2.7. Contextual fear conditioning test

The apparatus consisted of a transparent chamber inside an enclosure with an opening in the ceiling to allow video recordings. The chamber consisted of a steel grid floor connected to a shock generator scrambler. The test encompassed two sessions: conditioning and a context test. On the conditioning day, the mice were individually placed in the chamber and allowed to explore freely for 5 min during which, at the 180th second, they received a foot shock of 0.50–0.80 mA for 3 s through the bars of the floor. Twenty-four hours after conditioning, the mice were individually placed back in the chamber for 4 min, this time with no noxious stimuli. The mice were monitored for movement and freezing behaviour was recorded using computer software (Limelight, ActiMetrics, Wilmette, IL, USA). Exclusion criteria were set for freezing events of less than 2 s. This test was used to determine associative working memory. We explored the animal's ability to associate an environment with a noxious event that it experienced there. When the animal is returned to the same environment, it generally will demonstrate a freezing response if it remembers and associates that environment with the shock. Freezing is a species-specific response to fear, which is defined as “the absence of movement except for respiration”. This may last for seconds to minutes depending on the strength of the aversive stimulus and whether the subject is able to recall the shock [36,37].