Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Fear conditioning test
• Data

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

1. Mice are allowed to become acclimated to the testing room for about 20 min before the start of testing each day.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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:
  • First minute
• 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