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Further Evaluation of DNT Hazard Screening using Neural Networks from Rat Cortical Neurons on Multi-well Microelectrode Arrays
Citation:
Mundy, W., K. Wallace, D. Hall, AND J. Brown. Further Evaluation of DNT Hazard Screening using Neural Networks from Rat Cortical Neurons on Multi-well Microelectrode Arrays. Society of Toxicology Annual Meeting, New Orleans, LA, March 13 - 17, 2016.
Impact/Purpose:
This abstract evaluates an assay to screen and prioritize compounds for potential developmental neurotoxicity. It has a greatly reduced cost compared to in vivo guideline DNT studies and has a higher throughput.
Description:
Thousands of chemicals have not been characterized for their DNT potential. Due to the need for DNT hazard identification, efforts to develop screening assays for DNT potential is a high priority. Multi-well microelectrode arrays (MEA) measure the spontaneous activity of electrically excitable cells at multiple sites in a network (a single well) providing sufficient throughput as well as high-content data. The current study examined the ontogeny of spontaneous activity in cortical neural networks from Long Evans rat pups (0-1 day old) after developmental exposure to a set of compounds with in vivo evidence of developmental neurotoxicity. Primary cultures are comprised of both excitatory (glutamatergic) and inhibitory (GABAergic) neurons and glial cells. Activity was monitored over 12 days in vitro (DIV). Previous evaluation of this assay by screening 36 compounds indicated that MEAs may be useful for screening compounds for potential DNT hazard. The present study evaluated 12 additional compounds: 8 positives (amphetamine, chlordiazepoxide, colchicine, aminonicotinamide, bis(tri-n-butyltin)oxide, acrylamide, diethylstilbestrol, PBDE-47) and 4 negatives (Chloramben, cotinine, isoniazid, acetaminophen) at 0-30 µM. Cortical cells were exposed to compound 2 hours after seeding on a 48 well MEA plate. Network activity was recorded for 15 minutes on DIV 2, 5, 7, 9, and 12. Following the DIV 12 recording viability was assessed by Cell Titer Blue and LDH assays. On DIV 12, positives Bis(tri-n-butyltin)oxide, acrylamide, diethylstilbestrol, PBDE-47,) decreased the mean firing rate (MFR) at concentrations that were not cytotoxic, while chlordiazepoxide, colchicine, and aminonicotinamide decreased MFR concomitantly with cell death. Negative compounds chloramben, cotinine, and isoniazid also decreased MFR and viability. Acetaminophen and amphetamine showed no clear effect on either parameter. Combined with previous results, 31/39 compounds with in vivo evidence of DNT alter MFR and/or viability, 6/9 negative compounds did not affect either measure. These results indicate that this assay may be a useful screen for compounds with DNT potential (This abstract does not reflect EPA policy)