Freezeview automated analysis

Cue fear conditioning and extinction consisted of five training trials on day 1 in context A, a 24-h cue recall test in a novel context B, 3 days of cue extinction training (context B), a 30-day cue recall test (context B), and a 31-day contextual recall test (context A). Context B was a circular apparatus (11.2″H × 10.6″ diameter) with white walls and a blue plastic floor and was cleaned with ethanol with a 0.1% lemon scent between animals. On day 1, animals were conditioned as described for contextual fear conditioning. On day 2, mice received a 24-h cue recall test in a novel circular chamber with green flooring (context B). Following a 2-min habituation period, five 30-s tones were presented and freezing during each of the tones was measured. On days 3–5, mice underwent cued fear extinction following the same protocol as day 2. On day 30, mice received an additional cue recall test, and on the following day (day 31) were placed back into context A for a context recall test. Behavior was recorded using a camera mounted above the apparatus, and freezing was measured using FreezeView automated analysis (Coulbourn Instruments, Whitehall, PA). As all behavioral analyses were performed by objective computerized methods, investigators were not blinded to genotype. For all experiments, outliers were determined using Grubb’s outlier test and removed from the analyses.

Contextual fear conditioning and extinction consisted of five training trials on day 1 followed by a 24-h recall test on day 2, followed by 3 days of extinction training and then remote memory tests on days 30, 60, and 180. On day 1, mice were placed in the conditioning apparatus containing a shock chamber enclosed by a sound-attenuated box (Coulbourn, Whitehall, PA, USA). The conditioning apparatus (context A) was a square apparatus (7″W × 7″D × 12″H) with a rod floor and was cleaned with ethanol with a 0.1% peppermint scent between animals. Mice were given 2 min for habituation, followed by five conditioning trials, each consisting of a 30-s tone presentation (2.9 kHz, 84-dB) that co-terminated with a 1-s foot shock (0.7 mA). Conditioning trials were separated by increasing inter-trial intervals. Mice were returned to their home cage after conditioning. On day 2, mice were placed back into the shock chamber, and after 1 min of habituation, freezing was measured during a 4.5 min contextual recall test. Contextual fear extinction on days 3–5 and remote context memory tests on days 30, 60, and 180 followed this same protocol. Behavior was recorded using a camera mounted above the apparatus, and freezing was measured using FreezeView automated analysis (Coulbourn Instruments, Whitehall, PA).