Tracking system

Animals were habituated to the box (without objects) for 5 min 24 h before the first trial of the test (Nikiforuk et al. 2013 (link)). The test consisted of two trials (T1 and T2, 5 min each) separated by 1-h intervals. In the first trial (T1), two identical objects were placed in the box 15 cm from the walls. After T1, rats were returned to their home cages. In T2, one of the objects was replaced with a novel object (NO). The location of the novel object was randomly determined for each animal to avoid the occurrence of place preference.
The exploration of an object was defined as looking at, licking, sniffing or touching the object but not leaning against or sitting or standing on the object (Nikiforuk et al. 2016 (link)).
The behaviour of the animals was recorded using a camera placed above the apparatus and connected to the Any-maze® tracking system.

During fear conditioning, a conditional stimulus (CS; tone) is paired with an unconditional stimulus (US; footshock). With repeated CS-US pairings, mice display a conditional response (CR; freezing) to both the CS and training context. Fear conditioning was performed the week following Barnes maze at 15 weeks post-injury. The chamber was cleaned with 70% ethanol between mice. Total freezing time and freezing episodes were recorded daily via the ANY-maze tracking system. Default settings were used, including a minimum freeze duration of 1,000 ms, a “freezing on” threshold of 30, and a “freezing off” threshold of 40. Tone was set at 2 KHz, 80% volume. The protocol used consisted of 3 testing sessions over 3 days (Figure 1D).

The experiments were conducted in an open field arena (length × width × height: 57 × 67 × 30 cm) made of black Plexiglas. The arena was dimly illuminated with an indirect light of 18 Lux. Rats were individually placed in the arena for 5 min. The distance travelled were automatically scored using the Any-maze® tracking system. Additionally, the time spent in the center of the open field was used as a measure of anxiety. The number of rearing episodes was manually scored by an experimenter.

This test assesses cognition and sociability, namely the ability to recognize novel versus familiar animals. This test was performed according to the method outline in previous studies with minor modifications^{90 (link)}. Experiment mice were habituated in the test arena for 10 min/day for 2 days before testing. Age- and size-matched C57BL/6J male stimulus mice were used and habituated to a cylinder holder made of transparent acryl (30-cm high, 10.5-cm diameter, with 16 holes of 0.5-cm diameter) for 30 min before testing. In this test, the same stimulus animal was used for both acquisition and recognition phases (intruder 1). Over the course of multiple exposures, experiment mice became habituated to intruders and no longer regarded them as interesting as they did for completely novel intruders. During testing, each experimental mouse was given four 5-min exposures, with 15–20 min of intervals, in a transparent acrylic box (60 × 60 × 30 cm). In the fifth trial, the experimental mice encountered an entirely novel intruder (intruder 2). All test trials were video-recorded using the Any-maze tracking system, and the total investigation time was subsequently analyzed. The time spent by each mouse (nose-point) in close proximity (~2 cm) to the cylinder holder was measured as interaction time (i.e. sniffing time, approach) to estimate the approach behavior.