Contextual fear conditioning (CFC) task

As described previously⁵⁴, mice were subjected to the CFC task in three sessions, each distanced by 24 h: a training session (day 1) and two re-exposure sessions (day 2 and day 3). On day one, mice were subjected to a single fear conditioning training session of 5 min, in which they learned to associate the conditioned stimulus (CS: context) to the unconditioned stimulus (US: a foot-shock). To this end, each mouse was placed in a fear-conditioning chamber (18 × 18 cm², Noldus) with an electrified grid floor. After 120 s of free exploration, the mouse was subjected three times to a foot-shock (0.5 mA; 2 s) delivered through the grid-floor, every 1 min. Mice remained in the conditioning chamber for 1 min after the last shock delivery. On days two and three, each mouse was placed back in the conditioned chamber for 10 and 5 min, respectively, without delivering the electrical foot-shock to test their memory retention and memory extinction. Behavior during all experimental sessions was recorded by a video camera mounted above the ceiling of the cage and connected to a computer equipped with the Ethovision XT software (Noldus). Percentage of time spent freezing (absence of all but respiratory movements for at least 3 s) was scored to assess emotional reactivity during training (day 1) and fear memory during retention test (the first 3 min of day 2 session) and extinction test (first 3 min of day 3 session). All behavioral parameters were scored by an experimenter blind to the animal experimental condition. Pairs of control-mutant littermates were randomly tested in the morning and in the afternoon to control for circadian rhythm.