Institute of Physiology and Cell Biology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15/102, 30173, Hannover, Germany.
Arch Toxicol. 2020 Dec;94(12):4099-4113. doi: 10.1007/s00204-020-02929-6. Epub 2020 Oct 20.
Exposure to environmental chemicals during in utero and early postnatal development can cause a wide range of neurological defects. Since current guidelines for identifying developmental neurotoxic chemicals depend on the use of large numbers of rodents in animal experiments, it has been proposed to design rapid and cost-efficient in vitro screening test batteries that are mainly based on mixed neuronal/glial cultures. However, cell culture tests do not assay correct wiring of neuronal circuits. The establishment of precise anatomical connectivity is a key event in the development of a functional brain. Here, we expose intact embryos of the locust (Locusta migratoria) in serum-free culture to test chemicals and visualize correct navigation of identified pioneer axons by fluorescence microscopy. We define separate toxicological endpoints for axonal elongation and navigation along a stereotyped pathway. To distinguish developmental neurotoxicity (DNT) from general toxicity, we quantify defects in axonal elongation and navigation in concentration-response curves and compare it to the biochemically determined viability of the embryo. The investigation of a panel of recognized DNT-positive and -negative test compounds supports a rather high predictability of this invertebrate embryo assay. Similar to the semaphorin-mediated guidance of neurites in mammalian cortex, correct axonal navigation of the locust pioneer axons relies on steering cues from members of this family of cell recognition molecules. Due to the evolutionary conserved mechanisms of neurite guidance, we suggest that our pioneer axon paradigm might provide mechanistically relevant information on the DNT potential of chemical agents on the processes of axon elongation, navigation, and fasciculation.
在子宫内和出生后早期发育过程中接触环境化学物质会导致广泛的神经缺陷。由于目前用于识别发育神经毒性化学物质的指南依赖于在动物实验中使用大量啮齿动物，因此有人提议设计快速且具有成本效益的体外筛选测试电池，这些电池主要基于混合神经元/神经胶质培养物。然而，细胞培养测试并不能检测神经元回路的正确布线。精确的解剖连接的建立是大脑功能发育的关键事件。在这里，我们将蝗虫（Locusta migratoria）的完整胚胎暴露在无血清培养物中，以测试化学物质，并通过荧光显微镜观察鉴定的先驱轴突的正确导航。我们为轴突伸长和沿着刻板路径导航定义了单独的毒理学终点。为了将发育神经毒性（DNT）与一般毒性区分开来，我们在浓度反应曲线上量化轴突伸长和导航的缺陷，并将其与胚胎的生化确定活力进行比较。一组公认的 DNT 阳性和阴性测试化合物的研究支持这种无脊椎动物胚胎测定具有相当高的可预测性。类似于哺乳动物皮层中突起的 semaphorin 介导的导向，蝗虫先驱轴突的正确轴突导航依赖于来自这个细胞识别分子家族成员的导向线索。由于神经突引导的进化保守机制，我们建议我们的先驱轴突范例可能为化学物质的 DNT 潜力对轴突伸长、导航和聚集过程提供机制上相关的信息。
