3L ?? 29.5W ?? 29.5H cm; Coulbourn Instruments.). The total floor area of each chamber 17-AAG IC50 was 746 cm2. The chambers were constructed from aluminum (sidewalls and ceiling) and Plexiglas (rear and front walls). They were placed individually in sound-attenuated cabinets with black inside walls (interior dimensions: 43.3L ?? 55.3W ?? 58.5H cm; Coulbourn Instruments.), which were located in a dedicated room. A ventilation fan in each cabinet provided 50 dB of background noise, and a 24V DC white light, mounted on a wall of each chamber, provided illumination (65 lux at the floor level). A speaker mounted in the wall opposite to the light delivered an acoustic CS. The floor of each chamber, which consisted of 26 stainless steel rods (3 mm in diameter) spaced 11 mm center to center, was wired to a precision-regulated shocker (H13-15, Coulbourn Instruments).
A camera mounted above the chamber recorded mouse activity. Conditioning was assessed by the analysis of the fear response expressed as freezing behavior with the aid of the FreezeFrame program (v. 3.06, Actimetrics). Freezing was defined as the cessation of all movements other than respiratory activity [43]. Conditioning procedure and memory tests Mice were exposed to the context of a training chamber and a tone, both initially novel and neutral stimuli, in one training session. They were transported in squads of four in individual containers filled with home cage bedding and placed singly in the conditioning chamber. During training, the mice received two pairings between a tone (80 dB, pulse (six clicks per second (c.p.
s)), 30 seconds duration) and a 0.45 mA foot shock (two seconds duration, co-terminated with a tone). The first CS-US pairing was delivered at the end of 120 seconds of the initial exploration of the chamber, and the second following a 60-second interval. After the second CS-US pairing the mice were given a 60-second post-training period. The total duration of the training session was 300 seconds. After a day of recovery, the mice were returned to their respective conditioning chambers and tested for fear-induced freezing to the context in which they received foot shocks. The test, carried out in an extinction mode with no shock administered, lasted 300 seconds. The following day, the mice were tested for the association between the tone and the foot-shock in a modified chamber.
The floor and the walls of the chamber were replaced with plastic inserts (opaque white for the floor, and semi-transparent white at the front and opaque green at the back for the walls), which also eliminated Batimastat corners in the chamber. The total floor area selleck chemical of the modified chamber was about 671 cm2. A Petri dish containing a drop of a Pure Lemon Extract (McCormick) was placed underneath the floor of each chamber to provide a distinct novel odor in the chamber. The above modifications did not change the light intensity in the chamber. The tone test lasted 360 seconds.