A high-throughput test of anxiety for zebrafish (Danio rerio) larvae: the larval diving response (LDR) (2022)

Barbara D. Fontana, B.D. & Parker, M.O. (2022). A high-throughput test of anxiety for zebrafish (Danio rerio) larvae: the larval diving response (LDR). bioRxiv doi.org/10.1101/2022.05.16.492196

doi: 10.1101/2022.05.16.492196


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.


psychopharmacology; larval diving response; LDR; drug discovery; novel tank; zebrafish