Have I been here before? Complex interactions of age and test experience modulate the results of behavioural tests

Recently, a discussion about the reproducibility of results from behavioural phenotyping experiments has emerged. A huge emphasis has therefore been put on the identification of those factors that might limit the reproducibility of behavioural data. As a comprehensive phenotypic characterisation can involve testing of the same animal repeatedly over a specific time period, the aim of the present study was to systematically investigate effects of two potentially confounding variables, age of the animals and test experience. For this purpose, the behaviour of 48 male C57BL/6 J mice of two different ages (9 vs. 13 weeks) was assessed in a battery of common behavioural tests measuring anxiety-like and exploratory behaviour (Elevated Plus Maze, Dark-Light test, Open Field test, Novel Cage test). While half of the mice of each age group was naïve to the test battery, the other half had experienced the same tests before. Besides main effects of both age and test experience on anxiety-like and exploratory behaviour, the analysis also revealed profound interactions between these factors. More precisely, an effect of age was apparent in experienced but not in naïve mice. Furthermore, the effect of previous test experience was more pronounced in older than in younger mice. These findings clearly demonstrate that experimental factors, such as age and test experience, can influence behavioural data not just additively, but also in a complex, interactive way. To provide robust and reproducible results, it is thus fundamental to consider such factors systematically in the study design.