Ethovision xt tracking software

The EPM test was used to assess anxiety-like behaviors under stress (25 (link)). Mice were placed in the center of a plus maze that was elevated 50 cm above the floor with two opposite open arms and two opposite closed arms (each arm was 88 cm long, and 28 cm-height walls only on the closed arms) arranged at right angles. The number of entries and time spent in the closed and open arms were monitored for 10 min. The behaviors were video recorded and automatically scored using Ethovision XT tracking software (Noldus, Leesburg, VA).

For the Open Field assay the lights in the room were turned down and mice were allowed to acclimate for at least 2 h prior to testing. The Open Field chamber was constructed as previously described (Golden et al., 2011 (link)). Thirty minutes after injection of either CNO or saline, mice were placed into the open field chamber, next to the edge, and allowed to explore for 5 min while movement was recorded using EthoVision XT tracking Software (Noldus, Leesburg, VA, USA). The open field chamber was cleaned between each mouse with Minncare disinfectant to remove residual odors. During testing, the lights in the room were turned down, providing a dim light environment.

The open field behavioral test was performed as previously described. A 40 cm × 40 cm × 29.5 cm (L × W × H) opaque arena with an open top was centered under a Basler acA1300-60gm camera (Basler, Ahrensburg, Germany) inside of a noise canceling room (MDL 4848 S, Whisper Room Inc., Knoxville, TN, USA). An 8 cm-wide peripheral zone was defined along the edge of the arena (64% of the arena), and the remaining inner region (576 cm²) was defined as a central zone (36% of the arena area) (Supplementary Figure 4A). Each subject was placed mid-way along one of the arena walls, facing the wall at the start of the trial. The mouse was then allowed to explore the arena for 20 min, while Ethovision XT tracking software (Noldus Information Technology, Leesburg, VA, USA) recorded the total distance traveled, and the time elapsed in inner and peripheral zones in 5-min intervals. The arena was cleaned with 70% ethanol between subject trials.

Spontaneous activity of mice was recorded in a 40 × 20-cm open field (OF) arena for 20 min in the dark using an infrared video camera. EthoVision XT tracking software (Noldus) was used to measure the distance traveled in the OF (average centimeters traveled per second) and the normalized distance traveled in the center of the arena (5 cm from the wall), expressed as (distance traveled in the center of the OF)/(total distance traveled in the OF). For spontaneous activity over 30 h, mice were individually housed in clear cages (40 × 24 × 18 cm) with free access to food and water and maintained on their standard light/dark cycle. Three infrared beams passed the bottom part of the cage. Total beam breaks were recorded over 33 h, the first 3 h corresponding to a phase of habituation to the cages and the next 30 h to the testing phase. For the elevated plus maze (EPM), mice were placed for 5 min in a maze composed of four perpendicular 40-cm arms, two opposite arms with high walls, and two other opposite arms without walls. The exploration of the EPM was recorded by a computer-linked video camera located above the arena, and the time spent in the open arms was quantified using EthoVision XT.

All behavioral testing occurred in a dedicated behavior room, separate from the home room, as conducted previously [31 (link), 37 (link)]. The behavioral room is lit only by red light, allowing for minimal interruptions of the animals’ circadian cycle during behavioral testing [49 (link)]. Two days after 3SCSRTT testing, mice were brought to the behavioral room and allowed to acclimate for 1 hour before testing began. Mice were placed into the PhenoTyper (Noldus, Wageningen, the Netherlands) and allowed to explore for 15 minutes while movement was recorded using EthoVision XT tracking software (Noldus). The PhenoTyper was cleaned between each mouse with Minncare disinfectant to remove residual odors. We waited 5 minutes between each animal to allow for any residual odor from the cleaning agent to dissipate. During testing, a yellow light was turned on in the PhenoTyper, to provide consistent illumination of the arena. To ensure that arena novelty was not a confounding variable during the social interaction assay, all mice underwent this experiment before all other experiments conducted in the PhenoTyper.

An accelerated version of the Morris Water task was adapted from Stone et al. [45 (link)]. The Morris Water task equipment consisted of a white circular pool measuring 123.5 cm in diameter with 36 cm high walls and a transluscent square platform (12 × 12 cm) submerged 1.5 cm below the surface of the water. The pool was filled with water (21 °C) to a depth of 22 cm. For each of the three days, mice were trained on four trials. For each trial, the mouse was released from one of four starting locations, the order of which was pseudo-randomly determined. Once the mouse located the hidden platform, it remained there for 15 s before being removed from the pool and placed back in its home cage for the inter-trial interval (~5–10 min). If the platform was not located within 60 s, the mouse was manually guided to the platform, and a maximum latency score of 60 s was assigned. For each trial during the training period, latency to find the platform, path length, and swimming speed were tracked and measured with an overhead camera and Ethovision XT tracking software (Noldus, Wageningen, The Netherlands).