Contextual fear conditioning

The contextual fear conditioning test for Cohort S2 was performed for successive 2 d on the fear conditioning test system (O’HARA & CO., LTD). On day 1, the mice learned the association between the context and electric footshock (acquisition). The experimenter wearing a white lab coat moved mice homecage from the breeding rack to the standby rack in the experimental room, 30 min before the trial started. Immediately before the trial started, the experimenter moved each mouse from the homecage to the chamber by using the delivery cage filled by woodchip. The acquisition chamber was composed of an acrylic cube (17 × 15 × 12 cm), with a wall of black and white stripes. The trial was started after the mice entered the chamber, and lasted for 9 min. At 148 s from the trial start, an electric foot-shock (0.4 mA) was given from the grid floor for 2 s in duration, and was repeated five times with 90-s interval. After the trial ended, the mice were returned to the homecage via the delivery cage. The soundproof box, chamber, and the grid floor were cleaned with 70% ethanol before each trial started. The brightness in the chamber and experimental room was 70 and 80 lx, respectively.

Twenty-four hours after the acquisition, the mice were exposed to a novel context to confirm that the generalization of the fear memory did not occur (retrieval 1). In this context, the soundproof box and the chamber was cleaned with 70% propanol. The retrieval chamber was composed of an acrylic white cube (18 × 11 × 10.5 cm) paved by latex sheets. The room light was turned off, while the concealed light was turned on, such that the brightness in the chamber and the experimental room was 40 and 8 lx, respectively. The experimenter moved each mouse directly to the retrieval chamber from the homecage in the breeding rack, then trial was started. Each trial lasted for 6 min, and no foot-shocks were given. After the trial ended, the experimenter returned each mouse directly to the homecage from the chamber. Two hours after retrieval 1, the mice were re-exposed to the acquisition context (retrieval 2). The environment and procedure were the same as in the acquisition, except that the duration per trial was 6 min, and no foot-shocks were presented.

All behaviors in the chamber were recorded by a camera at 2 Hz. Freezing response as a conditioned response in each trial was detected when the number of pixels whose intensity changed between successive two frames were fewer than 30, and this state continued for >2 s. Freezing rate was calculated as a percentage of total duration of freezing response to total recording time for each mouse in each trial. We assume that contextual fear memory is formed if the freezing rate in the retrieval 2 was significantly higher than that in the retrieval 1.