Developmental neurotoxic compounds impair the developing human nervous system at lower doses than those affecting adults. Standardized test methods for assessing developmental neurotoxicity (DNT) require the use of high numbers of laboratory animals. Here, we use a novel assay that is based on the development of an intact insect embryo in serum-free culture. Neural pathways in the leg of embryonic locusts are established by a pair of afferent pioneer neurons, extending axons along a welldefined pathway to the central nervous system. After exposure to test chemicals, we analyze pioneer neuron shape with conventional fluorescence microscopy and compare it to 3D images, obtained by scanning laser optical tomography (SLOT) and processed by a segmentation algorithm. The segmented SLOT images resolve the 3D structure of the pioneers, recognize pathfinding defects and are thus advantageous for detecting DNT-positive compounds. The defects in axon elongation and pathfinding of pioneer axons caused by two DNT-positive reference compounds (methylmercury chloride; sodium(meta)arsenite) are compared to the biochemically measured general viability of the embryo. Using conventional fluorescence microscopy to establish concentration-response curves of axon elongation, we show that this assay identifies methylmercury chloride and the pro-apoptotic compound staurosporine as developmental neurotoxicants. The developing fetal and juvenile human brain is more sensitive to exposure to industrial chemicals than the adult. Investigations of developmental neurotoxicity (DNT) deal with any adverse effects on the structure or function of the nervous system during gestational-or lactation-periods 1. DNT positive chemicals pose a serious threat to the health of our children, because maldevelopment of the brain will eventually result in behavioral abnormalities, such as attention deficit, hyperactivity, and autism spectrum disorder 2,3. Since there is growing concern that the increase of those neurologic defects may, at least in part, be caused by a cocktail of DNT positive compounds used in our daily life, there is now an urgent demand for risk assessment of industrial chemicals. Until 2017, only 13 different substances have been identified as developmental neurotoxicants in humans by epidemiological approaches. In contrast, tens of thousands industrial compounds have not been studied so far and remain uncertain 2,4. To evaluate these potentially toxic compounds large numbers of vertebrates over long testing periods are needed. Due to limitations of traditional animal testing by guidelines of the OECD, alternative assays have to be developed. Alternative in vitro testing methods focus on key aspects in brain development that can be readily quantified in cell culture assays. They include, for example, measurements of cell viability, cell proliferation, neurite extension, development of neurochemical phenotype, and electrical activity in randomly formed neural network as toxicological endpoints 5-8. However, there are also non-mammalian organ...
