Fig. 8: Network analysis with micro-electrode arrays demonstrates basal hyperexcitability in neurons overexpressing eGFP-Tau^P301L.

All experiments were undertaken in DIV12 cultures cultured in CytoView micro-electrode array (MEA) multi-well plates using the Maestro-Pro MEA system (Axion Biosystems). A Each well contains 16 electrodes that can record local activity B Fluorescent microscopy shows DIV12 primary cortical neurons transduced with AAV-Syn1-eGFP and cultured on a micro-electrode plate. Scale bar = 275 µm. C An example of a raster plot, which shows firing over time, taken at baseline from untransduced neurons. Each row corresponds to an individual electrode. Action potentials are recorded as spikes (black lines) and this is termed ‘firing’. A burst (blue) occurs when multiples action potentials fire in rapid succession in the locality of an electrode; a minimum threshold is set at 50 spikes within 100 ms. A network burst was classified as at least 35% of electrodes recording bursts simultaneously; this shows that the neurons throughout the culture are connected. D Representative raster plots showing 60 s recordings of untransduced neurons at baseline and following treatment with 0.625 µM 4AP/12.5 µM Bicuculline (Bic). Baseline recordings show spontaneous firing and bursting. Treatment with 4AP/Bic stimulates synchronous and oscillatory firing across the well, as shown by a switch to network bursting. E 4AP/Bic treatment significantly increases activity and (F) burst strength (t-test, ****P < 0.0001, n = 3 technical repeats). G Representative raster plots comparing neurons overexpressing eGFP, eGFP-Tau^WT or eGFP-Tau^P301L at baseline and following treatment with 4AP/Bic. H At baseline, the activity and (I) burst strength of neurons overexpressing eGFP-Tau^P301L is significantly greater than those expressing eGFP or eGFP-Tau^WT. Following treatment with 4AP/Bic, activity but not burst strength is significantly greater with eGFP-Tau^P301L than eGFP or eGFP-Tau^WT (two-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 4 technical repeats).