Standardized Method for the Assessment of Behavioral Responses of Zebrafish Larvae.

Hideyuki Maeda,Noritoshi Fukushima,Akihiro Hasumi

doi:10.3390/biomedicines9080884

Abstract

Zebrafish are easy to breed in a laboratory setting as they are extremely fertile and produce dozens of eggs per set. Because zebrafish eggs and the skin of the early-stage larvae are transparent, their embryos and the hearts and muscles of their larvae can be easily observed. Multiple rapid analyses of heart rate and behavior can be performed on these larvae simultaneously, enabling investigation of the influence of neuroactive substances on abnormal behavior, death, and associated pathogenetic mechanisms. Zebrafish larvae are becoming increasingly popular among researchers and are used in laboratories worldwide to study various vertebrate life phenomena; more experimental systems using zebrafish will undoubtedly be developed in the future. However, based on the available literature, we believe that the conceptualization of a protocol based on scientific evidence is necessary to achieve standardization. We exposed zebrafish larvae at 6–7 days post-fertilization to 50 repeated light–dark stimuli at either 15-min or 5-min intervals. We measured the traveled distance and habituation time through a video tracking apparatus. The traveled distance stabilized after the 16th repetition when the zebrafish were exposed to light–dark stimuli at 15-min intervals and after the 5th repetition when exposed at 5-min intervals. Additionally, at 15-min intervals, the peak of the traveled distance was reached within the first minute in a dark environment, whereas at 5-min intervals, it did not reach the peak even after 5 min. The traveled distance was more stable at 5-min intervals of light/dark stimuli than at 15-min intervals. Therefore, if one acclimatizes zebrafish larvae for 1 h and collects data from the 5th repetition of light/dark stimuli at intervals of 5 min in the light/dark test, a stable traveled distance result can be obtained. The establishment of this standardized method would be beneficial for investigating substances of unknown lethal concentration.

Highlights

Zebrafish (Dario rerio) have various practical advantages that make them useful animal models, such as small size, optical transparency, and high fertility, which makes them easy to breed and allows for in vitro fertilization [1]
The larvae were left to acclimatize for 1 h, and the light–dark test was repeated 50 times at 15-min intervals (Figure 1a) [1]
The light/dark box test evaluates the responses of fish in an aquarium with a black and white background, taking advantage of the fact that fish feel safer in dark environments [13]

Summary

Introduction

Zebrafish (Dario rerio) have various practical advantages that make them useful animal models, such as small size, optical transparency, and high fertility, which makes them easy to breed and allows for in vitro fertilization [1]. The morphology and biology of zebrafish are sometimes homologous to those of mammalian species, making them an attractive animal model for studying human disorders [2]. A natural preference for dark environments (scototaxis) in adult zebrafish is a useful indicator of anxiety, which is reduced by anxiolytic drugs and increased by anxiogenic agents [3]. Because of their short generation time, permeability to small molecules, similarity of morphology, genes, and behaviors to other vertebrates, and ease of manipulation, the locomotor activity of zebrafish larvae in response to certain stimuli can be studied through

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