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).