Anxiety-related platform in zebrafish (Danio rerio) larvae: An integrated analysis of behavioural and molecular assay in drug screening research

Zebrafish larvae are increasingly used to study anxiety and stress, but it is unclear whether common behavioral tests and molecular stress markers give consistent results. This study tested several drugs with known anxiety- or depression-related effects, including diazepam, amitriptyline, and fluoxetine, along with other compounds like psilocybin. Diazepam showed the most consistent anxiety-reducing behavioral effects, but this was not accompanied by lower cortisol levels or normalized stress-related gene expression. Amitriptyline reduced cortisol but only partially affected behavior. Other drugs altered movement in ways that could reflect sedation rather than anxiety relief. The findings suggest that behavioral and molecular measures should not be treated as equivalent indicators of anxiety; instead, combining them provides a more nuanced profiling approach to identify specific drug effects and avoid misinterpretation.

Study at a glance

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.