Understanding Developmental Neurotoxicity: Insights from Zebrafish Behavioral Assays

Developmental neurotoxicity (DNT) has long been associated with the evaluation of environmental chemicals and pollutants, especially in ecotoxicology. However, the increasing evidence that early-life exposure to certain chemicals can lead to long-term cognitive, emotional, and behavioral impairments has also raised concern in pharmacology and drug development.

 Incorporating DNT screening early in drug discovery and development helps mitigate safety risks and supports more informed decision-making across the preclinical pipeline. 

What Is Developmental Neurotoxicity and Why Does It Matter in Drug Discovery

Developmental neurotoxicity refers to any adverse effect on the developing nervous system that results from exposure to a chemical agent. These effects can range from structural malformations to subtle changes in behavior, cognition, or neuroendocrine function. 

In the context of drug discovery, identifying compounds with potential neurotoxic effects early in the development process is essential. Exposure to different chemicals in the developing brain, either during prenatal or early postnatal stages, can cause different neurological and neuropsychiatric impairments, including attention deficit hyperactivity disorder, autism spectrum disorders, and Parkinson’s disease. 

Currently, DNT evaluation is not a mandatory requirement in the USA or the European Union for pharmaceuticals, not even for pesticides or industrial chemicals. It is only performed in specific cases, when the molecule’s structure suggests it might be harmful to the brain, or when earlier animal studies show signs of possible effects on the nervous system. 

For regulatory purposes, OECD TG 426 and OECD TG 443 dictate the identification of chemicals with DNT potential. These TG are based on animal studies, preferably using rats, which result in very intensive in terms of animals, time, and overall cost. As such, there is a growing demand for more sensitive, high-throughput, and ethically sustainable models to improve toxicity assessment approaches and guide compound prioritization in drug development. 

Zebrafish has emerged as a compelling alternative that meets these needs.

Zebrafish as a Model for Assessing Developmental Neurotoxicity

Zebrafish has emerged as a powerful model organism in DNT testing due to several key advantages: 

• Its embryos develop externally and are transparent, allowing for direct observation of neurodevelopmental processes.
• It shares approximately 70% of its genes with humans, including many involved in nervous system development, and expresses orthologs for 85% of human drug targets. Development processes and mechanisms of the central nervous system (CNS)  and other vertebrates are well-conserved, including the development of the blood brain barrier (BBB). Many subdivisions found in the developing mammalian brain are morphologically identifiable in the developing zebrafish. 
• Zebrafish embryos are small and can be raised in microtiter plates, enabling high-throughput screening (HTS) of multiple compounds and concentrations.
• Compounds are administered via water immersion. The tested compound is added to the water and penetrates by passive diffusion through the embryo’s external membrane.   
• Zebrafish neural development is fast; the brain and CNS develop within 3 days post-fertilization (dpf) (Figure 1), making them ideal for short-term studies that capture critical windows of neurodevelopment.

Figure 1. Zebrafish neurodevelopment. (a) At 72 h post fertilization (hpf), major subdivisions of the zebrafish brain are present; Zebrafish axonal networks visualized by acetylated α-tubulin staining (b) at 72 hpf, (c) at 96 hpf, and (d) at 120 hpf of development. Scale bar = 100 μm. (C, cerebellum; H, hindbrain; M, midbrain; F, forebrain).  Source: Lee J, Freeman JL. Zebrafish as a Model for Developmental Neurotoxicity Assessment: The Application of the Zebrafish in Defining the Effects of Arsenic, Methylmercury, or Lead on Early Neurodevelopment. (2014). Toxics. 2(3):464-495. 

Behavioral Assays in Zebrafish Larvae: Tools for Functional Neurotoxicity Screening

Among the various endpoints used in DNT studies, behavioral readouts are considered particularly sensitive and integrative. Behavioral assays in zebrafish larvae are capable of detecting functional alterations resulting from developmental exposures, even in the absence of gross morphological defects.

A widely used one is the gold standard light/dark transition behavioral assay, which evaluates locomotor activity in response to changes in light intensity. Typically performed at 5 dpf, zebrafish are incubated with several concentrations of the compound of interest and exposed to alternating periods of light and dark. Locomotion patterns are assessed using an automated system such as DanioVision^TM, together with the Ethovision XT software (Noldus IT) that records and analyzes locomotive patterns (Figure 2). 

