High-resolution bioelectrical imaging of Aβ-induced network dysfunction on CMOS-MEAs for neurotoxicity and rescue studies

Neurotoxicity and the accumulation of extracellular amyloid-beta1–42 (Aβ) peptides are associated with the development of Alzheimer’s disease (AD) and correlate with neuronal activity and network dysfunctions, ultimately leading to cellular death. However, research on neurodegenerative diseases is hampered by the paucity of reliable readouts and experimental models to study such functional decline from an early onset and to test rescue strategies within networks at cellular resolution. To overcome this important obstacle, we demonstrate a simple yet powerful in vitro AD model based on a rat hippocampal cell culture system that exploits large-scale neuronal recordings from 4096-electrodes on CMOS-chips for electrophysiological quantifications. This model allows us to monitor network activity changes at the cellular level and to uniquely uncover the early activity-dependent deterioration induced by Aβ-neurotoxicity. We also demonstrate the potential of this in vitro model to test a plausible hypothesis underlying the Aβ-neurotoxicity and to assay potential therapeutic approaches. Specifically, by quantifying N-methyl D-aspartate (NMDA) concentration-dependent effects in comparison with low-concentration allogenic-Aβ, we confirm the role of extrasynaptic-NMDA receptors activation that may contribute to Aβ-neurotoxicity. Finally, we assess the potential rescue of neural stem cells (NSCs) and of two pharmacotherapies, memantine and saffron, for reversing Aβ-neurotoxicity and rescuing network-wide firing.

(a) Number of active electrodes increases for control groups (black lines) and decreases after 26 h of exposure to 0.1 μM Aβ-oligomers (red line). **Denotes p < 0.01 when compared to untreated ctr and veh, and ### denotes p < 0.001 when compared to scrambled-Aβ, ANOVA with Tukey's post hoc test. (b) Mean bursting rate (MBR) indicates a decreasing tendency of the network synchronization and its significant suppression after 26 h of Aβ-oligomers treatment (red line), with respect to control groups (black lines). ***p < 0.001, ANOVA with Tukey's post hoc test. (c) Representative layouts of the full array (left) vs. the down-sampled network on 60 electrodes layout (right), to compare their averaged firing rates. (d) MFRs of Aβ-treated group plotted for the full-array vs. the down-sampled network recordings, where both are significantly different over 7 phases of recordings. p < 0.001, Kolmogorov-Smirnov test. Down-sampled recordings show higher variability compared to full-array recordings. (e) Number of active electrodes of Aβ-treated group plotted for the full-array vs. the down-sampled network recordings, where both are significantly different over 7 phases of recordings. p < 0.001, Kolmogorov-Smirnov test. Down-sampled recordings show high-variability (zoomed in; right) and preclude the characterization of active-electrodes trend upon Aβ-treatment.     Network-wide activity responses evaluated in terms of number of active electrodes and mean bursting rate (MBR) for two tested compounds co-administrated with Aβ-oligomers.
(a) Memantine at 10 µM and saffron at 25 µg/ml both show a potential neuroprotective effect by preserving the number of firing electrodes compared to Aβ-treated cultures. *p < 0.05, ANOVA with Tukey's post hoc test. ns denotes "not significant when compared to the Aβ-treated group". (b) Both memantine and saffron significantly maintain the network synchronization as indicated by the MBR compared to Aβ treated networks. *p < 0.05, ANOVA with Tukey's post hoc test. ns denotes "not significant when compared to the Aβ-treated group". Network-wide activity responses evaluated in terms of number of active electrodes and mean bursting rate (MBR) for two tested compounds administrated 26 h after Aβ-oligomers induced neurodegeneration.
(a) Saffron at 25 µg/ml shows a potent effect over 52 h to rescue the dysfunction of the neuronal network by preserving the number of firing electrodes compared to Aβ-treated cultures (0.1 µM), while when the degeneration is in-place, no significant rescuing effect is observed with memantine treatment. *p < 0.05, ANOVA with Tukey's post hoc test. ns denotes "not significant when compared to the Aβ-treated group". (b) Similarly, only saffron shows a potent effect over 52 h to rescue the network synchronization properties as indicated by the MBR compared to the Aβ-treated group. ** Denotes p < 0.01 when compared to Aβ, and ## denotes p < 0.01 when compared to control, ANOVA with Tukey's post hoc test. ns denotes "not significant when compared to the Aβ-treated group ". Network-wide activity responses evaluated in terms of number of active electrodes and mean bursting rate (MBR) upon cell-therapy using NSCs administrated 12 h after Aβ-oligomers induced neurodegeneration.
(a) NSCs integrating into pre-existing networks treated with 0.  (a) Dot plots for changes in the group treated with co-administered Aβ+memantine, between baseline and after 26 h. Single units are classified as described in (the methods section) to (left) unchanged, (right, green) increased, (right, red) healthy networks (Neuron+NSCs). (d) As in (a), but for changes in the group, where NSCs added to a diseased pre-existing networks, 12 h after toxicity was induced by Aβ-oligomers (Aβ+NSCs). (e) Quantification of data reported in (a-d). * and + denote p < 0.05 when compared to control and Neuron+NSCs, respectively. # Denotes p < 0.05 when compared to Aβ, ANOVA with Tukey's post hoc test. ns, ns β , and ns c denote not significant when compared to control, Neuron+NSCs, and Aβ, respectively.
Figure S13 | Schematic of the CMOS-MEA platform and the acquisition system.