Effect of evaluation timing and duration of anxiety-like behaviors induced by conditioned fear in rats: Assessment using the triple test

The fear conditioning-based models of anxiety disorders have been widely used for assessing anxiolytic action. The triple test is an evaluation method for anxiety-like behaviors that integrates the open field, elevated plus maze, and light/dark box, which can comprehensively reflect the anxiety-like behaviors state of animals. However, there are many factors that can affect the evaluation results of anxiety-like behaviors, especially the time of day (morning or afternoon), which are often ignored by most experimental studies. Accordingly, in the present study, we first measured the anxiety-like behavior in the fear conditioning-based model rats by using the triple test in the morning and afternoon, respectively (experiment 1). In experiment 2, the anxiety-like behaviors were evaluated on the 1st, 7th, 10th and 14th day after experiencing foot shock for 4 consecutive days, respectively. The fear-conditioned rats exhibited increased anxiety-like behaviors only in the afternoon and prolonged freezing times either in the morning and afternoon. Furthermore, model rats exhibit increased anxiety-like behaviors in open field, elevated plus maze, and light/dark box on the 1st day; in open field and light/dark box on the 7th day; in elevated plus maze on the 10th day; and in light/dark box on the 14th day after the final foot shock. The results suggested that the time of day had a significant effect on anxiety-like behaviors caused by conditioned fear, and increased in the afternoon vs morning. Moreover, the lasting increased in anxiety-like behavior for 14 days was appropriated to explore the effects of long-term administration of anxiolytics.