The light-dark task in zebrafish confuses two distinct factors: Interaction between background shade and illumination level preference.

The light-dark preference task has been commonly used in rodents to screen for anxiogenic and anxiolytic drugs. However, recent adaptations of the light-dark preference test for zebrafish have produced inconsistent results. Several studies have reported that zebrafish exhibit a preference for light, while others have found a preference for black. We suggest the inconsistencies may be the result of confusing certain parameters of the test leading to improper interpretation. For example, researchers often use "light" interchangeably with "white" and "dark" with "black" when these are two distinct factors: level of illumination vs. background shade. In the current study, we use specifically designed preference tanks to investigate the influence of background shade (i.e. white vs. black) and level of illumination (i.e. light vs. dark) on preference and anxiety-like behaviour. Furthermore, we pharmacologically validate our results by quantifying the effects of ethanol, a drug with known anxiety-altering properties, on anxiety-like behaviours. Here we report that zebrafish's preference varies depending upon background shade and level of illumination. We also found that ethanol administration altered behavioural responses in an illumination- and background shade-dependent manner. Our findings reinforce the need to correctly differentiate between these factors when interpreting results obtained with this behavioural paradigm. Lastly, our results show that simple modifications to the experimental tank in which anxiety-related responses are measured can significantly alter behaviour of zebrafish, supporting the need for standardized testing procedures and/or for detailed description of experimental procedures and the apparatus.