Anxiety-related platform in zebrafish (Danio rerio) larvae: An integrated analysis of behavioural and molecular assay in drug screening research
Małgorzata Potoczna, Natalia Kasica, Małgorzata Chmielewska-Krzesińska, Joanna Dybalska-Szczepanek, Krzysztof Wa̧sowicz, Ewa Sokołowska, Piotr Podlasz
Biomedicine & Pharmacotherapy June 17, 2026 DOI: 10.1016/j.biopha.2026.119680 (opens in new tab)
Study at a glance
AI-extracted from the abstract| Characteristics | Experimental study Peer reviewed |
|---|---|
| Population | Zebrafish larvae |
| Interventions | diazepam amitriptyline fluoxetine TP003 DOI 5-MeO-DMT psilocybin |
| Topics | Anxiety |
| Keywords | Zebrafish Drug discovery Screening techniques Phenotypic screening High-content screening Computational biology Pharmacology Bioinformatics |
| Key findings | Behavioral and molecular endpoints in larval zebrafish anxiety-related drug screening provide complementary but not interchangeable evidence, with diazepam showing the strongest behavioral anxiolytic-like consistency without corresponding molecular stress marker changes. |
Abstract
Zebrafish larvae are increasingly used in behavioural pharmacology because of their translational relevance and suitability for anxiety- and stress-related studies. However, it remains unclear whether commonly used behavioural assays and molecular stress markers provide convergent evidence of anxiolytic-like drug activity. This study therefore aimed not to validate a single predictive screening platform, but to assess the concordance and interpretative value of behavioural and molecular endpoints in larval zebrafish anxiety-related drug screening. Reference compounds with established anxiolytic or antidepressant activity, including diazepam, amitriptyline, and fluoxetine, were used to anchor assay performance. TP003, a GABAergic modulator, and three serotonergic psychedelic compounds, DOI, 5-MeO-DMT, and psilocybin, were included to challenge the assays with pharmacologically diverse mechanisms relevant to stress- and anxiety-related responses. Diazepam showed the strongest cross-assay behavioural consistency compatible with an anxiolytic-like profile. However, this response was not accompanied by reduced cortisol levels or straightforward normalisation of stress-related gene expression. Amitriptyline reduced cortisol but produced only partial behavioural effects, whereas DOI affected selected behavioural preference endpoints and transcriptional markers without significantly reducing cortisol. Fluoxetine and 5-MeO-DMT altered locomotor activity in patterns requiring cautious interpretation, as reduced movement may reflect locomotor suppression or sedation rather than anxiolysis alone. These findings indicate that behavioural and molecular endpoints should not be treated as interchangeable measures of a single anxiety construct. Instead, combined behavioural and molecular assessment is most useful as a profiling framework to identify endpoint-dependent responses, detect sedative or nonspecific locomotor confounds, and support more cautious interpretation of putative anxiolytic-like effects in larval zebrafish.