten wire.
   Notes:
   1. The grid cage needs to be high enough to prevent subject mice from climbing.
      
   2. It is helpful to cover the upper half of the grid cage with an overhead projector (OHP) film to prevent the subject mouse from climbing, and to add a white opaque partitioning in the middle of the grid cage to block the target mouse from climbing (Figure 1A).
      
2. The U-shaped two-choice field (Figures 1B and 1C)
   The U-shaped two-choice field is a modified open field (45 cm in width x 45 cm in depth x 35 cm in height) that is partially partitioned with a wall (20 cm in width x 35 cm in height) to the central point, so that a ‘U-shaped’ field that contains two closed quadrants and two open quadrants of the same size is formed (Figures 1B and 1C).
   Notes:
   1. The U-shaped two-choice field is a modified open field made of cream colored FOAMEX panel (1 cm in thickness) (Expanded PVC; LG Ltd., Republic of Korea).
      
   2. The floor and walls of the U-shaped two-choice field are made of the same type of FOAMEX panel described above.
      
   3. Alternatives for the walls and floor might be workable, but reflective plastic materials or uncoated veneer plywood is not recommended. The floor should not be slippery.
      
   4. The U-field two-choice test is performed in an isolated room in the absence of potentially disrupting environmental factors. If an enclosed spacious room is not available, we recommend padding the outside walls of the U-shaped field with Styrofoam boards.
      
   5. The behavior test room is lit with 2 or 4 indirect lighting sources to achieve 20 lux on the floor of the U-shaped field.
      
      
      Figure 1. Photos showing the grid cages and U-shaped two-choice field. A. The circular grid cage. The upper half of the grid is covered with a transparent film. A white, opaque-circular partitioning is added in the middle of the grid cage. B. The open field is partially partitioned with a wall projecting to the center. C. A grid cage is placed in each outer corner of the closed quadrants making up the U-shaped two-choice field.
      
      
3. Brightness-adjustable indirect light sources
   
4. Digital Lux meter (TES Electrical Electronic, catalog number: TES-1350A )
   
5. White noise generator (HDT Korea, Genius mate)
   
6. Sound level meter (TES Electrical Electronic, catalog number: TES-1330A )
   
7. Stainless steel large forceps (20 cm in length) (Fine Science Tools, catalog number: 11000-20 ) with protective silicone tip guards (optional)
   
8. Automatic recording systems
   1. A computerized video tracking system (SMART, Panlab, Spain) and PC
      
   2. CCD camera (Samsung, catalog number: SDC-410 ) for video tracking
      
   3. Webcam camera (Logitech, catalog number: C210 ) (used as a supplemental and independent recording system)
      

Software

1. A computerized video tracking system (SMART, Panlab, Spain)
   
2. GraphPad PRISM 6.0 software (GraphPad Software. Inc., San Diego, CA, USA) for data analysis
   

Procedure