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.