Ethovision 14

All samples were imaged live in 0.75× MMR at 20 °C using a Nikon SMZ-1500 microscope equipped with both top and substage illumination. Still images were captured on a QImaging Retiga 2000R CCD camera and videos were captured using a Sony IMX234 at a sample rate of 30 frames per second. XY movement tracks were extracted for each run using Noldus Ethovision 14 software, and smoothed using a one-dimensional Gaussian filter (SI Appendix, section S9.1). The tdTomato lineage tracer was imaged using a standard tetramethylrhodamine isothiocyanate (TRITC) filter cube and fluorescent light source to verify cardiac muscle cell location, and GFPIII signal was imaged with a standard fluorescein isothiocyanate (FITC) filter cube to verify epidermal cell location (SI Appendix, section S9.2).

Following a 10 minute exploration of a 50 × 50 cm open-field arena, mice were returned to the arena the next day, which contained 2 identical objects. Mice were allowed to explore the objects and the arena 3 times for 10 minutes, with an inter-trial interval of 10 minutes. The following day, one of the objects was replaced with a novel object and the mice were returned to the arena and allowed to explore for 10 minutes. Mouse movement, exploration, and nuzzling was automatically recorded with an Ikegami ICD-49E camera with EthoVision 14 (Noldus Information Technology). Discrimination of the novel object was assessed by calculating (time exploring novel object – time exploring familiar object)/(time exploring novel object + time exploring familiar object).

MWM was carried out as previously described (57 (link)). Mice were trained 4 times a day, using 60 second trials every 30 minutes during which they were placed into the pool, each time from a different quadrant, and allowed to swim to find the escape platform. All trials were filmed with a video tracking system using EthoVision 14 (Noldus Information Technology), and escape latency, or time to find the submerged escape platform, was determined by video analysis.

The elevated plus maze (EPM) was used to assess anxiety‐like behavior. The maze consisted of four 60 cm long, 10 cm wide arms located 60 cm above the ground. Two “closed arms” of the maze were surrounded by 60 cm high opaque walls while the other two “open arms” had no walls. Each animal was placed in the center of the maze and allowed to freely roam for 4 min while being tracked by a motion camera at 15 frames/s (Noldus Ethovision 14).

The open field (OF) test was also used to assess anxiety‐like behavior. The OF arena consists of an opaque, square Plexiglass chamber measuring 90 cm × 90 cm with 40 cm high walls. Rats were placed in the same corner to begin each trial and were allowed to roam the field for a 4‐min period while being tracked by a motion camera at 15 frames/second (Noldus Ethovision 14).

CPP was performed as described(Land et al., 2008 (link)). The apparatus consisted of two chambers with distinct visual cues (vertical or horizontal black and white stripes) and one center chamber. Each chamber was filled with bedding, and the chambers were wiped down with water between sessions. Habituation and baseline chamber preference: On days 1 and 2, animals were allowed access to all three chambers for 30 min and the baseline values for time spent in each chamber were derived from day 2. Vehicle/drug chamber pairings for each mouse were then randomly assigned. Pairing: Days 3-5 served as chamber pairing days. On these days animals were first injected with vehicle in the AM and confined to the designated chamber for 30 min. At least 4 hrs later animals were injected with the assigned drug conditioned and were confined to the opposite chamber for 30 min. Post-test: On day 6, animals were again allowed free access to all three chambers for 30 min.
Video was recorded from the top of each chamber at rate of 25 fps using Bonsai open-source software(Lopes et al., 2015 (link)). Videos were then analyzed using Ethovision 14 (Noldus Information Technology). Ethovision was used to calculate the amount of time spent in each chamber and the amount of distance travelled during conditioned sessions.