Procedure performed Context Stimulus Duration Data collected 1 Pre-training observation Standard none 150 s Baseline freezing behavior 1 Conditioned stimulus (CS) paired with unconditioned stimulus (US); done twice within 30s interval Standard Tone (CS), foot shock (US) 30 s none 2 Measured contextual freezing behavior Standard none 150 s Contextual freezing behavior 3 Measured altered contextual freezing behavior Altered none 150 s Altered contextual freezing behavior 3 Measured freezing behavior during CS and without US; done twice within 30s interval Altered Tone 30 s Freezing to tones
Fear-conditioning chambers obtained from Med Associates (St. Albans, VT) had inside dimensions of 29 cm x 19 cm x 25 cm with metal walls on each side, clear plastic front and back walls and ceilings, and stainless steel bars on the floor. A fluorescent light provided dim illumination (~3 lux) and a fan provided a low level of masking background noise.
Behavior was recorded with digital video and analyzed with FreezeFrame software from Actimetrics (Evanston, IL).
Disinfectant or cleaning solutions and paper towels: for cleaning testing chamber before and after use.
Acclimation to test conditions
Testing is conducted during the light phase between 08:00 and 16:00. Mice are brought into the testing room in their home cages and allowed to adapt for a minimum of 30 min before testing.
a. 7-12-wk-old chromosome substitution strain (CSS) mice of both sexes are first brought into the testing room to acclimate for at least 30 min before any testing is done.
b. Fear conditioning chambers are cleaned and made odor-free, and prepared for testing: a white fluorescent light bulb is dimly illuminated and a fan is turned ON to provide a low level of background noise.
c. On the 1st day (Day 1) of testing, the mice are brought to the fear-conditioning chamber in transfer cages with bedding material and picked-up by their tails with neutral color gloved hands.
d. After allowing the mice to freely investigate the fear-conditioning chamber for 30 s, subsequent baseline (pre-training) freezing behavior is recorded for duration of 150 s.
e. Immediately after measuring pre-training freezing (t = 3 min), the mice are trained to associate a previously innocuous tone (conditioned stimulus, CS) with an aversive foot-shock (unconditioned stimulus, US).
f. The 85 dB, 3 KHz tones are presented for 30 s and then paired with 0.5-mA foot shocks for 2 s, wherein both the tone and the foot shock are simultaneously terminated together, and with a 30 s inter-trial interval.
g. The mice are allowed to recover for an additional 30 s before removing them from the fear conditioning chambers.
h. The following day (Day 2) the same mice above are brought back to the testing room and allowed to acclimate as before. They are also brought to the fear-conditioning chamber in transfer cages with similar bedding material and picked-up by their tails with the same color gloved hands as before.
j. After allowing the mice to freely investigate the fear-conditioning chamber for 30 s, their freezing to context behavior (learned fearfulness attributed to memory of painful foot shock the day before) is measured for duration of 150 s. On this Day 2 of testing, the mice are presented to the familiar fear-conditioning chamber, but in the absence of tones and foot shocks for a maximum duration of 5 min.
k. On the last day (Day 3) of testing, the context of the fear-conditioning protocol is altered: the fear-conditioning chamber is cleaned, but made odorous with 0.1% acetic acid solution, the background noise from the fan is slightly muffled by partially obstructing the air vent, the light bulb is covered with a yellow filter, the metal shock grid is hidden with white plastic flooring, and a bent plastic wall is placed within the chamber.
l. The mice are allowed to acclimate as usual, but brought into the fear-conditioning chamber in plain (no bedding) transfer cages, and are picked-up by their tail in differently colored-gloved hands.
m. Following a 30 s acclimation, mice freezing behavior to the altered context of the test chamber is measured for 150 s.
n. After observing the mice for 3 min in the test chamber, the same 85 dB, 3 KHz tones in Day 1 are presented alone without foot shock for 30 s with an inter-trial interval of 30 s.
o. During the presentation of the 2 tones above, the strength of fear-conditioning in Day 1 is reflected by the heightened freezing behavior of the mice in response to the tones that is no longer associated with foot shock.
p. Both the freezing behavior during the conditioned stimulus tones and freezing behavior in between trials are measured.
Pre-training, baseline, freezing behavior, freezing to context, freezing to altered context, and freezing to tones and in between tones.
Definitions & formulas
Conditioned stimulus (CS) is usually an innocuous cue (i.e. tone) that is paired with an aversive stimulus (US) in a classical fear-conditioning paradigm.
Unconditioned stimulus (US) is usually an aversive intervention (i.e. foot shock) that is paired with previously benign stimulus in a classical fear-conditioning paradigm.
Freezing to context = (freezing_shock) – (baseline freezing or pre-training freezing)
Average freezing to tones = [(freezing_tone1) + (freezing_tone2)] ÷ 2