Behavioral assay
Behavior was assessed using open field test (OFT), social interaction test (SIT), novel object recognition test (NORT) and forced swim test (FST). Behaviors were analyzed using an automated video tracking software (EthoVision) and/or Behavioral Observation Research Interactive Software (BORIS; Friard & Gamba, 2016). For manual scoring, raters were blind to mouse subject identification. Due to feasibility, testing order was maintained. Three identical testing chambers (24 x 54.5 x 40 cm3) were used and modified to fit each test, excluding FST. Chambers and all objects used in each test were cleaned with 70% ethanol between trials.
OFT: Hyperactivity, interpreted as psychomotor agitation, was assessed as total distance traveled in centimeters. Mice were placed in the chamber and were recorded for 10 minutes, consisting of 5 minutes of habituation and 5 minutes of behavioral analysis.SIT: To test spontaneous sociability and preference, Crawley’s sociability test was used (Kaidanovich-Beilin et al., 2011). Spontaneous social behavior was measured in phase 1 by dividing time spent with a stranger conspecific with time spent with a stranger mouse to produce a social behavior index (SI). Social memory preference index (SMI) was measured in phase 2 by dividing time spent with a stranger conspecific with time spent with a familiar conspecific. NORT: In phase 1, two identical red plastic bulbs (2.5 x 2.5 cm3) were mounted at each end of the testing chambers using Velcro. Mice were placed in the center chamber, dividers were removed, and recording commenced for 5 minutes. In phase 2, one bulb was replaced with a gold bell or cone (2.5 x 2.5 cm3). FST: Mice were placed in a glass (8.5 x 20 cm) that was filled partway with water (22 – 25 degrees Celsius). A 2-minute period of habituation occurred before recording behavior for 4 minutes.