Abstract: Neurological disorders affect approximately 20% of the world population and are among the top ten leading causes of disability and death. Excitotoxicity has been associated with numerous neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Alzheimer’s. Excitotoxicity is caused by an excess of extracellular neurotransmitters like glutamate that will induce an overreaction of glutamate receptors in neurons. Excitotoxicity caused by excess extracellular glutamate could lead to cell damage and/or cellular death. Existing testing strategies are expensive, have low reproducibility and do not account for the localization (soma vs. axon) of cellular excitotoxicity. Our technology enables rapid neuronal growth on a chip, precisely organizing neuronal networks at high throughput for safety and efficacy evaluations (Magdesian et al., Biophys J. 2016; JOVE 2017). NeuroHTSTM is compatible with standard automation equipment to reproducibly test over 3,000 neurons per plate. NeuroHTSTM enables compartmentalization of neurons and supports evaluation of toxicity exposure independently to the soma as well as axon regions. Upon exposure, a suite of 7 morphological features can be evaluated in addition to biochemical screens with over 85% plate to plate reproducibility. Here we exposed soma and axons of human motor neurons grown in NeuroHTSTM to different concentrations of glutamate to model differential toxicity exposure. We measured 8 parameters per assay including: cell number, nucleic aggregation, axonal fibre thickness, axon length, branching, number of branching junctions, axon fragmentation index and neurite straightness. We observed significant differences between the two treatments in degrees of neuronal degeneration in 3 of the 8 measures: neurite length, fragmentation index, and nucleic aggregation. Neuronal degeneration is much less severe when glutamate toxicity is applied to axon only. In this exposure, toxicity is largely localized. We demonstrated the potential of a top-down screening approach using the NeuroHTSTM. The NeuroHTSTM is the first multi-well microplate to enable compartmentalized testing of axons and soma and generate robust big data for neuronal analysis. The paradigm shift top down screening approach provides key efficiencies needed for high throughput compound screening for safety and toxicity.
