This manuscript was supported by R01NS127829 NIH/NINDS (LTD). Figure 1 has been generated using biorender.com.

Recording Network Activity in Brain Assembloids: Multi-Electrode Array Approach

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The authors have no relevant disclosures.

MEA-based methods for electrophysiological recordings of network activity in iPSC-derived assembloids have been used for in vitro modeling of epilepsy22,23. This proposed platform integrating excitatory and inhibitory synaptic connections has the potential to address mechanisms of neuronal hyperexcitability and the role of cortical interneurons in the process of epileptogenesis. Additionally, this platform allows for the stabilization of assembloids and collection of longitudinal electrophysiological data for about 50 days in vitro, even in the presence of pharmacological perturbations, thereby providing an approach for testing compounds with potential therapeutic targets.
Stabilizing mature, relatively large assembloids on a hydrophobic surface for a long period in vitro has been technically challenging. To overcome this obstacle, the twice-coating strategy is applied with conversely charged chemicals to facilitate short- and long-term adherence of organoids to the electrode surface. This protocol optimizes the previously used method24 by switching the plate-coating reagents from poly-L-ornithine (PLO)/laminin to PEI/BMM, achieving solid attachment with high success rates and low toxicity. Furthermore, this stabilized long-term culture system has the potential to yield high-throughput data. For example, pre-labeling with dual viruses can complement spike sorting by individual electrodes to localize and compare activity from different regions. The proposed platform also provides a stable tool for recording local field potentials, which is highly relevant to interneuron function without disrupting the intact structure of assembloids25. The major limitation of this protocol is that assembloids are flattened after plating, which raises concerns about whether the initial 3D structures are preserved or not. Growing organoids around the electrodes using more advanced MEA platforms that envelope an organoid with electrodes26 could potentially overcome the limitation. 
One of the main obstacles of MEA-based methods for electrophysiological recording of organoids is the variability and reproducibility of experimental findings across differentiation protocols and even batches of differentiation. Previous studies show that the network activity of neurons measured by MEA is dependent upon the neuronal maturation status of both dorsal and ventral organoids27. Recording from assembloids at a later timepoint with relatively mature neurons and circuits is one way to improve consistency. Furthermore, the choice of multi-well MEA systems generating high throughput data will allow statistical comparisons in light of experimental variability. If the electrophysiological activity of a culture is inconsistent with what is expected at a given time point, collecting the intact culture from the functional platform for quality control measures, including immunocytochemical and transcriptomic analysis, will be important and helpful.
Paramount to the proposed platform is to functionally integrate regionally specified excitatory cortical neurons on the dorsal side and inhibitory interneurons on the ventral side. Protocols for the generation of brain region-specific organoids found in the literature differ extensively in relation to media conditions, the timing of cultures, yield, and maturation profiles, among other factors28. While this protocol is cortex- and ganglionic eminence-specific in its ability to generate excitatory neurons and GABAergic interneurons, well-established differentiation techniques can be used to generate iPSC-derived neurons displaying other identities, such as motor neurons29 and Purkinje cells30. Thus, the proposed platform has the potential to model neural circuits between the cerebral cortex and the spinal cord/ cerebellum. After some optimization related to the timing of plating and visualization of cell types with viral labeling, this method can be translated to other disease models aside from epilepsy, like Amyotrophic Lateral Sclerosis (ALS)17.

