In vitro neurotransmitters release to study developmental neurotoxicity (DNT) in hiPSCs-derived neurons

Human induced pluripotent stem cells (hIPSCs) and their neuronal/glia derivatives are considered a valid resource to set up an in vitro model to study developmental neurotoxicity (DNT). Within this field, an in vitro battery to assess DNT through the evaluation of neuronal specific endpoints, such as NSC proliferation and apoptosis, differentiation into neurons and glia, neuronal migration, synaptogenesis, and neuronal network formation, is proposed by the official guidelines. However, none of these tests includes the possibility to assess how neurotoxicants may hinder neurotransmitters (in particular glutamate) release and clearance, creating a gap in the biological applicability of such battery. Hence, we implemented an high-performance liquid chromatography (HPLC)-based methodology to measure glutamate and aspartate release in a previously characterized model of hIPSC-derived NSC at different stages of differentiation towards a mixed neuron/glia culture. The analytical method involves precolumn derivatization with o-phalaldehyde, followed by separation on a C18 reverse-phase chromatography column and fluorimetric detection. We found that hiPSC-derived neurons acquired Ca²⁺-dependent glutamate release properties as a hallmark of neuronal maturation. Then, we determined the ability of the cell culture of releasing the neurotransmitters in basal conditions or upon depolarization, as well as after repeated exposure to known neurotoxicants, alone or in three different chemical mixtures. Concurrently, the effect of the toxic molecules was determined through non-conventional electrophysiology on Multi Electrode Arrays, in order to evaluate any alteration on neuron-glia networks. Our results suggest that hIPSC-derived neurons are able to release glutamate in a vesicular manner, and that the treatment with the different chemicals induces alterations at this level. In conclusion, our methodology provides essential information about hIPSC-derived cultures in the context of DNT, helping clarifying the mechanisms underlying neurotoxicants-induced damage. Moreover, given its sensitivity, the analysis of neurotransmitters release should be considered a reliable in vitro test to define functional neuronal endpoints.