Effects of cup handling on emotional and cognitive behaviour in mice

Animal handling is an essential but often underestimated variable in behavioural neuroscience. Traditionally, laboratory mice are picked up by the tail, a method suspected to elicit anxiety, aversion, and stress-related behaviours. Recent studies suggest that non-aversive handling techniques, such as tunnel or cup handling, can reduce such responses and improve the reliability of behavioural outcomes. Tunnel handling is advantageous when minimal direct contact with the animal is required. Cup handling, in contrast, requires frequent and consistent interaction to gradually habituate the mouse to being touched and lifted by the experimenter. This makes it particularly valuable in experimental paradigms that require precise control; for instance, when placing the animals into the arena for memory tasks without inducing anxiety or when connecting head-mounted devices for photometry, electrophysiology, or two-photon imaging. In such contexts, the use of tunnels can be impractical or even disruptive. Furthermore, while tunnel handling has been well-studied, cup handling remains comparatively underexplored, particularly in emotionally and cognitively complex behavioural paradigms.This study aims to assess how cup handling, compared to conventional tail handling, influences behavioural outcomes in mice across domains such as anxiety, exploration, memory, and social behaviour. We hypothesise that non-aversively handled mice will show reduced anxiety-like behaviour, increased exploratory drive, and more consistent performance across behavioural tests. By examining these behavioural shifts, we also aim to evaluate whether such refinement can contribute to lower variability and improved reproducibility in experimental results.C5BL/6J mice will be randomly assigned to either a five-day cup handling protocol or a tail handling condition. Following this, mice will undergo a behavioural test battery. Handling will be consistent with current refinement guidelines, and all behavioural scoring will be performed by observers blinded to group allocation.Through this study, we seek to contribute to the broader refinement efforts in line with the 3Rs by providing detailed behavioural evidence on the benefits of gentle handling techniques. Our findings aim to support best practices that improve both animal welfare as well as maintain the scientific integrity of behavioural research.