Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Procedure: Observational fear learning (OFL)
• Procedure: Pavlovian fear conditioning (FC)
• Procedure: Open field test (OFT)
• Procedure: Elevated plus maze (EPM)
• Procedure: Sociability testing
• Data

Workflow and sampling

Test	Procedure	Equipment	Data collected
1	Observational fear learning (OFL)	Observational fear apparatus	Percent time freezing during conditioning and during contextual exposure (contextual memory)
2	Pavlovian fear conditioning (FC)	Fear-conditioning chamber	Percent time freezing during contextual exposure (contextual memory)
3	Open field test (OFT)	Open field arena	Locomotor activity and percent time spent in center
4	Elevated plus maze (EPM)	Elevated plus maze	Percent time in open arms
5	Social behavior	Three-chambered social approach apparatus	Time spent with novel mouse 1 (stranger 1), time spent with novel object, time spent with stranger 1 (mouse 1), time spent with stranger 2

Equipment, software and supplies

• Observational fear learning apparatus
  • Contains two identical chambers -- (each 18x17.5x38 cm) containing a transparent Plexiglas partition in the middle and a stainless-steel rod floor (5-mm diameter rods, spaced 1 cm apart) -- which are modified using a passive avoidance cage (Coulbourn Instruments, Whitehall PA)
• Animal shocker (Coulbourn Instruments, Whitehall PA)
• FreezeFrame software (v3.32; Coulbourn Instruments, Whitehall PA)
• FreezeView software (Coulbourn Instruments, Whitehall PA)
• Fear-conditioning chamber (Coulbourn Instruments, Whitehall PA)
• Open field box
  • 50x45x40 cm, made of gray plastic walls and the center defined as a square area (20x20 cm)
• Video camera
• MATLAB
• Elevated plus maze
  • Contains two white open arms (25x8x20 cm), two black enclosed arms (25x8x20 cm) and a central platform (8x8x8 cm) in the form of a cross; 50 cm above the floor
• Three-chambered social approach apparatus
  • Contains an opaque-white walled Plexiglas arena (60x40x22 cm) divided into a center chamber and two side chambers; retractable doors built into the two dividing walls allows access to the side chambers
• EthoVision XT software v9 (Noldus, Wageningen NETHERLANDS)

Procedure: Observational fear learning (OFL)

1. Naive mice (one observer, one demonstrator; non-siblings, non-cage mates) are individually placed in a chamber for 5 min.
2. For conditioning (training), a two-second foot shock (1 mA) is delivered every 10 s for 4 min to one of the mice (demonstrator) via a computer-controlled animal shocker.
3. For contextual memory, the observer mouse is placed back into the training context 24 h after training (without demonstrator mouse); freezing behavior is observed for 4 min.
4. Behavior is recorded and analyzed with software (FreezeFrame and FreezeView).

Procedure: Pavlovian fear conditioning (FC)

1. Naive mice are placed in the fear-conditioning chamber on training day.
2. After a 5-min exploration period, three foot shocks (0.7 mA/s) separated by 1-min intervals are delivered. After an additional 1-min, mice are returned to their home cage.
3. After 24 h, mice are placed back into the FC chamber and behavior is recorded for 5 min and analyzed with FreezeFrame software.

Procedure: Open field test (OFT)

1. Naive mice are placed in the periphery of the field and the paths of the freely exploring mice are recorded for 30 min by a video camera.
2. Videos are analyzed using custom made software based on MATLAB.

Procedure: Elevated plus maze (EPM)

1. Naive mice are placed on the center section with their heads directed toward one of the closed arms.
2. Total time spent in each arm or center was analyzed by video monitoring for one 5-min trial.

Procedure: Sociability testing

1. Naive mice are placed in the apparatus and allowed to acclimate for 10-min (access to all three chambers).
2. Retractable doors built into the dividing walls of the chambers are then closed with the test mouse left in the center chamber.
3. A novel object (inverted steel-wire cage) is placed in one side chamber and a novel mouse (stranger 1) contained in a similar inverted cage is placed in the other chamber.
4. After both wire cages are positioned, the two side doors are lifted and the test mouse is allowed access to all three chambers for 10 min.
5. At the end of the 10-min sociability test, each mouse is further tested in a third 10-min session to quantify preference to spend time with a new stranger (stranger 2).
6. Movement of test mice is video-recorded and the amount of time spent in each chamber is analyzed using EthoVision XT software.
7. Note that mice used as stranger 1 and stranger 2 are age- and sex-matched mice of the same strain as the test mouse.

Data collected by investigator

• Observational fear learning: percent time freezing during conditioning phase
• Observational fear learning: percent time freezing during contextual exposure
• Fear conditioning: percent time freezing during contextual exposure
• Open field test: locomotor activity (distance per second)
• Open field test: percent time in center of arena
• Elevated plus maze: percent time in open arms
• Sociability test: time spent with novel mouse (stranger 1)
• Sociability test: time spent with novel object
• Sociability test: time spent with stranger 1
• Sociability test: time spent with novel stranger 2