ESTABLISHMENT OF EMBRYONIC ZEBRAFISH NEUROSPHERES AS A NOVEL MODEL FOR DEVELOPMENTAL NEUROTOXICITY TESTING

The field of developmental neurotoxicity testing (DNT) is in need of high-throughput alternative methods to current animal testing. DNT animal testing protocols are resource intensive, require large animal numbers and are unable to cover the growing chemical market demands raising ethical concerns. Hence, the OECD/EPA have recently developed a DNT In-Vitro Testing Battery (DNT-IVB) comprising multiple in vitro assays assessing fundamental neurodevelopmental processes and capable of screening large numbers of chemicals. Zebrafish embryos have been recognized by the OECD as a promising model organism for DNT-testing due to the possibility of studying the impact of chemicals on behaviour within the scope of complete brain development in a whole organism model. In order to perform a risk assessment on human DNT for new chemicals by bridging human cell-based assays of the DNT-IVB with zebrafish whole-organism studies, our aim is to include an embryonic zebrafish neurosphere model to the testing battery. In preliminary experiments, we successfully generated zebrafish neurospheres by isolating and dissociating zebrafish brains at 48 hours post-fertilization. Assessment of cell composition using cryosectioning showed that embryonic zebrafish neurospheres incorporate several cell populations, including stem cells and neural progenitor cells and the absence of differentiated cell types. Upon plating on an extracellular matrix, we observed that zebrafish neurospheres exhibit radial migration and differentiation to neuronal cells. To date, we have developed an optimized protocol for isolating embryonic zebrafish neural tissue followed by neurosphere proliferation, migration and differentiation, while including positive controls for each endpoint and assessing the variability of the assay.