Evaluation of neurotoxicity for pesticide-related compounds in human iPS cell-derived neurons using microelectrode array.

In vivo evaluations of chemicals in neurotoxicity have certain limitations due to the considerable time and cost required, necessity of extrapolation from rodents to humans, and limited information on toxicity mechanisms. To address this issue, the development of in vitro test methods using new approach methodologies (NAMs) is important to evaluate the chemicals in neurotoxicity. Microelectrode array (MEA) allows the assessment of changes in neural network activity caused by compound administration. However, studies on compound evaluation criteria are scarce. In this study, we evaluated the impact of pesticides on neural activity using MEA measurements of human iPSC-derived neurons. A principal component analysis was performed on the electrical physiological parameters obtained by MEA measurements, and the influence of excessive neural activity due to compound addition was defined using the standard deviation of neural activity with solvent addition as the reference. By using known seizurogenic compounds as positive controls for neurotoxicity in MEA and evaluating pesticides with insufficient verification of their neurotoxicity in humans, we demonstrated that these pesticides exhibit neurotoxicity in humans. In conclusion, our data suggest that the neurotoxicity evaluation method in human iPSC neurons using MEA measurements could be one of the in vitro neurotoxicity test methods that could replace animal experiments.

Large-Area Field Potential Imaging Having Single Neuron Resolution Using 236 880 Electrodes CMOS-MEA Technology.

The electrophysiological technology having a high spatiotemporal resolution at the single-cell level and noninvasive measurements of large areas provide insights on underlying neuronal function. Here, a complementary metal-oxide semiconductor (CMOS)-microelectrode array (MEA) is used that uses 236 880 electrodes each with an electrode size of 11.22 × 11.22 µm and 236 880 covering a wide area of 5.5 × 5.9 mm in presenting a detailed and single-cell-level neural activity analysis platform for brain slices, human iPS cell-derived cortical networks, peripheral neurons, and human brain organoids. Propagation pattern characteristics between brain regions changes the synaptic propagation into compounds based on single-cell time-series patterns, classification based on single DRG neuron firing patterns and compound responses, axonal conduction characteristics and changes to anticancer drugs, and network activities and transition to compounds in brain organoids are extracted. This detailed analysis of neural activity at the single-cell level using the CMOS-MEA provides a new understanding of the basic mechanisms of brain circuits in vitro and ex vivo, on human neurological diseases for drug discovery, and compound toxicity assessment.

In Vitro Pain Assay Using Human iPSC-Derived Sensory Neurons and Microelectrode Array.

Drug-induced peripheral neuropathy occurs as an adverse reaction of chemotherapy. However, a highly accurate method for assessing peripheral neuropathy and pain caused by compounds has not been established. The use of human-induced pluripotent stem cell (hiPSC)-derived sensory neurons does not require animal experiments, and it is considered an effective method that can approach extrapolation to humans. In this study, we evaluated the response to pain-related compounds based on neural activities using in vitro microelectrode array (MEA) measurements in hiPSC-derived sensory neurons. Cultured sensory neurons exhibited gene expression of the Nav1.7, TRPV1, TRPA1, and TRPM8 channels, which are typical pain-related channels. Channel-dependent evoked responses were detected using the TRPV1 agonist capsaicin, a TRPA1 agonist, allyl isothiocyanate (AITC), and TRPM8 agonist menthol. In addition, the firing frequency increased with an increase in temperature from 37°C to 46°C, and temperature sensitivity was observed. In addition, the temperature of the peak firing rate differed among individual neurons. Next, we focused on the increase in cold sensitivity, which is a side effect of the anticancer drug oxaliplatin, and evaluated the response to AITC in the presence and absence of oxaliplatin. The response to AITC increased in the presence of oxaliplatin in a concentration-dependent manner, suggesting that the increased cold sensitivity in humans can be reproduced in cultured hiPSC-derived sensory neurons. The in vitro MEA system using hiPSC-derived sensory neurons is an alternative method to animal experiments, and it is anticipated as a method for evaluating peripheral neuropathy and pain induced by compounds.