Anymaze 6

Three days after hypoxic stress, mice were placed in the open field arena (65 × 45 × 65 cm) and video recorded for their locomotor activity over a 30 min period. Time spent in center, total distance, and average speed were tracked and measured using the ANY-maze 6.1 automated software (Stoelting, Wood Dale, IL). Experimenters were blinded during video and statistical analysis.

The number of nose poke responses was counted by a software (MED-PC V, Med Associates, Inc). To quantify individual preferences of altruistic over selfish responses or left and right nose pokes we calculated a decision preference score, as following: (number of altruistic responses - number of selfish responses) / total number of responses. Video images were analyzed a posteriori for scoring of exploratory behavior using Anymaze 6.2 (Stoelting, UK) and Boris.⁴⁶ We measured the time spent by the recipient and the actor in social exploration in the area (highlighted in red, Fig. 1h) in the proximity of the adjacent compartment, where they could explore each other.

The apparatus consisted of two identical and adjacent fear conditioning chambers (Ugo Basile, 24×20×30 cm) separated by a transparent Plexiglas partition. Olfactory and auditory cues could be transmitted between the chambers. A demonstrator mouse (previously recipient in the SDM task) and an observer (previously actor in the SDM task) were individually placed in the two chambers and allowed to explore the chambers for 5 min (baseline). Then, a 2-s foot shock (0.7 mA) was delivered every 10 s for 4 min to the demonstrator mouse using a behavior tracking software (Anymaze 6.0, Stoelting). The same pairs of mice tested in the SDM were used. Based on previous studies,^{28 (link)} we used 10-s intervals for foot shocks and a 4-min training. At the end of the procedure mice returned to their home-cage.

Experiments were conducted in a standard operant chamber (actor’s compartment, L: 24 cm x W: 20 cm x H: 18,5 cm; ENV-307W-CT; Med Associates, Inc.) fused with a custom-made small triangle-shaped chamber that hosted the recipient (L: 18 x W: 14 cm x H: 18,5). The separation wall between the compartments (operant chamber and recipient chamber) was replaced by a metal mesh with 1cm holes that allowed social exploration and nose-to-nose interaction. The actor’s compartment was equipped with two nose poke holes and a food magazine between them, for delivery of food rewards (14 mg; Test Diet, 5-TUL). The recipient’s compartment presented only a food magazine connected to a food dispenser. The setup was placed inside a sound attenuating cubicle (ENV-022V, Med Associates, Inc) homogeneously and dimly lit (6 ± 1 lux) to minimize gradients in light, temperature, sound, and other environmental conditions that could produce a side preference. All tasks were controlled by custom scripts written in MED-PC IV (Med Associates, Inc.). A digital camera (Imaging Source, DMK 22AUC03 monochrome) was placed on top of the setup to record the test using a behavioral tracking system (Anymaze 6.0, Stoelting).