A high-throughput test of anxiety for zebrafish (Danio rerio) larvae: the larval diving response (LDR)

Abstract

A high level of anxiety is a characteristic symptom across the anxiety spectrum disorders and several other affective disorders, and is often detected early in a child's development. Zebrafish are widely used in translational studies for drug discovery in anxiety research, where the species naturalistic diving response to a new environment is a reliable and validated marker for anxiety-like behavior. Here, we present for the first time a method for examining diving behavior in zebrafish larvae to assess anxiety-like behaviors at early-stages (7 days-post-fertilization). Anxiogenic (caffeine 100 mg/L) and anxiolytic (diazepam 5 mg/L) drugs were used to pharmacologically validate the protocol. Larvae were pretreated in drug (or fresh water) for 30-min, then transferred to cuvettes containing fresh water and habituated for a further 30-min. Larval diving response (LDR) was then recorded using an automated system (Zantiks) for 30-min. As with adult fish, diazepam was anxiolytic, significantly reducing the time spent in bottom of the novel environment, and increasing the time spent in the top. Meanwhile, caffeine induced anxiogenic-like effects by increasing the time spent in the bottom zone of the environment. This new automated and high-throughput screening tool has the potential use for screening of anxiogenic and anxiolytic compounds, and for studies aiming to understand the mechanisms underlying affective disorders.

Larval Zebrafish as a Model for Mechanistic Discovery in Mental Health

“…A variation of the NTD has been proposed for younger larvae. Seven dpf larvae were shown to display depth preference in the first 30 min of introduction to a novel tank, with caffeine enhancing, and diazepam alleviating bottom-dwelling behavior (Fontana and Parker, 2022).…”