Cognitive bias, the altered information processing resulting from the background emotional state of an individual, has been suggested as a promising new indicator of animal emotion. Comparable to anxious or depressed humans, animals in a putatively negative emotional state are more likely to judge an ambiguous stimulus as if it predicts a negative event, than those in positive states. The present study aimed to establish a cognitive bias test for mice based on a spatial judgment task and to apply it in a pilot study to serotonin transporter (5-HTT) knockout mice, a well-established mouse model for the study of anxiety- and depression-related behavior. In a first step, we validated that our setup can assess different expectations about the outcome of an ambiguous stimulus: mice having learned to expect something positive within a maze differed significantly in their behavior towards an unfamiliar location than animals having learned to expect something negative. In a second step, the use of spatial location as a discriminatory stimulus was confirmed by showing that mice interpret an ambiguous stimulus depending on its spatial location, with a position exactly midway between a positive and a negative reference point provoking the highest level of ambiguity. Finally, the anxiety- and depression-like phenotype of the 5-HTT knockout mouse model manifested - comparable to human conditions - in a trend for a negatively distorted interpretation of ambiguous information, albeit this effect was not statistically significant. The results suggest that the present cognitive bias test provides a useful basis to study the emotional state in mice, which may not only increase the translational value of animal models in the study of human affective disorders, but which is also a central objective of animal welfare research.

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The ongoing debate on the reproducibility crisis in the life sciences highlights the need for a rethinking of current methodologies. Since the trend towards ever more standardised experiments is at risk of causing highly idiosyncratic results, an alternative approach has been suggested to improve the robustness of findings, particularly from animal experiments. This concept, referred to as “systematic heterogenisation”, postulates increased external validity and hence, improved reproducibility by introducing variation systematically into a single experiment. However, the implementation of this concept in practice requires the identification of suitable heterogenisation factors. Here we show that the time of day at which experiments are conducted has a significant impact on the reproducibility of behavioural differences between two mouse strains, C57BL/6J and DBA/2N. Specifically, we found remarkably varying strain effects on anxiety, exploration, and learning, depending on the testing time, i.e. morning, noon or afternoon. In a follow-up simulation approach, we demonstrate that the systematic inclusion of two different testing times significantly improved reproducibility between replicate experiments. Our results emphasise the potential of time as an effective and easy-to-handle heterogenisation factor for single-laboratory studies. Its systematic variation likely improves reproducibility of research findings and hence contributes to a fundamental issue of experimental design and conduct in laboratory animal science.