Repeated elevated plus maze trials as a measure for tracking within-subjects behavioral performance in rats (Rattus norvegicus)

The elevated plus maze (EPM) is routinely used in neuroscience research to evaluate emotional behavior in rodents by measuring general exploratory performance and avoidance of the aversive open arms of the maze. According to standard practice, behavior on the EPM is evaluated during a single trial to avoid the possibility of habituation to the apparatus that would result in lost sensitivity of key outcome measures. However, this possibility has not been systematically evaluated across repeated trials or across different environmental conditions. In the current study, we assessed within-subject behavior on the EPM in adult male rats over thirteen trials (tested twice weekly) repeated under identical conditions. We also assessed within-subject behavior on the EPM in adult male rats under dim (1 lux in the closed arm) and lit (246 lux in the closed arm) environmental conditions. We found that measures of general performance (basic movements and total distanced travelled throughout the maze) were stable across repeated trials and environmental conditions. We found that measures of open arm avoidance (distance travelled in, time spent in and entries in to the open arm) varied across trials and environmental conditions and were sensitive to the lighting conditions of the initial test. Though measures of open arm avoidance did show a linear trend indicative of habituation across repeated trials, this effect was variable across trials. Notably, preference for the open arm over the closed arm (measured as% of time spent in the open arm) assessed among individual animals occurred rarely and was never observed on the group level across the thirteen repeated trials. Together, these data demonstrate that measures of general performance such as basic movements and total distance traveled are robust to repeated testing and changing environmental lighting conditions. In contrast, measures of open arm avoidance show habituation with repeated testing and are sensitive to changing environmental lighting conditions. Based on these results, we suggest that withinsubjects repeated testing on the EPM is valid in well-controlled studies that include an untreated control group to account for inter-trial variability and habituation.