|Observational fear learning (OFL)
||Observational fear apparatus
Percent time freezing during conditioning and during contextual exposure (contextual memory)
|Pavlovian fear conditioning (FC)
Percent time freezing during contextual exposure (contextual memory)
||Open field test (OFT)
||Open field arena
||Locomotor activity and percent time spent in center
||Elevated plus maze (EPM)
||Elevated plus maze
||Percent time in open arms
||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
- 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
- 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)
- Naive mice (one observer, one demonstrator; non-siblings, non-cage mates) are individually placed in a chamber for 5 min.
- 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.
- 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.
- Behavior is recorded and analyzed with software (FreezeFrame and FreezeView).
Procedure: Pavlovian fear conditioning (FC)
- Naive mice are placed in the fear-conditioning chamber on training day.
- 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.
- 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)
- 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.
- Videos are analyzed using custom made software based on MATLAB.
Procedure: Elevated plus maze (EPM)
- Naive mice are placed on the center section with their heads directed toward one of the closed arms.
- Total time spent in each arm or center was analyzed by video monitoring for one 5-min trial.
Procedure: Sociability testing
- Naive mice are placed in the apparatus and allowed to acclimate for 10-min (access to all three chambers).
- Retractable doors built into the dividing walls of the chambers are then closed with the test mouse left in the center chamber.
- 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.
- 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.
- 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).
- Movement of test mice is video-recorded and the amount of time spent in each chamber is analyzed using EthoVision XT software.
- Note that mice used as stranger 1 and stranger 2 are age- and sex-matched mice of the same strain as the test mouse.
- 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