2.2. Fear conditioning, fear extinction, and extinction recall

Fear conditioning experiments were conducted using eight High Response to Training (HRT) and eight Low Response to Training (LRT) mature male rats. Fear conditioning, extinction, and extinction recall were performed as previously published (experiment 3 from Knox et al. [2012]). All fear conditioning, extinction, and extinction recall experiments were performed in four identical Coulbourn Instruments Rat Test Cages (12"W × 10"D × 12"H) (Whitehall, PA) containing a Shock Floor with 18 current‐carrying metal bars, a wall‐mounted speaker and in‐chamber lighting. Test cages were housed in wooden sound‐attenuating boxes. Tones were delivered via speakers mounted in the housing of the test cages and controlled by FreezeFrame data acquisition software (Coulbourn Instruments). Shocks were delivered through precision animal shockers (Coulbourn Instruments) also controlled by FreezeFrame software. Ceiling mounted cameras recorded behavior for analysis and FreezeFrame was used to assess freezing levels.

As previously published (Knox et al., 2012), two unique contexts were created using two different sets of olfactory and visual cues. Context A consists of 50 ml of 1% acetic acid solution placed in a small dish above the test cage and standard lighting which illuminates the chamber walls of the Rat Test Cages. Context B consists of 50 ml of a 1% ammonium hydroxide solution placed in a small dish above the test cage along with patterned paper placed on the chamber walls to alter the visual context. Other labs, as well as our own, have used these specific methods repeatedly in the past and found no evidence of increased stress with these concentrations of acetic acid or ammonium hydroxide, that is, they show no effects on behavior or HPA response (Knox et al., 2012).

Fear conditioned animals were exposed to five, 1 mA, 1‐s foot‐shocks paired with the cessation of a 10 s 80 dB tone in Context A. The first tone was presented 180 s after the animal was placed in the test cage and the subsequent tones occurred with a 60‐s inter‐tone interval. Sixty seconds after the last tone, animals were removed to their home cages. Fear extinction was conducted 24 hr after fear conditioning and was performed in the distinctly different Context B. Fear extinction consisted of 180 s acclimation to the new context and presentation of 30 ten‐second tones without the paired foot‐shock with each tone followed by a 60‐s inter‐tone interval. Extinction recall was assessed 24 hr after extinction and consisted of the animals being placed back into the same fear extinction context (Context B) for 180 s acclimation followed by 10 tones (60‐s inter‐tone interval), again without foot shock. The percent time spent immobile (freezing) within each 70‐s long block (the 10‐s tone and 60‐s inter‐tone interval combined) was assessed by setting threshold values of movement (number of pixels which moved between frames) via FreezeFrame Software. Threshold values were verified to be similar to experimenter‐confirmed immobility times.