Conditioning chamber

Conventional Pavlovian fear conditioning was performed as previously described^{58 (link)}, with minor modifications. One day before the conditioning day, each mouse was habituated in a cued recall box for 20 min. On the conditioning day, the mouse was placed in the conditioning chamber (Coulbourn Instruments). After 3 min of exploration, a 30-sec (86 dB, 3000 Hz) auditory conditioned stimulus (CS) was delivered. In the last 2 sec of the CS, an aversive unconditioned stimulus (US, 1 sec foot shock at 0.7 mA) was delivered. For the conditioning, the mice underwent four CS-US pairs separated by 150-sec intervals. Twenty-four hours after training, the contextual fear memory was tested in the same chamber for 5 min in the absence of the auditory stimulus and shock. After 3 hrs, the auditory fear memory was tested in a cued recall box, which differed from the conditioning chamber. After 10 min of exploration time, the 30-sec auditory CS was delivered. The freezing behavior of the mouse was recorded and automatically analyzed with FreezeFrame software (Actimetrics) using the significant motion pixels (SMP) algorithm.

Rats were trained to associate a sensory stimulus with a foot shock in an conditioning chamber12 (link) (Coulbourn Instruments). The floor was made of stainless steel rods connected to a shock generator set to deliver 0.7 mA current. The chamber was fitted with a loudspeaker connected to a tone generator set to deliver an 80 dB, 1,000 Hz pure tone (auditory CS), and a 12 W fluorescent light bulb (rise and decay times, 100 ms; visual CS); both the loudspeaker and light bulb were located 20 cm above the floor. One animal at a time was placed inside the chamber and left undisturbed for 2 min. Then, it was exposed to a series of seven consecutive auditory or visual CSs, each lasting 8 s and paired, during the last 1 s (for auditory conditioning) or 2 s (for visual conditioning), respectively, with an electric foot shock; the seven sensory stimuli were separated by intervals of 22 s.

We conducted the trace-fear conditioning test, which reflects hippocampal-dependent memory, without surgery (Phillips and Ledoux, 1992 (link)), to confirm the cognitive dysfunction in 6-month-old SAMP8 in comparison with SAMR1. Each mouse was placed in a commercially available conditioning chamber (Panlab Inc., Barcelona, Spain), and underwent a training session consisting of eight cycles with a 0.8-mA foot shock interspersed by a randomly selected 1–4 min interval. The mice were returned to the same chamber 3 days after training and underwent the 4-min memory retention test. Subsequently, the behavior of mice was analyzed by an investigator who was blinded to the mouse species, and the duration of the freezing behavior was measured. Freezing was defined as the lack of movement, except for breathing. The freezing behavior was expressed as a percentage of the 4-min retention period.

—On Day 1 (PND 27), the previously operated rats, as described earlier, were placed in a conditioning chamber (Panlab, Spain) with a grid floor, black methacrylate walls, and transparent front door. For conditioning, they received three 0.35- to 0.4-mAMP, 0.5-second foot shocks delivered through the grids and administered 90, 210, and 330 seconds after the beginning of the session.
For recent CFC tests, the animals were placed in the context in which conditioning took place for 5 minutes on the day after conditioning (PND 28; T1) for a retrieval test and for 10 minutes on the following 2 days (PND 29–30; T2–T3) to induce extinction.
For remote CFC tests, a month after conditioning, the cannulated animals, as described earlier, were placed in the context where conditioning took place for a 5-minute retrieval test and for 10 minutes on the following 2 days for extinction (T2–T3).
The level of freezing (i.e., cessation of mobility except for breathing) was measured by means of a high-sensitivity weight-transducer system connected to the grid floor using an analog signal generated in response to animal movement and transmitted to the software module for analysis. Freezing was represented as a percentage of the time the animal was in a state of freezing during the test. After each behavior session, the shock grids and walls were cleaned with 70% ethanol and dried with a paper towel.

Contextual FC tests were performed using a conditioning chamber (Panlab). In the training trial, mice were allowed to move freely in the chamber for 3 minutes and then given a shock (2 seconds, 0.75 mA), and the mice were then allowed to stay in the chamber for another 2 minutes. Twenty-four hours after training, the mice were subjected to testing of the hippocampus-dependent memory. Mice were put into the same chamber as the previous day for 5 minutes, and the freezing time was recorded by a tracking system (Panlab).