Human pluripotent stem cell (hPSC)-derived brain organoids are spatially self-organized 3D structures mirroring the tissue architecture and developmental trajectories in vivo. They are composed of multiple cell types, including progenitors (neuroepithelial cells, radial glia, neuronal progenitors, glial progenitors), neurons (cortical-like excitatory neurons and inhibitory interneurons), and glial cells (astrocytes and oligodendrocytes)1,2. Assembloids represent the next generation of brain organoids, capable of integrating multiple brain regions and/or cell lineages within a 3D culture. They provide a useful tool for modeling connections between various brain regions resembling the in vivo counterparts, capturing interactions between neurons and astrocytes to better mimic mature and complex neural networks, and to study the assembly of neural circuits. Therefore, assembloids are becoming a widely used tool to recapitulate hallmarks of epilepsy pathophysiology, in which functional measures are necessary to interrogate aberrant neural networks that may underlie the cause of the disease3,4,5,6.
To model interactions between cortical glutamatergic neurons and GABAergic interneurons, several groups developed separate organoids resembling the dorsal and ventral brain and then fused them together into a multi-region assembloid7,8,9,10. Here, a previously described assembloid culture protocol with regionally specific neural subtypes was applied9. However, a significant barrier is the lack of reproducible functional assays to monitor neural network activity during neurodevelopment. Many functional tests of the networks in organoids yield results that have high variability among batches of differentiations and cell lines. Techniques that involve slicing or dissociating organoids alter their inherent networks by severing synaptic connectivity8.
Multi-electrode arrays (MEA) provide a large-scale view of network activity over time with high temporal resolution to characterize electrophysiological properties of organoids without disrupting culture conditions or cell membrane integrity11. Compared to patch clamp electrophysiology, MEA enables high-throughput data acquisition based on large populations of neurons rather than single cells. MEA platforms vary in their electrode density, catering to different needs in brain organoid research12. The widely used systems, as shown in this protocol, record from 8 to 64 electrodes per well13,14,15. High-density MEAs with up to 26,400 electrodes per well enable increased spatial and temporal resolution, quantification of action potential propagation speed, and combination with optogenetic stimulation14,16,17. Therefore, MEA serves as a powerful tool for modeling epilepsy in vitro and a translational paradigm for anti-seizure medication screening.
One major challenge is to stabilize the large-sized assembloids on a hydrophobic metal surface for long-term recordings. This protocol outlines a detailed methodology for plating intact assembloids on MEA plates for long-term longitudinal recording together with pharmacological assays. Unique advantages of the protocol include stable attachment of assembloids to the electrode surface without losing electrical activity, use of commercially available neurophysiological basal media to accelerate functional network maturation after plating, feasibility to conduct downstream functional assays like drug treatment, and wide application to organoids generated with other region-specific protocols.
The goal is to provide a functional assay with high temporal resolution to investigate network activity, examine disease-specific changes, and test drugs with therapeutic potential in epilepsy. Video instructions are provided for the most challenging steps of this protocol, showing the techniques for plating assembloids on MEA plates, as well as representative recordings from these cultures.

