Animal models enable investigation of the impact of stress on emotional-behavioral performance to facilitate understanding of posttraumatic stress symptoms experienced in humans. Refinement of animal stress models could lead to a reduction in the number of subjects needed to detect statistically significant stress effects, in accordance with Russel and Burch’s three Rs of research. We assessed whether performance of experimental procedures (that is, stress exposure and poststress behavioral testing) during the dark or light phases of the 12-h light/12 h-dark cycle is a refinement that could accomplish this reduction. At 3 h into either the light or dark phase, male and female adult Sprague–Dawley rats underwent a single-day traumatic stress exposure. Rats then underwent behavioral testing for exploratory behaviors, startle responses, and conditioned fear responses at 2 h, 1 d, and 9 d after stress exposure. Distance traveled in the elevated plus maze (EPM) by both male and female rats was significantly reduced in the dark phase compared with the light phase. Male rats of the dark phase group also spent less time in the open arms of the EPM, and traveled less, spent less time in the center, and spent more cumulative time freezing in the open field. Female rats of the dark phase group spent more cumulative time freezing in the EPM and exhibited significantly more tone-cued conditioned freezing responses. Our results suggest that performing experimental procedures during the dark phase of the light cycle may be a useful refinement mechanism, as procedures performed during this period had the greatest effect on behavioral outcomes in both males and females. Light cycle phase is an experimental variable that should be considered when designing experiments to maximize behavioral effects, including those in response to stress.
Study timeline representation. Day −14 is the arrival at the facility of the rats. All were allowed 14 d of acclimation to both facility and applicable light cycle group (light compared with dark; day −14). Day 0 was onset of procedures with stress exposure (PE, IES, UWS), followed immediately by blood collection (BC). On day 0, 2 h after BC, behavior testing (EPM, OF, SR) was performed and repeated 24 h later (day 1). Behavior procedures were repeated on day 9 with the addition of conditioned freezing (FRZ) to conclude the study.
Behavior in the elevated plus maze (EPM) in females and males during the light or dark phase and under control or stress conditions (n = 8 to 12 per group). (A, B) Total distance traveled in the EPM. (C, D) Time spent in the open arms of the EPM. (E, F) Distance traveled in the open arms of the EPM. (G, H) Time spent freezing in the EPM. Significant main effects for each factor are shown. *, P < 0.05. Significant post hoc analyses resulting from significant interactions between timepoint and light cycle factors are denoted. †, P < 0.05. All data are shown as means ± SEM.
Behavior in the open field (OF) in females and males during the light or dark phase and under control or stress conditions (n = 8 to 12 per group). (A, B) Total distance traveled in the OF. (C, D) Time spent in the center of the OF. (E, F) Distance traveled in the center of the OF. (G, H) Time spent freezing in the OF. Significant main effects for each factor are shown. *, P < 0.05. Significant post hoc analyses resulting from significant interactions between timepoint and stress condition factors are denoted. ‡, P < 0.05. All data are shown as means ± SEM.
Startle behavior in the startle response (SR) chambers in females and males during the light or dark phase and under control or stress conditions (n = 8 to 12 per group). (A, B) Average startle force across all trials. (C, D) Startle reactivity across the first 5 trials. Significant main effects for each factor are shown. *, P < 0.05. No significant post hoc analyses resulted from significant interactions between factors. All data are shown as means ± SEM.
Conditioned fear responding in the conditioned freezing (CF) chambers in females and males during the light or dark phase under stress conditions (n = 10 to 12 per group). (A, B) Percent of time spent freezing during baseline (BL) and each conditioned stimulus trial. Significant main effects for each factor are shown. *, P < 0.05. Significant post hoc analyses resulting from significant interactions between light cycle and CS trial factors are denoted. #, P < 0.05. All data are shown as means ± SEM.
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