Journal: Frontiers in Neuroscience

Article Title: Harnessing the potential of human induced pluripotent stem cells, functional assays and machine learning for neurodevelopmental disorders

doi: 10.3389/fnins.2024.1524577

Figure Lengend Snippet: Comparison of functional assays for hiPSC-based disease study.

Article Snippet: KCNQ2-associated epilepsy (R581Q variation) , , 2D cortical excitatory neurons (NGN2 transduction + dual SMAD inhibition) , 12-well LD-MEA plate (Axion Biosystems) , Daily recording from days 15 to 31 , Neurobasal media with supplement , Patch clamping , Number of bursts, number of spikes per burst, % of spikes that occur within a burst, MFR, ISI, burst frequency, burst duration, IBI , KCNQ2-mutated neurons started exhibiting spontaneous activity earlier than the isogenic control cells, while showing an increasingly phasic bursting pattern with more and higher percentage of spikes per burst and shorter interval between spikes. Applying apamin (a SK channel antagonist) and paxilline (a BK antagonist) to KCNQ2-mutated neurons rescued the network activity to the same level as the control neurons, while the control neurons with chronic XE-991 treatment, M-current inhibitor, suggesting the role of reduced M-current and ion channel dysfunction in altered network behavior in KCNQ2-mutated neurons..

Techniques: Comparison, Functional Assay, Transferring, Microscopy, Imaging, Fluorescence

Journal: Frontiers in Neuroscience

Article Title: Harnessing the potential of human induced pluripotent stem cells, functional assays and machine learning for neurodevelopmental disorders

doi: 10.3389/fnins.2024.1524577

Figure Lengend Snippet: How MEA was used to study NDD with hiPSC model.

Article Snippet: KCNQ2-associated epilepsy (R581Q variation) , , 2D cortical excitatory neurons (NGN2 transduction + dual SMAD inhibition) , 12-well LD-MEA plate (Axion Biosystems) , Daily recording from days 15 to 31 , Neurobasal media with supplement , Patch clamping , Number of bursts, number of spikes per burst, % of spikes that occur within a burst, MFR, ISI, burst frequency, burst duration, IBI , KCNQ2-mutated neurons started exhibiting spontaneous activity earlier than the isogenic control cells, while showing an increasingly phasic bursting pattern with more and higher percentage of spikes per burst and shorter interval between spikes. Applying apamin (a SK channel antagonist) and paxilline (a BK antagonist) to KCNQ2-mutated neurons rescued the network activity to the same level as the control neurons, while the control neurons with chronic XE-991 treatment, M-current inhibitor, suggesting the role of reduced M-current and ion channel dysfunction in altered network behavior in KCNQ2-mutated neurons..

Techniques: Control, Transduction, Activity Assay, Disruption, Mutagenesis, Imaging, Inhibition, In Vitro

Journal: bioRxiv

Article Title: BrainPhys neuronal medium optimized for imaging and optogenetics in vitro

doi: 10.1101/2020.09.02.276535

Figure Lengend Snippet: A-E, optogenetic testing of human iPSC-derived neurons with whole-cell patch-clamp recordings. A, live image of typical neurons expressing the optogene synapsin:ChETA-YFP and filled with rhodamine from the patch pipette. B, optogenetic responses of patch-clamped human neurons to blue LED stimuli. Whole-cell patch-clamped traces of example neurons stimulated with 0.4 mW 10 × 5 ms flashes of blue light at 10 Hz in BPI and ACSF. Top traces show ChR2-mediated currents recorded in voltage-clamp at −70 mV. Bottom traces show action potential evoked by ChR2-evoked membrane depolarization in current-clamp. Corresponding raster plots highlight consistent light-evoked firing over 10 sweeps. C, quantitative comparison shown in the graphs on the right were performed from a total of 8 neurons recorded in ACSF and BPI and tested with identical light stimulation parameters as shown in the trace-examples. Symbols represent human neurons tested first (triangles) or second (circle) in either medium. D , optogenetic responses from the same patch-clamped neuron under 0.1 mW of blue light while alternating perfusate from BPI to NEUMO and back to BPI for recovery. E, quantitative comparison shown on the graphs on the right were performed from a total of 22 neurons across four coverslips. The perfusate was alternated between BPI and NEUMO. The neurons were stimulated with 10 × 5 ms flashes of blue light at 10 Hz under three different blue LED power intensities (0.1, 0.2, 0.4 mW at 475 nm). F-I, optogenetically evoked and spontaneous firing rates of human iPSC-derived neurons expressing synapsin:ChETA-YFP were recorded in a 48-well multielectrode array (MEAs) plate at 37 °C with 5% CO 2 in either BP, BPI or NEUMO basal media with identical supplements. Spontaneous and light-evoked firing rates were recorded for 10 minutes after feeding. Neurons in this dataset were cultured in standard BP for 82 days before testing different media. 240 electrodes across 15 wells were recorded over 40 days during media testing. F-G, recordings collected from one 48-well MEA plate were split into two groups of wells represented in the graph by ‘circles’ (8 wells) and ‘triangles’ (7 wells); Both groups were maintained in BP from neural maturation until they were changed to BPI on day 0. Days 7-13 were the ‘test’ period in which half the wells (7) were switched to NEUMO. From day 14, both groups were cultured in BP to recover for one week and then were switched back to BPI. H-I, optogenetics responses of human iPSC-derived neurons recorded with MEAs. The wells in NEUMO and BPI were optically stimulated with blue light (Lumos) at increasing intensities while recording on MEA. Stimulation lasted 1.1s and consisted of 10 flashes of light, each lasting 10ms at 10 Hz. The cumulative number of spikes was summed in binning windows ( H ): 27.5 ms bins ; ( I ): 2s bins starting at the onset of the first light stimulus) over three replicates. In ( H) the cumulative number spikes were plotted with quadratic non-linear curves at various LED intensity (% of max power: 3.9mW/mm 2 ). Values are shown as mean ± SEM. Significance determined via two-tailed non-parametric unpaired (Mann Whitney) tests. P-values are annotated as follows: ns for P>0.05.

Article Snippet: The 48-well Lumos MEA plate was maintained at 37°C, 5% CO 2 environment within a Maestro Pro MEA system (Axion Biosystems).

Techniques: Derivative Assay, Patch Clamp, Expressing, Transferring, Cell Culture, Optogenetics, Two Tailed Test, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: Tau P301L disengages from the proteosome core complex and neurogranin coincident with enhanced neuronal network excitability

doi: 10.1038/s41419-024-06815-2

Figure Lengend Snippet: 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).

Article Snippet: Twenty-four and 48-well CytoView MEA Plates (Axion Biosystems) were coated with 0.1% polyethylenimine (PEI) in borate buffer (100 mM Boric acid, 75 mM NaCl, 25 mM Sodium tetraborate, pH 8.4).

Techniques: Cell Culture, Activity Assay, Microscopy, Transduction, Expressing