<table><tbody><tr><td>10 cm Corning Non TC-treated culture dishes</td><td>Corning</td><td>08-772-32</td><td>For suspension culture on the shaker</td></tr><tr><td>100 mL Beaker&amp;nbsp;</td><td>Fisher Scientific</td><td>FB100100</td><td></td></tr><tr><td>100% Ethanol</td><td>Fisher Scientific</td><td>BP28184-4L</td><td></td></tr><tr><td>2-Mercaptoethanol (&amp;beta;-ME)</td><td>Thermo Fisher</td><td>21985023</td><td>Working concentration 100 &amp;mu;M</td></tr><tr><td>48-well cell culture plate</td><td>Fisher Scientific</td><td>50-202-140</td><td></td></tr><tr><td>6-well cell culture plate</td><td>Fisher Scientific</td><td>07-200-83</td><td></td></tr><tr><td>Aggrewell 800</td><td>Fisher Scientific</td><td>501974754</td><td></td></tr><tr><td>Allegra X-14R Refrigerated Centrifuge</td><td>Beckman Coulter&amp;nbsp;</td><td>BE-AX14R</td><td></td></tr><tr><td>Allopregnanolone</td><td>Cayman</td><td>16930</td><td>Suspended 5mg into DMSO to get 1 mM stock solution. Aliquot and freeze at &amp;minus;80 &amp;deg;C. Dilute at 1:10,000 for use. Working concentration 100 nM.</td></tr><tr><td>Automated cell counter</td><td>Thermo Fisher</td><td>AMQAX2000</td><td></td></tr><tr><td>Axion CytoView MEA 6-well plates&amp;nbsp;</td><td>Axion Biosystems</td><td>M384-tMEA-6B</td><td></td></tr><tr><td>Axion Maestro MEA platform</td><td>Axion Biosystems</td><td>Maestro</td><td>With temp environmental control</td></tr><tr><td>B-27 supplement (regular, with Vitamin A)</td><td>Thermo Fisher</td><td>21985023</td><td></td></tr><tr><td>B-27 supplement (without Vitamin A)</td><td>Thermo Fisher</td><td>12587010</td><td></td></tr><tr><td>Basement membrane matrix- Geltrex</td><td>Thermo Fisher</td><td>A1569601</td><td></td></tr><tr><td>Bead bath</td><td>Fisher Scientific</td><td>10-876-001</td><td>Isotemp</td></tr><tr><td>Benchtop inverted microscope</td><td>Olympus</td><td>CKX53</td><td>Kept in laminar flow clean bench</td></tr><tr><td>Bicuculline</td><td>Sigma-Aldrich</td><td>14340</td><td>Working concentration 10 &amp;mu;M</td></tr><tr><td>Bleach</td><td>CLOROX</td><td>67619-26</td><td></td></tr><tr><td>Borate buffer 20x</td><td>Thermo Fisher</td><td>28341</td><td>Working concentration at 1x</td></tr><tr><td>BrainPhys media</td><td>StemCell Technologies</td><td>5790</td><td></td></tr><tr><td>Cell dissociation reagent (StemPro Accutase)</td><td>Thermo Fisher</td><td>A1110501</td><td></td></tr><tr><td>Celltron orbital shaker</td><td>HT-Infors</td><td>&amp;nbsp;I69222&amp;nbsp;</td><td></td></tr><tr><td>Detergent/enzyme (Terg-A-Zyme)</td><td>Sigma-Aldrich</td><td>Z273287</td><td>Working concentration 1% m/v</td></tr><tr><td>DMEM/F12 + HEPES/L-Glutamine</td><td>Thermo Fisher</td><td>113300</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>67685</td><td></td></tr><tr><td>Dorsomorphin</td><td>Sigma-Aldrich</td><td>P5499</td><td>Dissolve 5mg into DMSO to get 10 mM stock solution. Aliquot and freeze at &amp;minus;20 &amp;deg;C. Dilute at 1:2000 for use. (working concentration 5 &amp;mu;M)</td></tr><tr><td>D-PBS w/o calcium or magnesium</td><td>Thermo Fisher</td><td>14190144</td><td></td></tr><tr><td>Glial cell line-derived neurotrophic (GDNF)</td><td>Peprotech</td><td>450-10</td><td>Dissolve 100 &amp;mu;g in 1mL of PBS to 100 &amp;mu;g/mL. Aliquot and freeze at &amp;minus;20 &amp;deg;C. Dilute at 1:5000 for use. (working concentration 20 ng/mL).</td></tr><tr><td>GlutaMAX supplement</td><td>Thermo Fisher</td><td>35050061</td><td></td></tr><tr><td>Hemacytometer</td><td>Election Microscopy Sciences</td><td>63510-20</td><td></td></tr><tr><td>HEPES</td><td>Thermo Fisher</td><td>15630080</td><td></td></tr><tr><td>Heraguard ECO Clean Bench</td><td>Thermo Fisher</td><td>51029692</td><td></td></tr><tr><td>Humidity controlled cell culture incubator</td><td>Thermo Fisher</td><td>370</td><td>set to 37 &amp;deg;C, 5% CO2</td></tr><tr><td>IWP-2</td><td>Selleckchem</td><td>S7085</td><td>Aliquot and freeze at &amp;minus;80 &amp;deg;C. It will precipitate if thawed at room temp. Frozen aliquots should be placed directly into 37 &amp;deg;C before use.</td></tr><tr><td>Knockout serum replacement (KOSR)</td><td>Thermo Fisher</td><td>10828010</td><td></td></tr><tr><td>mTeSRplus (medium + supplements)</td><td>StemCell Technologies</td><td>100-0276</td><td>cGMP, stabilized feeder-free medium for human iPSC cells</td></tr><tr><td>N2 supplement</td><td>Thermo Fisher</td><td>17502048</td><td></td></tr><tr><td>Neurobasal A</td><td>Thermo Fisher</td><td>21103049</td><td></td></tr><tr><td>Non-essential amino acids (NEAA)</td><td>Thermo Fisher</td><td>11140050</td><td></td></tr><tr><td>NT3</td><td>Peprotech</td><td>450-03</td><td>Dissolve 100 &amp;mu;g in 1mL of PBS to 100 &amp;mu;g/mL. Aliquot and freeze at &amp;minus;20 &amp;deg;C. Dilute at 1:5000 for use. (working concentration 20 ng/mL).</td></tr><tr><td>NuFF Human neonatal foreskin fibroblasts</td><td>MTI-GlobalStem</td><td>GSC-3002</td><td></td></tr><tr><td>Parafilm</td><td>PARAFILM</td><td>P7793</td><td></td></tr><tr><td>Penicilin/Streptomycin</td><td>Thermo Fisher</td><td>15140122</td><td></td></tr><tr><td>Pipette (P10, P200, P1000)</td><td>Eppendorf</td><td>EP4926000034</td><td>Autoclaved cut P1000 tips for organoid collection</td></tr><tr><td>Poly (Ethyleneimine) (PEI)</td><td>Sigma-Aldrich</td><td>P3143</td><td>Dilute stock in sterile borate buffer. Working concentration 0.07%. See details in manuscript.</td></tr><tr><td>Recombinant human epidermal growth factor (EGF)</td><td>R&amp;amp;D Systems</td><td>236-EG-200</td><td>Suspended in PBS. Aliquot and freeze at -20 &amp;deg;C.</td></tr><tr><td>Recombinant Human fibroblast growth factor (FGF)-basic&amp;nbsp;</td><td>Peprotech</td><td>100-18B</td><td>Suspended in PBS. Aliquot and freeze at -20 &amp;deg;C.</td></tr><tr><td>Recombinant human-brain-derived neurotrophic factor (BDNF)</td><td>Peprotech</td><td>450-02</td><td>Centrifuge briefly before reconstitution. Dissolve 100 &amp;mu;g in 1 mL of PBS to 100 &amp;mu;g/mL. Aliquot and freeze at &amp;minus;20 &amp;deg;C. Dilute at 1:5000 for use. (working concentration 20 ng/mL).</td></tr><tr><td>Retinoic acid (RA)</td><td>Sigma-Aldrich</td><td>R2625</td><td>Dissolve 100 mg into 3.3 mL of DMSO to get 100 mM stock solution. Aliquot the stock 100 &amp;mu;L/tube and freeze at &amp;minus;80 &amp;deg;C. Take 200 &amp;mu;L of 100 mM stock and dilute 10x (add 1.8 mL of DMSO) to make 10 mM stock. Aliquot 50 &amp;mu;L/tube and store at &amp;minus;80 &amp;deg;C. Dilute at 1:100,000 for use. (working concentration 100 nM).</td></tr><tr><td>ROCK inhibitor Y-27632</td><td>Tocris</td><td>1254</td><td>1:200 from 10 mM stock</td></tr><tr><td>SAG (smoothened agonist)</td><td>Selleckchem</td><td>S7779</td><td>Aliquot and freeze at &amp;minus;80 &amp;deg;C. Stock concentration 1mM. Use at 1:10,000 dilution (working concentration 100 nM).</td></tr><tr><td>SB-431542</td><td>Tocris</td><td>1614</td><td>Dissolve 5mg into 1.3 mL of DMSO to get 10 mM stock solution. Aliquot and freeze at &amp;minus;80 &amp;deg;C. Dilute at 1:1000 for use. (working concentration 10 &amp;mu;M)</td></tr><tr><td>Serological pipette filler</td><td>Fisher Scientific</td><td>14-387-166</td><td></td></tr><tr><td>Steriflip vacuum tube top filter</td><td>Sigma-Aldrich</td><td>SE1M179M6</td><td></td></tr><tr><td>Sterile cell culture hoods</td><td>Baker Company</td><td>SG-600</td><td></td></tr><tr><td>Trypan blue solution (0.4%)</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Thermo Fisher</td><td>25200056</td><td></td></tr><tr><td>Zoom stereomicroscope</td><td>Olympus</td><td>SZ61/SZ51</td><td>Kept in laminar flow clean bench</td></tr></tbody></table>

