Videofreeze software

Training consisted of exposing mice for 120 s to the context to assess the baseline level of activity. This period was followed by a 5 kHz 80 dB tone (conditioned stimulus (CS)) for 10 s. Immediately after the tone, a 2 s 0.4 mA foot shock (unconditioned stimulus (US)) was applied. The tone and foot shock (CS-US pairing) were repeated once more after a 10 s resting interval. The second foot shock was followed by 30 s period without any sound/shock; this was done to avoid the formation of an aversive association to the handling procedure in the mouse. Mice were trained within the same session for both contextual and cued fear conditioning. The contextual memory test was performed 24 h after the CS-US training. Mice were monitored for 120 s for freezing in the same context as used for training without any tone or foot shock. The cued memory test was performed 4 h after the contextual memory was tested in a new chamber. First, mice were monitored for freezing over a 120 s precue period with no tone to assess freezing in the new context. Next, a 120 s cue period followed in which the tone was presented. Duration of freezing behavior, defined as absolute lack of movement (excluding respiratory movements), was recorded by a video camera and a PC equipped with “Video Freeze” software (MED Associates, St. Albans, Vermont, USA).

On the acquisition day, mice were placed in a near-infrared video fear conditioning chamber (Med Associates, Inc.) for a total of 14 min. During the first 3 min mice explored the chamber, and then a series of 5 tones (3 kHz, 80 dB, 20 s) were played each co-terminating with a shock (0.75 mA, 1 s) administered via the floor rods. There was a 120 s intertrial interval between tone/shock presentations. 24 h later, cue-evoked responses were assessed in a novel context in which lighting, odor, and floor texture had been altered. Mice were in this context for a total of 10 min with the tone continuously presented during minutes 4–6. A no-shock control group was performed with identical methods as described above except that foot shocks were omitted. VideoFreeze software (Med Associates, Inc.) was used to quantify freezing during acquisition and testing. Freezing is quantified during the entirety of acquisition and during the time that the tone is played during the cued test, with freezing normalized to the air-treated condition. Freezing in seconds (mean ± SEM) for each group is reported in Supplementary Table 1.

On the acquisition day, mice were placed in a near-infrared video fear conditioning chamber (Med Associates, Inc.) for a total of 8 min. During the first 3 min mice explored the chamber, and then a series of 5 shocks (0.75 mA, 1 s) were administered via the floor rods with a 60 s intertrial interval. 24 h later, mice were returned to the acquisition context for 6 min in the absence of foot shocks. As with cued conditioning, freezing was quantified during acquisition and testing using VideoFreeze software (Med Associates, Inc.) and normalized to the freezing in the air-treated condition. Freezing in seconds (mean ± SEM) for each group is reported in Supplementary Table 1.