Ethovision video tracking software

Testing was conducted in open-field arenas as described previously^{80 (link)}. Mice were introduced to the arena and allowed to explore undisturbed for 30-min. They were briefly removed from the open fields, during which four identical objects were fixed in the center of each arena. Mice were re-introduced to the arena and tested for an additional 30 min. All sessions were recorded using the Ethovision video tracking software (Version XT 8.5, Noldus Technology, The Netherlands) equipped with three-point (nose-point, center-point, and tail-base) detection settings. Arenas and objects were cleaned with 70% EtOH between trials. As an index of active object exploration, the time spent in contact with the object was scored when the mouse´s nose was located within a 2-cm radius around the object. Latency to approach was indexed as the time elapsed between introducing the object and first contact with the object. Locomotor activity was indexed as the distance moved as a function of 5-min consecutive bins.

We identified sensitive mice using the social interaction test (SIT). Mice were placed in a unique, open-field arena with an interaction zone for two 150-s trials. An empty cage was placed in the interaction zone in the first ‘no target’ trial. Next, a new CD-1 mouse was placed inside the cage in the interaction zone for a ‘target trial’. Ethovision video tracking software was used to track the time spent in the interaction zone (Noldus Technology). We calculated the social interaction ratio by dividing the time spent in the interaction zone in the ‘target trial’ by that of the ‘no target’ trial. Susceptible mice were defined as having a social interaction ratio < 1.

The social approach/avoidance test was conducted in a three‐chamber container (58 × 38 × 41 cm³), with lightweight metal cages on either side (Figure 5a). Experimental rats were allowed to habituate to the chamber for 5 min and then were reintroduced to the “neutral” zone when a novel Long‐Evans rat was placed under the containment cage on one side of the three‐chamber container. The behaviour of the rat was recorded using Ethovision video tracking software (Noldus), which separated the chamber into three zones: the interaction zone (the side that contains the novel rat), the neutral zone (the middle) and the avoidance zone (the side that contains an empty containment cage). The number of respective entries into the avoidance and interaction zones were recorded, as well as the total distance travelled (cm).

For the NORT, a white, cubic box (60 × 60 × 60 cm³) was used as the open field arena. Three identical, cylindrical, blue objects were used (O1, O2, and O3). One orange, square object was used as the novel object (N). A video camera was installed on top of the apparatus and was connected to EthoVision video tracking software (version xt8; Noldus Information Technology, Wageningen, The Netherlands) [30 (link)].
The test was performed in three stages: habituation, familiarization, and test stages. The duration of each stage was 10 min, separated by 6 h intervals. The entire procedure was performed on all rats in the four groups.
In the habituation stage, the animals were allowed to explore the empty box. In the familiarization stage, two objects (O1 and O2) were placed at two poles of the box, about 5 cm from the walls. The animals were allowed to explore them for 10 min. The procedure was repeated in the test stage, but O1 and O2 were replaced with O3 and N. Exploration was considered if the rats touched or sniffed the object with the nose [31 (link)]. Rats with normal memory functions explored N more. The box was cleaned between each stage.
The total exploration time in the test stage was calculated as the total time consumed exploring both objects using the discrimination index (DI) [32 (link)]: $$DI=\frac{\left(N-O3\right)\times 100}{\left(N+O3\right)}$$