Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Activity in a novel environment using a monitoring system
• Procedure: Novel environment preference using a two chamber apparatus
• Procedure: Head dipping on the hole board
• Procedure: Object preference on the hole board
• Procedure: Spontaneous alternation using the Y-maze
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Activity in a novel environment	Automated activity monitor	Distance traveled, number of rears
2	Novel environment preference	Two chamber apparatus	Activity in familiar and novel compartments
3	Head dipping	Hole board	Distance traveled, activity, number of head dips, time spent head dipping
4	Object preference	Hole board with objects in two of the holes	Distance traveled, activity, number of head dips, time spent head dipping
5	Spontaneous alternation	Y-maze	Alternations

Equipment and supplies

• Automated activity monitor (AccuScan Instruments, Columbus OH)
  • Equipped with infrared beams
• Two chamber box
  • Acrylic
  • 40 x 40 cm total area
  • One black-walled chamber with a white floor; one white-walled chamber with a black floor; separated by a vertically sliding door
  • Equipped with infrared beams
• Hole board
  • 40 x 40 cm board
  • Four 2.9-cm diameter holes, spaced equidistant from one another
  • Equipped with infrared beams
  • Centrifuge caps, black binder clips, or marbles used as objects (each ~1.5 cm across)
• Y-maze
  • Each arm 15 cm tall and 30 cm long and 5 cm wide clear acrylic
  • Angles between arms = 120°
  • Movable, opaque partition

Reagents and solutions

• Solution for cleaning equipment between tests

Procedure: Activity in a novel environment using a monitoring system

1. Mice are placed in the apparatus and allowed to freely explore for 30 min.

Procedure: Novel environment preference using a two chamber apparatus

1. Mice are placed into one compartment of the two chamber apparatus.
2. Initial placement into the black or white chambers is counterbalanced within all genotypes.
3. After a 15-min exposure to one of the compartments, the door is opened, and the mouse is allowed to freely explore for 20 min.
4. Chance performance is indicated by a time of 600 s spent in the novel environment.

Procedure: Head dipping on the hole board

1. Mice are placed in the apparatus and allowed to freely explore for 10 min.

Procedure: Object preference on the hole board

1. Two small objects are placed into two of the holes at opposite corners of the apparatus.
2. Mice are placed in the apparatus and allowed to freely explore for 10 min.
3. Preference is calculated as the amount of time spent head dipping in holes that had objects divided by the total time spent head dipping (percent preference).

Procedure: Spontaneous alternation using the Y-maze

1. Mice are forced to choose one of the two arms by occluding one arm with an opaque partition. Mice are confined to the chosen arm for ~ 30 s and then placed into holding cages while the apparatus is cleaned and both partitions removed.
2. Mice are then placed back into the stem of the apparatus and allowed to choose either the arm they had previously entered or the novel arm.
3. An entrance is defined as all four paws being within the arm.

Data collected by investigator

• Activity in a novel environment
  
  • Distance traveled
  • Number of rears
  • Rears residual
• Novel environment preference
  • Activity in familiar compartment
  • Activity in novel compartment
  • Time in novel compartment
  • Number of transitions between familiar and novel compartments
  • Novel time residual
• Head dipping
  • Distance traveled
  • Activity
  • Number of head dips
  • Time spent head dipping
  • Head dipping residual
• Object preference
  • Distance traveled
  • Activity
  • Number of head dips
  • Time spent head dipping
  • Object preference, % time
  • Object preference residual
• Spontaneous alternation
  • Alternations