Research reportAutomated analysis of behavior in zebrafish larvae
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.
Section snippets
Zebrafish larvae
Adult wild type zebrafish were obtained from Carolina Biological. The fish were maintained in a mixed population on a 14 h light/10 h dark cycle and were fed with a combination of brine shrimp and flake food. Embryos were collected from the tanks at ‘dawn’ and were raised at 28 °C in a culture medium, containing 60 mg/l sea salt (Instant Ocean) in deionized water and 0.25 mg/l methylene blue as a mold inhibitor. Embryos were grown at a density of 25 embryos per 50 ml culture medium in plastic 8.5 cm
The zebrafish imaging system
The zebrafish imaging system was built in an upright and inverted configuration, each with specific pros and cons (Fig. 1). The upright configuration is well suited for presenting visual stimuli to the larvae. Culture dishes and multiwell plates were placed directly on the LCD screen, bringing the larvae in close proximity to the visual stimuli. Due to the 9 megapixel resolution of the camera, it is possible to collect detailed images of the larvae in a large field of view. In large culture
Automated imaging systems
When we set out to build the zebrafish imaging system, we aimed for a high-resolution imaging system that could: (1) carry out automated measurements of the location and orientation of zebrafish larvae in a large field of view, (2) image standard culture dishes and multiwell plates, and (3) present visual stimuli to the larvae. In addition, we aimed for a user-friendly low-cost system that would be easy to replicate in other laboratories and could be scaled up for high-throughput applications.
Acknowledgements
I thank Jill Kreiling and Ruth Colwill for their feedback on the behavioral assays, Christina Parodi for her work on the physiology of left–right asymmetry, and Nicole Fuerst, Xiaoxuan Chen, Charles Kambe, and Elena Miyoko Carver for studying various aspects of zebrafish behavior. This work was funded in part by the National Science Foundation (0421654).
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