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

Abstract

Neurotoxicity and the accumulation of extracellular amyloid-beta_1-42 (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.

Figure 1

Schematic illustration of the experimental neurodegeneration model on-CMOS-MEA chips. (a) Preparation and optimization of Aβ-oligomers. (b) Preparation and seeding of embryonic hippocampal neurons on CMOS chips. (c) Monitoring the spontaneous firing activity from 4096 electrodes on CMOS chips at baseline and after 0.1 µM Aβ-oligomer treatment.

Figure 2

Quantification of activity-dependent changes in neuronal network induced by early Aβ-neurotoxicity. (a) Large-scale array electrical readouts (MFR) of control groups (black lines) versus the 0.1 μM Aβ-induced toxicity group (red line). ***p < 0.001, ANOVA. (b) Confocal micrographs showing cellular c-fos expression after 26 h, cross-validated at the cellular level the electrical readouts. Scale bar represents 50 μm. (c) Quantification of c-fos⁺ nuclei ratio shows a significant decrease in expression 26 h after Aβ-exposure (red) compared to that in control groups (black and gray). ***p < 0.001, ANOVA. At the considered time points, the c-fos and electrical readouts show similar significant changes. (d) Intensity quantification of caspase-3 activity 26 h after Aβ-increased concentration shows no apoptotic activation at 0.1 μM, whereas such activation is significantly present at 10 µM. *p < 0.05, ANOVA. (e) Quantification of MTT assay shows no significant cell death at 12 and 26 h after 0.1 μM Aβ-exposure. (f) Distributions of firing rates (baseline and after 26 h) for the untreated condition, showing a significant peak shift toward higher frequencies of the preserved lognormal-like distributions, p < 0.05, Kolmogorov-Smirnov test. (g) Distributions of firing rates (baseline and after 26 h) for 0.1 μM Aβ-exposure, showing a significant peak shift toward lower frequencies and loss of the lognormal-like distribution, p < 0.05, Kolmogorov-Smirnov test. (h) Quantification of single active units showing increased, decreased or unchanged fractions of firing rates after 26 h, with respect to baseline, for untreated and Aβ-treated cultures. **p < 0.01, ANOVA.

Figure 3

Testing the NMDAR target mechanism hypothesis to emulate early Aβ-neurotoxicity. (a) Concentration-response curve of MFR for (0.1, 0.3, 0.5, and 1 µM) Aβ and (0.1, 0.5, 1, 5, 10, 30, 50, and 100 µM) NMDA after 26 h treatment. The goodness R² values of these fit curves are 0.98 for Aβ-data, and 0.89 for NMDA-data, respectively. (b) Arrows from the borderline that is indicated at 10 µM NMDA on the normalized MFR distinguish between effects of low NMDA doses (left, green) responsible for neuroprotection and high NMDA doses (right, red) responsible for neurotoxicity. (Insets: Spontaneous extracellular signal traces before and after 26 h of NMDA treatment (left) with 10 µM and (right) with 24.5 µM. (c) Distribution of firing rates after 26 h of exposure to a neuroprotective NMDA dose (10 µM) shows a similar lognormal-like distribution to baseline, with a slight non-significant peak shift toward higher frequencies, p = 0.22, Kolmogorov-Smirnov test. (d) At 24.5 µM NMDA, i.e., a concentration reported in this study to link Aβ excitotoxicity and extrasynaptic NMDARs activation, the firing distribution shows abnormal network dynamics, loss of the lognormal-like distribution and a peak shifted toward low rates, p < 0.05, Kolmogorov-Smirnov test. (e) Quantification of single active units showing increased, decreased or unchanged fractions of their firing rates after 26 h with respect to baseline, for cultures treated with a neuroprotective dose (10 µM) and a neurotoxic dose (24.5 µM) of NMDA. *p < 0.05, ANOVA.

Figure 4

Rescue strategies using biochemical molecules (memantine) and (saffron) to neuroprotect and reverse Aβ-induced neurotoxicity on CMOS chips. (a) Schematic of the 1^st scenario of rescue therapies using 10 µM memantine and 25 µg/ml saffron co-administered with 0.1 µM Aβ. (b) MFR responses of the 1^st scenario rescue therapy. *Denotes p < 0.05 compared to Aβ, ANOVA. ns, not significant. (c) Lognormal-like distributions of firing frequencies after 26 h in control and treated groups. p < 0.05, Kolmogorov-Smirnov test for biochemical treatments (memantine and saffron) versus Aβ. (d) Quantification of single active units. **Denotes p < 0.01 compared to Aβ, ANOVA. ns, not significant. (e) Schematic of 2^nd scenario rescue therapies, as in (a) but with molecules administered 26 h after 0.1 µM Aβ-oligomers and responses monitored for 52 h. (f) MFR responses of the 2^nd rescue strategy. **Denotes p < 0.01 compared to Aβ, ^##denotes p < 0.01 compared to control group, ANOVA. (g) As in (c) but corresponding to treatment in (e). p < 0.05 and p = 0.18, Kolmogorov-Smirnov test for saffron versus 0.1 µM Aβ and memantine versus 0.1 µM Aβ, respectively. (h) Quantification of single units as in (d) but for the 2^nd scenario treatment. *Denotes p < 0.05 compared to control, ^#denotes p < 0.05 compared to Aβ, ANOVA. ns and ns^β denote not significant compared to control and Aβ, respectively.

Figure 5

Cell-based therapeutic strategy using neural stem cells (NSCs) to reverse Aβ-induced neurotoxicity on CMOS chips. (a) Schematic of therapeutic treatment using NSCs administration on-chip 12 h after 0.1 µM Aβ. (b) Confocal micrographs of diseased neuronal culture (left), network of NSCs (middle), and mixed populations of matured neurons and NSCs (right). Scale bars represent 50 μm. (c) On-chip MFR upon rescuing therapy using NSCs monitored for 120 h. *Denotes p < 0.05 compared to Aβ; ^# and ⁺denote p < 0.05 compared to control, ANOVA. ns^c denotes not significant compared to control. (d) Lognormal-like distributions after 48 h recording corresponding to rescuing strategy in (a). p < 0.05, Kolmogorov-Smirnov test for rescued networks (Aβ + NSC) versus diseased networks (Aβ). (e) Quantification of single-unit analysis for NSCs rescue strategy, * and ⁺denote p < 0.05 compared to control and Neuron + NSCs, respectively. ^#Denotes p < 0.05 compared to Aβ, ANOVA. ns, ns^β, and ns^c denote not significant compared to control, Neuron + NSCs, and Aβ, respectively.