Automated analysis of behavior in zebrafish larvae

Abstract

Zebrafish larvae have become a popular model system to examine genetic and environmental factors that affect behavior. However, studying complex behavior in large numbers of fish larvae can be challenging. The present study describes a novel high-resolution imaging system that is unique in its ability to automatically analyze the location and orientation of zebrafish larvae in multiwell plates. The system revealed behaviors in zebrafish larvae that would have been missed by more manual approaches, including a preference to face a threatening stimulus from a distance and a clockwise orientation in a two-fish assay. The clockwise orientation of the larvae correlates with a clockwise orientation of molecular structures during early development. Larvae with reversed embryonic asymmetries display a counter-clockwise orientation in the two-fish assay, suggesting that embryonic asymmetry and chiral behavior are regulated by the same developmental mechanisms. The developed imaging techniques may be used in large-scale screens to identify genes, pharmaceuticals, and environmental toxicants that influence complex behaviors.

Introduction

Although there is no substitute for looking at behavior with the naked eye, automated imaging systems have become valuable tools for the analysis of behavior in a variety of organisms. Large data sets can be obtained while avoiding observer bias or fatigue. In addition, automated imaging systems have been developed to facilitate high-throughput genetic, pharmacological, and environmental screens [14], [27], [38]. The images in a high-throughput screen can contain a significant amount of information and image-based screens are often referred to as ‘high-content screens’. Large-scale screens and particularly high-content behavioral screens are impractical in most vertebrates due to the number of animals that need to be examined. A notable exception is zebrafish, which has become a powerful model system for medium to high-throughput applications [7], [9], [10], [12], [13], [19], [20], [24], [28], [39], [46]. Zebrafish are small, maintenance costs are low compared to other vertebrates, and a modest colony of fish can produce hundreds or even thousands of embryos on a daily basis. These embryos develop rapidly into free-swimming fish. At 24 h of development, the embryos have a beating hart, moving tail, eyes, and a primitive brain [29], [45]. Embryos hatch from their chorion between 2 and 3 days of development. After hatching, the free-swimming 3–7-day-old zebrafish larvae display a range of behaviors that are important for finding food and avoiding predators. Some of these behaviors are robust and suitable for large-scale screening. For example, two robust behaviors of zebrafish larvae that have been examined by mutagenesis screens are the optokinetic and optomotor response [10], [11], [36]. The optokinetic response is an eye movement in response to a moving object. The optomotor response is a swimming behavior in the same direction as a moving pattern of stripes. While the optokinetic response needs to be evaluated in individual zebrafish larvae, the optomotor response can be examined in groups of larvae. For example, Muto et al. imaged groups of 10–40 larvae in special ‘racetracks’ [36]. Twelve racetracks were imaged at once, which greatly accelerated the analysis of the optomotor response. In the course of 3 years, more than 500,000 F₃ zebrafish larvae were examined for either the optokinetic or optomotor response [36]. The analysis of behavior in groups of larvae may be particularly advantageous if larvae could be imaged in multiwell plates. These plates are ideally suited for large-scale pharmacological or environmental studies, since one can make use of robotic fluid handlers. In addition, libraries of small molecules can be conveniently added to the culture medium. By imaging larger numbers of individual larvae in multiwell plates, it may be possible to expand the types of behaviors that can be examined in high-throughput screens. However, imaging zebrafish behavior in multiwell plates remains challenging. First, the optics of a multiwell plate is sub-optimal, in particular along the walls of the wells. Second, it is difficult to provide visual stimuli to individual wells of a multiwell plate. Third, it is difficult to measure behaviors other than activity in a multiwell plate. Driven by our own interests in measuring spontaneous and stimuli-induced activity, asymmetric behavior, social behavior, learning, and memory, we developed a novel high-resolution imaging system that is well suited for imaging zebrafish larvae in large culture dishes and multiwell plates. The system measures the location as well as the orientation of zebrafish larvae and visual stimuli can be presented to the larvae via a LCD screen. The system was tested by imaging the optomotor response and by imaging asymmetric behavior in a two-fish assay.