Truscan software

Open field testing was done to assay activity and anxiety-like behaviors in response to a novel environment. Mice were placed in the center of a plexiglass open field arena (Coulbourn Instruments, Holliston, MA, USA) and allowed to explore for 30 min. Total distance traveled as well as distance traveled in the center of the chamber were recorded by TruScan software (Coulbourn Instruments) in five-minute epochs. Anxiety-like behavior was assessed by the ratio of distance traveled in the center to the total distance traveled; the center to total distance ratio is inversely proportional to anxiety levels. For open field testing, nine WT mice were not included (one due to missing data, three due to equipment malfunction, and five were statistical outliers); eight Fmr1 KO mice were not included (one due to missing data, three due to equipment malfunction, and four were statistical outliers); eight Fxr2 Het mice were not included (three due to equipment malfunction and six were statistical outliers); and ten Fmr1 KO/ Fxr2 Het mice were not included (two due to missing data, three due to equipment malfunction, and five were statistical outliers.

The evaluation methodology was based on previous studies, with slight modifications (26 (link)–28 (link)). Rodents display characteristic of thigmotaxis to a new environment. Therefore, the central zone represents a threatening situation, while the peripheral area is relatively safe. Experiments were conducted in a location shielded from incidental noise. The space is a circular arena with a diameter of 97 and 42 cm high walls. Lighting with 850 nm far-red LEDs was used and any anxiety induced by the bright illumination was minimized. The room temperature was maintained at 25°C± 2°C during data acquisition. Each mouse was recorded for 10 min and then returned to the group housing. Mice were observed by video recording (FDR-AX700, Sony, Tokyo, Japan), and the time spent in the center, frequency of entering the center, and time spent standing were recorded for 10 min. An increase in the frequency and time spent staying in the center of the space indicated a decrease in anxiety. Video analysis was performed with TruScan software (Version 2.2; Coulbourn Instruments, Allentown, PA, USA). After each test, the space was cleaned with a 70–75% alcohol solution to avoid the effect of the odor of the previous animal on the next one.

Rats were placed into an open-field arena (Coulbourn Instruments, Allentown, PA). Animals’ spontaneous locomotor activity was recorded for 30 min before being injected with D-amphetamine sulfate (0.5 mg/kg, i.p.) after which their locomotor activity was recorded for another 90 min. Locomotor activity was measured via beam breaks and recorded with TruScan software (Coulbourn Instruments). The arena was cleaned between rats with ethanol 70% v/v.