Prenatal polysubstance exposure alters behaviour in zebrafish larvae

Substance use during pregnancy has been linked to various adverse outcomes in infants, including congenital disabilities, neurodevelopmental delays, and long-term effects such as learning difficulties. An additional concern is that newborns are often exposed to multiple substances in utero. The biological consequences of such exposure remain largely unknown. Zebrafish offer an exciting alternative to fill this gap and deepen our understanding of the biological impact of prenatal multidrug exposure. We utilized zebrafish's scalability to expose embryos to some of the most commonly used substances: nicotine, alcohol, opioids, and all their possible combinations. After embryonic drug exposure, we conducted a detailed behavioural analysis across three developmental stages. Our results revealed drug-specific outcomes, including both synergistic and antagonistic effects. Furthermore, we identified distinctive effects across development, highlighting potential developmental shifts and individual differences in resilience. Overall, these findings demonstrate that prenatal polydrug exposure results in complex, stage-dependent effects, sometimes antagonistic, which cannot be predicted from single-drug outcomes. Our study emphasizes the value of zebrafish as a model for investigating polydrug interactions and provides a framework for exploring biomarkers of vulnerability and resilience in offspring. • Zebrafish provides a scalable platform to assess behavioural outcomes of neonatal drug exposure throughout development. • Prenatal exposure to multiple drugs results in complex, stage-dependent neurobehavioral effects. • Nicotine, alcohol, and morphine interact in vivo, leading to both synergistic and antagonistic outcomes. • Effects of individual drugs do not reliably predict phenotypes resulting from combined drug exposure. • Variability between individuals suggests different resilience and vulnerability trajectories following exposure.