Fear conditioning chamber

The fear conditioning training was conducted according to previously described methods^{9 (link)}. Each mouse was placed individually in the fear conditioning chamber (Coulbourn Instruments), which was positioned at the centre of a sound-attenuating cubicle. Prior to each session, the chamber was cleaned with 10% ethanol to provide a background odour, while a ventilation fan generated background noise at around 55 dB. The training began with a 2-min exploration period, after which the mice received three tone-foot shock pairings separated by 1-min intervals. Each tone, an 85 dB 2-kHz sound, lasted for 30 s, and was followed by a 2-s foot shock of 0.75 mA, with both ending simultaneously. Following each pairing, the mice remained in the chamber for an additional 60 s before being returned to their home cages. For context recall, the mice were reintroduced to the original conditioning chamber for 5 min, 16 days after the training. Injections of 4-hydroxytamoxifen injections were administered immediately prior to the recall experiments, within 30 min. In the HC and NR groups, 4-hydroxytamoxifen was injected at a similar time to the other two groups during the recall. The behaviour of the mice was recorded and analysed using FreezeFrame software (version 4; Coulbourn Instruments), with motionless bouts lasting over 1 s being considered as freezing.

Open‐field testing was used to measure the overall locomotor activity, novelty seeking, and anxiety levels of mice. In a white 32 cm × 32 cm × 40 cm arena, mice were placed at the center (20 cm × 20 cm) and tracked for 15 min using a computerized video tracking system. Anxiolytic and exploratory behaviors were characterized by exploring the center of the field rather than staying in the corner. Data were analyzed using the Ethovision 14 software (Noldus). Fear conditioning was performed as previously described with slight modifications.^[51] Briefly, on day one, after exploring a fear conditioning chamber (Coulbourn Instruments) for 3 minutes, mice received three pairs of electrical footshocks (0.5 mA, 2 s) and a conditioned acoustic stimulus (CS, 80 dB, 2800 Hz, 30 s), which were co‐terminated. The CS was delivered with 30 s‐inter‐tone interval. On day two, the mice were placed in the same fear conditioning chamber for 120 s. On the same day, the mice were placed in a novel cylinder chamber and received the CS for 120 s after 120 s baseline period. During all sessions, freezing behavior for at least 1 s was automatically measured using the Freeze Frame software (ActiMetrics, IL, USA).

The mice were put into a standard fear-conditioning chamber (25 × 25 × 25 cm, Panlab) for conditioning training. Firstly, the mice were allowed to habituate for 120 s without any stimulation (acclimation), then they were given 3 consecutive footshocks (0.7 mA, 1 s duration each) through a stainless steel grid floor. Each foot shock was separated by 60 s interval. After the last shock, mice were left in the chamber for additional 60 s before back to the home cages. One (Short-term memory, STM) or 24 hours after training (LTM), mice were placed back to the previous conditioning chamber where training occurred without foot shock. The times of mice with freezing behavior were recorded during the 5 min test.

Contextual fear conditioning was conducted in fear conditioning chamber (Panlab, Barcelona, Spain). In training session, mice were allowed to freely explore conditioning chamber for 2 min. After 2 min, sound pulse (amplitude 1, frequency 400 Hz) was given for 30 seconds and 0.8 mA electric footshock was delivered for 2 sec. After 24 hours, mice were put in the same conditioning chamber to test contextual fear memory for 3 min. The freezing time was recorded and analyzed by software (Panlab, Freezing 2.0 version).

On the training day, the mice were put into a standard fear-conditioning chamber (panlab). Each mouse was in the conditioning context for 2 min, at the end of which, three times of a 1 s, 0.5 mA shocks were given with an intertrial interval of 59 s. Following the last shock, mice remained in the chamber for 59 s before being moved back to their home cages. Twenty-four hours after training, mice were placed back to the previous conditioning chamber where training occurred and the freezing responses were recorded for 5 min without foot shock.

Mice were placed in a fear conditioning chamber (Panlab S.L.) and left to explore for 2 min. Afterwards, they were footshocked with an intensity of 0.4. Freezing (Panlab S.L.) software was set to consider events longer than 2 s of immobility as freezing, as measured by a piezoelectric accelerometer connected to a transducer. Memory for the context in which the animal received the shock was assessed 24 h later as previously described [17 (link)].