Behavioral Testing: Forced Swim Test and Elevated-Plus Maze

The FST was used to assess the response to acute inescapable stress which reflects the activity of neuronal networks that may be impaired in depression, ASD, and other disorders (Commons et al., 2017). More specifically, the increased immobility time during forced swim was used in our study as a measure for stress-coping behavior in rodents. In this study we used a repeated-trial procedure similar to the original FST protocol (Porsolt et al., 1977). Since rats remain immobile for a higher proportion of time in the second trial, this approach provides a more suitable baseline for measuring a decrease or increase in immobility duration. An interval of 24 h between the pre-test and the test trial was used. On day one, rats were individually immersed in a water-filled (23–25°C) plastic cylinder (46 cm high × 20 cm diameter) filled with tap water to 30 cm water depth which prevents them from touching the bottom. The animals were exposed to the forced swim for 15 min. After 24 h, the test trial was conducted similarly, however, in this trial animals were subjected to the forced swim for only 5 min and time spent immobile during each one min interval over the 5-min period was recorded along with swimming activity. All sessions were videotaped and recorded for analysis using ANY-MAZE video-tracking system and time spent immobile, swimming, or climbing for the four treatment groups was analyzed using a two-way ANOVA [maternal diet X post-weaning diet].

General exploratory and anxiety-like behaviors were evaluated using EPM apparatus consisting of a black acrylic cross with a shared central square (12 cm × 12 cm) with two opposite open arms and two opposite closed arms (50 cm × 12 cm). The closed arms were enclosed by 50-cm high walls. The test rat was placed on the central square facing a corner which allowed equal choice of entering an open or closed arm. The animal explored the apparatus during a 5 min session. The behavior was recorded using ANY-MAZE tracking system. Time spent and distance traveled in the open and closed arms were quantified. At the end of each session, the number of fecal boli were counted and the apparatus was thoroughly cleaned.

Upon completion of behavior testing the animals were maintained in their specific groups for 10–14 days prior to sacrifice to ensure that all animals had returned to a stable physiological baseline after behavioral testing. At 150 days of age, individual animals from each group were decapitated in a staggered fashion (CC, CHF, HFC, HFF, repeat). Immediately upon death, trunk blood was drained into serum collection tubes containing gel activator, whole hippocampi were rapidly dissected from each side of the brain and rapidly frozen using liquid nitrogen. Body weight was recorded and visceral fat pads, both retroperitoneal and gonadal, were excised and weighed.