2.5 Fear conditioning test
Fear conditioning procedures were performed according to previous protocols, with several modifications (Nassireslami, Nikbin et al., 2013). The tests took place in a 26.5 cm × 27.5 cm × 41.5 cm chamber (UGO BASILE SRL, Gemonio, VA, Italy) with clear Plexiglas walls and a ceiling with holes to allow ventilation. The floor was wired to a shock source and solid-state grid scrambler for the delivery of the foot shock. The chamber was illuminated by a single house light positioned at the top centre of one wall. A video camera was suspended on top of the chamber to record the behaviour of the animals, and an audio generator was mounted at the corner of the back wall. Before conditioning was initiated, the rats were brought to the behavioural assessment room for 1 h, placed individually in the conditioning box for 10 min, and returned to their home cages. This habituation procedure was introduced thoroughly to familiarise the rats with the stimuli of the experimental room, thus preventing any interference from uncontrolled novel stimuli during the experiments. The rats were trained and tested for three days, placed in the chamber on the day of conditioning, and allowed to explore freely for 2 min. After the habituation procedure, an electric foot shock (0.5 mA, 2s) was delivered immediately after a tone (3600 kHz, 20 s). The second cue tone and foot shock were delivered 2 min later. After termination of the shock, the rats were allowed to stay in the chamber for another 2 min before returning to their home cage. The contextual cued fear test was performed on day 2. The rats were, subsequently, returned to the conditioning box. Their behaviours were recorded for 3 min without a tone or foot shock, and the freezing time was automatically recorded. Freezing was defined as the complete absence of movement except for respiratory motion. The auditory-cued fear test was performed on day 3 in an altered chamber. Each rat was placed in a modified chamber and allowed to explore for 3 min, the cue tone (3600 kHz, 20 s) was delivered, and the freezing time was recorded for an additional 3 min. Freezing was presented as a percentage of the observation period. Data were recorded and analysed using a video tracking software (ANY-MAZE, Stoelting Co., Wood Dale, IL, USA; RRID: SCR_014289).