Project
protocol
-
Contents
Workflow
and
sampling
Step |
Procedure |
Equipment |
Data
collected |
1 |
Day 1: Conditioning (training)
|
Fear conditioning monitor |
Beam breaks before and after each tone-shock pairing |
2 |
Day 2: Fear conditioning to context |
Fear conditioning monitor |
Beam breaks over a 5 min period |
3 |
Day 2: Fear conditioning to altered context |
Fear conditioning monitor |
Beam breaks over a 3 min period |
4 |
Day 2: Fear conditioning to cue |
Fear conditioning monitor |
Beam breaks over a 3 min period |
Equipment
and
supplies
- Fear conditioning monitors (San Diego Instruments, San Diego CA)
- Inside dimensions: 25 (L) x 21 (W) x 18 (H) cm
- Grid floor connected to a shock generator (San Diego Instruments, San Diego CA)
- Equipped with a 12 x 10 photobeam array (spaced 2 cm apart)
- Enclosed in sound-attenuating chambers (63 (L) x 40 (W) x 41 (H) cm) with 15 W shielded light bulb and small fan
- PC computer and software (San Diego Instruments, San Diego CA)
Reagents
and
solutions
- 5% ammonium hydroxide (for cleaning chambers and to reduce residual stress odors)
Procedure: Fear conditioning test
- Mice are allowed to become acclimated to the testing room for about 20 min before the start of testing each day.
- During the first day of testing (training), the mouse is removed from its home cage and placed in the center of the fear-conditioning monitor, and the computer program is initiated.
- Conditioning consists of three pairings of an auditory stimulus with mild foot shock:
- The tone (90 dB) is delivered 150 s after the session starts and is 7 s in duration.
- The foot shock (0.5 mA scrambled) is administered only during the last 2 s of the auditory stimulus presentation.
- This process is repeated two more times over the 10-min session, with an inter-trial interval of 150 s.
- The mouse is removed from the fear-conditioning monitor 143 s after the last presentation of the foot shock and returned to its home cage and all mice returned to the colony room after the last mouse is tested.
- The chamber is cleaned with a solution of 5% ammonium hydroxide between each mouse tested.
- Fear conditioning to context:
- Approximately 24 h after conditioning (training), mice are placed in the fear conditioning monitor for 5 min, and the number of beam breaks is recorded per minute.
- Fear conditioning to altered context:
- One to two hours after conditioning to context, mice are tested for their behavior in an altered context:
- Monitor is altered by placing a sheet of gray Plexiglas over the grid floor, two sheets of off-white poster board over the beige walls of the sound attenuating chamber, and a striped panel on the back wall; fan is turned off and a novel odor (4-5 drops of McCormick orange oil extract in a cup suspended from the top left-hand side of the chamber) is added.
- Each mouse is placed in the altered context monitor, and the number of beam breaks is recorded over a 3-min period.
- Fear conditioning to cue:
- The tone is presented at 180 s time point in relation to the start of altered context testing for a duration of 5 s and again at 240 s and 300 s.
- After the last presentation of the tone, the mouse remains in the altered monitor for an additional 55 s.
- The number of beam breaks is recorded during a 3 min period.
- At the end of the session, the mouse is returned to its home cage.
Data
collected
by
investigator
- Conditioning (training), number of beam breaks:
- Prior to first tone-shock pairing (baseline)
- After first tone-shock pairing
- After second tone-shock pairing
- After third tone-shock pairing
- Mean
- Percentage of baseline activity that occurred after third shock of training period
- Fear conditioning to context, number of beam breaks:
- First minute
- Second minute
- Third minute
- Fourth minute
- Fifth minute
- Mean over 5 minutes
- Percentage of baseline activity that occurred after contextual conditioning
- Fear conditioning to altered context, number of beam breaks:
- Second minute
- Third minute
- Mean
- Fear conditioning to cue (tone), number of beam breaks:
- First minute
- Second minute
- Third minute
- Mean
- Percentage of altered context activity that occurred after cued conditioning
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