Cued Fear Conditioning in Zebrafish (Danio rerio)

Abstract

This protocol describes the cued fear conditioning task, designed to test the acquisition of the association between a fearful stimulus (unconditioned stimulus, US) and a cue (conditioned stimulus, CS) in zebra fi sh (Danio rerio). Here we use electrical shock as a US, and red light as a CS, both of which are easily manipu-lated and can be applied to fi sh swimming in the water, under the control of the strength, timing, and duration of the stimuli. While fi sh can display different forms of fear responses (e.g., freezing behavior and fl ight behavior), this protocol allows the researchers to analyze both freezing and fl ight behaviors in a quantitative manner, by using a video-/software-based analyses. Under threatening environments, the choice of a suitable response is critical for an animal's survival. The study of the regulation of the behavioral responses to fearful and stressful environments is also important in the fi elds of the human mental disorders. Animals exposed to natural predators elicit various behaviors, including the freezing-or-fl ight response, with the choice between the two depending on multiple factors (including species, environment, age, and experiences) (1– 3) . Recent studies have suggested that freezing and fl ight are encoded by distinct neural networks (1, 4, 5) . However, in spite of the progress made in the study of the respec-tive networks, the mechanisms underlying the decision of which circuit to elicit the behavioral choice remain unclear. To cast the deep insight into these issues, the usage of animal models is key. The zebra fi sh (Danio rerio) has recently started to attract attention as a novel model organism for the study of fear-related states. Here, we describe the protocol for the cued fear