Figure 2. Larvae videotracking of locomotion activity acquired by Noldus IT automatic tracking system. Red lines show larval movements. 

Behavioral alterations are subsequently associated with specific drug-induced defects in the CNS development. In healthy larvae, sudden transitions to darkness usually elicit increased activity, a defensive response. Neurotoxic compounds may either blunt this reaction, indicating CNS suppression, or exaggerate it, suggesting hyperexcitability or altered sensory processing (Figure 3). Readouts obtained include total distance moved under light/dark conditions and symptomatic movement patterns, distance moved per minute, velocity, acceleration, timing, and area of movement. 

Figure 3. Representative plot of locomotion response to light/dark stimulus of 96 larvae at 120 hpf treated with 7 concentrations of a compound of interest. The higher concentration (0.5uM) of compound X induces hypermobility.

In a recent study by Tombari et al., larval zebrafish were used to screen 13 psychoactive compounds, including psychedelics and psychostimulants. The study revealed that several of these compounds altered behavior without causing visible anatomical malformations, emphasizing the utility of behavioral endpoints in detecting neurofunctional deficits that structural assessments might miss.

Integrating Zebrafish Neurotoxicity Data into Preclinical Decision-Making

The data generated from zebrafish DNT models can play a pivotal role in the early-stage drug development process. By providing rapid, cost-effective, and mechanistically relevant insights, these assays help triage compounds with potential neurotoxic liability before committing to more resource-intensive rodent studies.

At ZeClinics, we have developed a Developmental Neurotoxicity assay, a rapid and biologically relevant in vivo screening approach for the prediction of the impact of drug or chemical exposure on the developing nervous system. We provide the well-validated light/dark transition zebrafish behavioral assay as a service for DNT assessment. 

Our commitment goes beyond offering the best services. We want to improve toxicity assessment approaches, and to do so, we participate in an international initiative to develop DNT Integrated Approaches to Testing and Assessment (IATA), as a member of the OECD Expert Group on Developmental Neurotoxicity. The project aims to draft a new guidance document to provide a DNT in vitro battery (DNT-IVB) that could be used immediately for the screening of all chemicals.

If you're interested in learning more about how zebrafish behavioral assays can fit into your preclinical pipeline, contact us!

References

Bal-Price A, Pistollato F, Sachana M, Bopp SK, Munn S, Worth A. Strategies to improve the regulatory assessment of developmental neurotoxicity (DNT) using in vitro methods. (2018). Toxicol Appl Pharmacol. 354:7-18. doi: 10.1016/j.taap.2018.02.008.

d'Amora M, Giordani S. The Utility of Zebrafish as a Model for Screening Developmental Neurotoxicity. (2018 ). Front Neurosci. 12:976. doi: 10.3389/fnins.2018.00976. 

Haigis AC, Ottermanns R, Schiwy A, Hollert H, Legradi J. Getting more out of the zebrafish light dark transition test. (2022). Chemosphere. 295:133863. doi: 10.1016/j.chemosphere.2022.133863. 

Lee, J.; Freeman, J.L. Zebrafish as a Model for Developmental Neurotoxicity Assessment: The Application of the Zebrafish in Defining the Effects of Arsenic, Methylmercury, or Lead on Early Neurodevelopment. (2014).Toxics. 2: 464-495. doi.org/10.3390/toxics2030464

Tombari RJ, Mundy PC, Morales KM, Dunlap LE, Olson DE, Lein PJ. Developmental Neurotoxicity Screen of Psychedelics and Other Drugs of Abuse in Larval Zebrafish (Danio rerio). (2023). ACS Chem Neurosci. 14(5):875-884. doi: 10.1021/acschemneuro.2c00642.

By Miriam Martinez

Miriam is a Human Biologist with a strong background in neuropharmacology and a passion for bridging science and innovation. After earning a master’s degree in the Pharmaceutical and Biotech Industry, she completed her PhD in Biomedicine at Pompeu Fabra University (Barcelona), where her research focused on the behavioral analysis of animal models for neurophenotypical characterization. Following her doctoral studies, Miriam transitioned into the healthcare marketing and communication sector, where she played a key role in developing impactful marketing strategies and educational campaigns for leading pharmaceutical brands. She now leverages her scientific expertise, strategic thinking, and creative communication skills in her current role at ZeClinics.