A video fear-conditioning system and software (Med Associates, Burlington, VT, USA) were used to measure freezing in the ICR mice while in a conditioning chamber (dimensions: 30.5 × 24.1 × 21.0 cm), which was cleaned in between testing with Vimoba, a chlorine dioxide solution. Freezing was defined as no movement other than respiration.28 (link) Mice were moved to a holding room adjacent to the test room and acclimated for 30 min (min) under 175–177 lux room lighting before testing.
For fear-conditioning procedures, mice were moved to the test room under the same conditions (175–177 lux) and then placed in the conditioning chamber for 30 s. After an initial one-half min of habituation (baseline), ICR mice were presented with six conditioned stimulus–unconditioned stimulus (CS–US) pairings (for example, tone–footshock pairings) separated by a 30-s interval. Each tone (80 dB, 3000 Hz) lasted 30 s. Mice were presented with the electric footshock at 0.7 mA during the last 2 s of each 30-s tone. The number of CS–US pairings and footshock intensity was used because ICR mice only present an appreciable increase in freezing behavior by the fourth CS–US presentation, demonstrating acquisition of conditioned fear learning (see Figures for illustrated examples). At the end of the fear-conditioning protocol, mice were placed in their home cages in a separate holding room before being returned to their housing facility.
The following day, mice were placed in the conditioning chamber with a white smooth floor contextual insert that was positioned over the grid floor and a white curved wall contextual insert (context B). Vanilla extract (McCormick, Sparks, MD, USA) was used as a distinctive olfactory cue in the conditioning chamber for the short-term extinction training. After 30 s of habituation in the chamber, mice were presented with 20 tones (80 dB, 3000 Hz) with durations of 30 s, each separated by a 30-s interval. After 20 min and 30 s, mice were returned to a holding room and their home cages.
The extinction protocol was repeated the following day using the same procedure and drug administration. For contextual fear conditioning, an identical paradigm was used except that all tones were omitted on all days of the experiment, and the extinction context was identical to that of the conditioning context. In overtraining experiments, mice were conditioned as described above for 5 consecutive days with the exact same procedure. The short-term extinction training procedure thereafter was identical to that described for the single conditioning experiments.
For fear-conditioning procedures, mice were moved to the test room under the same conditions (175–177 lux) and then placed in the conditioning chamber for 30 s. After an initial one-half min of habituation (baseline), ICR mice were presented with six conditioned stimulus–unconditioned stimulus (CS–US) pairings (for example, tone–footshock pairings) separated by a 30-s interval. Each tone (80 dB, 3000 Hz) lasted 30 s. Mice were presented with the electric footshock at 0.7 mA during the last 2 s of each 30-s tone. The number of CS–US pairings and footshock intensity was used because ICR mice only present an appreciable increase in freezing behavior by the fourth CS–US presentation, demonstrating acquisition of conditioned fear learning (see Figures for illustrated examples). At the end of the fear-conditioning protocol, mice were placed in their home cages in a separate holding room before being returned to their housing facility.
The following day, mice were placed in the conditioning chamber with a white smooth floor contextual insert that was positioned over the grid floor and a white curved wall contextual insert (context B). Vanilla extract (McCormick, Sparks, MD, USA) was used as a distinctive olfactory cue in the conditioning chamber for the short-term extinction training. After 30 s of habituation in the chamber, mice were presented with 20 tones (80 dB, 3000 Hz) with durations of 30 s, each separated by a 30-s interval. After 20 min and 30 s, mice were returned to a holding room and their home cages.
The extinction protocol was repeated the following day using the same procedure and drug administration. For contextual fear conditioning, an identical paradigm was used except that all tones were omitted on all days of the experiment, and the extinction context was identical to that of the conditioning context. In overtraining experiments, mice were conditioned as described above for 5 consecutive days with the exact same procedure. The short-term extinction training procedure thereafter was identical to that described for the single conditioning experiments.
Free full text: Click here
