Semi-Automated Image Analysis of Xenopus Laevis Behavioral Response to Visual Stimuli

Abstract

High-throughput screens to measure the behavioural effects of various genetic or pharmacological manipulations will require the development of assays that do not rely on subjective human observer input. The Xenopus laevis tadpole is a low-cost, easily accessible vertebrate animal model for studying brain development. We present a new semi-automated image analysis method to quantify avoidance swimming behaviour by multiple tadpoles in parallel. In our setup, Xenopus tadpoles swim in a water-filled Petri dish placed on a computer screen which displays moving dots as escape-evoking visual stimuli on a grey background. Each experiment is recorded by a camera as an image time series. An algorithm was developed to precisely track tadpole movements in space and time. In the control experiments, presenting only the background grey field, we measured the speed distribution of tadpole displacements per image interval. This distribution was used as a reference to assess the tadpole behavioural responses to contrast varying visual stimuli. In experiments where stimuli are brighter or darker with respect to the grey background intensity, we mapped the spatial distribution of responses as a function of the closest dot position with respect to tadpole orientation. We observed a greater response concentration within an area of 5 mm radius around the tadpole. By defining potential reaction events within this threshold minimal distance, we could evaluate the response probability for each tadpole. Our results show a higher tadpole response probability to darker dots than to brighter dots. Furthermore, we show an angular dependence of the response probability that varies with dot contrast. We conclude that this tracking and analysis technique can be applied to a variety of behavioural studies, enabling a rapid and more objective, automated evaluation of visuomotor responses under a wide range of experimental manipulations.