Application to developmental toxicity testing of a novel method for whole-brain imaging of microglia in zebrafish

Microglia, parenchymal macrophages resident in the central nervous system, regulate brain development by dynamically changing their functional and morphological states in a spatiotemporal-dependent manner. Because the function of microglia may differ depending on their location, their status should ideally be assessed within discrete brain regions. In this study, we developed a novel whole-brain imaging method to visualize microglial morphology in the forebrain, midbrain, and hindbrain of live zebrafish larvae at 5-6days post-fertilization, and quantified various morphological parameters using MorphoLibJ, a publicly available tool for mathematical analysis of three-dimensional images. We applied this method to assess the developmental toxicity of ethanol and valproic acid on microglial morphology in the zebrafish larvae, and were able to detect marked differences in the spatiotemporal effects of each compound. The duration of exposure required to detect significant changes in microglial morphology was shorter for ethanol than for valproic acid, and microglia in the forebrain diencephalon region were more susceptible to toxicity induced by ethanol compared with valproic acid. These results suggest that our whole-brain microglial imaging and modeling method may be a versatile tool to assess the developmental toxicity of chemicals in zebrafish.