Ethovision xt 12

The open field test was used to evaluate spontaneous activity and exploration behaviors. Mice moved freely in the box (60 cm × 60 cm × 25 cm) for 10 min, and then the distance traveled, the time spent in the center, and the number of entrances into the center area was recorded using the videotracking software EthoVision XT 12 (Noldus). Short-term memory was assessed by Y-maze test. The maze included the starting arm, the NA, and the other arm. Before the test, mice underwent a 5-min training period with a block of the NA in the maze. Two hours later, the NA was opened, and the mice were allowed to travel freely throughout the three arms, with the percentage of time spent in the NA and the number of entries into the NA in 5 min recorded by ANY-maze (Stoelting, America).

In order to control for the potentially confounding effects of hypo- or hyperactivity on the other behavioral assays, exploratory activity in a novel open arena was evaluated over a 30 min session. Rats were placed in the center of the arena at the start of the testing session. Using methods similar to those previously described^{48 (link),54 (link)}, total distance traveled and time spent in the center of the arena were measured using one of two comparable automated systems: an opaque matte black arena (54.1 cm l × 54.1 cm w × 34.3 cm h) equipped with video tracking software (EthoVision XT 12; Noldus Information Technology, Wageningen, Netherlands) or the fully automated Digiscan Animal Activity Monitors with Integra software (Omnitech Electronics, Columbus, OH, USA). The testing room was illuminated to ~30 lux.

In embryos at 30 hpf, coiling behavior was measured as recently described by Iacomino et al. [68 (link)] using the Danioscope software (Noldus Information Technology, Wageningen, The Netherlands). At 120 hpf, larval locomotion (distance and velocity) and thigmotactic behavior were measured using the Daniovision system connected with Ethovision XT12 (Noldus Information Technology), a specific video tracking software. Briefly, single larvae were taken from the rearing dishes and transferred into a 24-well plate along with 1 mL of egg water per well (1 larva/well). The plate was then placed in the Daniovision system and larval behavior was monitored for a total of 10 min, following Schnörr et al. [54 (link)]. The procedure was performed in two steps, including a 6-min acclimatization phase (minutes 0–6) and a 4-min interaction phase (minutes 7–10). In the acclimatization period, lights were kept ON (intensity level: 100%) and at minute 7 lights were turned OFF abruptly and were kept OFF until the end of the procedure. In order to measure thigmotaxis, a distinction was made between the inner and outer zone of each well. The width of the outer zone was set at 4 mm from the border of the well, while the diameter of the inner zone was set at 8 mm (see Figure 9).

The foraging track consisted of an elevated long linear chamber (150 × 32 × 25, L × W × H in cm) that was divided into 3 zones: trigger zone, corridor, and reward zone. The ends of the track were designated as the trigger zone and reward zone, while the center served as the corridor. This task consisted of 60 min sessions of self-paced trials. For each trial, food restricted (85% FFW) mice were trained to wait in the trigger zone for an auditory cue that signaled reward availability. Mice were then required to run from the trigger zone to the reward zone to retrieve a food reward (strawberry Ensure). Lastly, mice returned to the trigger zone to initiate another trial. The movement of the mice was tracked with an overhead camera and by digital distance sensors 15 cm (Pololu Robotics and Electronics) located throughout the track and at the entrance of the reward, trigger zone, and food port. Video tracking was performed using Ethovision XT 12 (Noldus) software. Strawberry Ensure solution was delivered through a liquid dispenser (Med-associates Inc.) localized at the end wall of reward zone, 2 cm above the floor of track. Experimental schedule and data acquisition were implemented through the Abet II software for operant control (Lafayette Instruments Neuroscience) and through the Whisker multimedia software (Lafayette Instruments Neuroscience).

The protocol used in the present study was the ‘modified version of the FST’ (Cryan et al., 2002 (link)), adapted from the original version described by Porsolt et al. (1977 (link), 1978 (link)). Swimming sessions were conducted by placing animals in individual Plexiglas cylinders (Malaga, Spain; 26×10 cm), containing 15 cm deep water at 23-24°C. The 6 min test session was recorded using a video camera positioned in front of the tank. The test was conducted as described in Moreno-Fernández et al. (2017) (link). As previously described (Juszczak et al., 2008 (link); Wong et al., 2017 (link)), automatic measurements of floating/immobility (immobility threshold of <12%) and climbing/struggling (threshold of >18.5%) were obtained twice per second using the software EthoVision XT 12 (Noldus Information Technology, Wageningen, The Netherlands).