Abstract
New environments are known to be anxiogenic initially for many animals including the zebrafish. In the zebrafish, a novel tank diving (NTD) assay for solitary fish has been used extensively to model anxiety and the effect of anxiolytics. However, studies can differ in the conditions used to perform this assay. Here, we report the development of an efficient, automated toolset and optimal conditions for effective use of this assay. Applying these tools, we found that two important variables in previous studies, the direction of illumination of the novel tank and the age of the subject fish, both influence endpoints commonly measured to assess anxiety. When tanks are illuminated from underneath, several parameters such as the time spent at the bottom of the tank, or the transitions to the top half of the tank become poor measures of acclimation to the novel environment. Older fish acclimate faster to the same settings. The size of the novel tank and the intensity of the illuminating light can also influence acclimation. Among the parameters measured, reduction in the frequency of erratic swimming (darting) is the most reliable indicator of anxiolysis. Open source pipeline for automated data acquisition and systematic analysis generated here and available to other researchers will improve accessibility and uniformity in measurements. They can also be directly applied to study other fish. As this assay is commonly used to model anxiety phenotype of neuropsychiatric ailments in zebrafish, we expect our tools will further aid comparative and meta-analyses.