a multi-electrode array platform for modeling epilepsy using human pluripotent stem cell-derived brain assembloids

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal brain development. The fusion of dorsal with ventral regionally specified brain organoids in vitro generates assembloids, which have functionally integrated microcircuits with excitatory and inhibitory neurons. Due to their structural complexity and diverse population of neurons, assembloids have become a useful in vitro tool for studying aberrant network activity. Multi-electrode array (MEA) recordings serve as a method for capturing electrical field potentials, spikes, and longitudinal network dynamics from a population of neurons without compromising cell membrane integrity. However, adhering assembloids onto the electrodes for long-term recordings can be challenging due to their large size and limited contact surface area with the electrodes. Here, we demonstrate a method to plate assembloids onto MEA plates for recording electrophysiological activity over a 2-month span. Although the current protocol utilizes human cortical organoids, it can be broadly adapted to organoids differentiated to model other brain regions. This protocol establishes a robust, longitudinal, electrophysiological assay for studying the development of a neuronal network, and this platform has the potential to be used in drug screening for therapeutic development in epilepsy.

Human dorsal-ventral assembloids were plated on a 6-well MEA plate (n = 6 per differentiation, 3 differentiations), with each well containing 64 electrodes (Figure 2A). Nine out of ten assembloids planned for longitudinal recording were firmly attached to the electrodes for over 50 days in vitro (Figure 2D). 6 out of 8 assembloids designated for pharmacological assays were successfully settled through the wash-out phase with development stage-appropriate network activity (Figure 2E). Phase contrast and fluorescence images from one representative well are shown (Figure 2B), in which the AAV-CAG-tdTomato-labeled dorsal and AAV-mDLX-GFP-labeled ventral organoids are easily distinguished7,9.
Activity of neurons on an MEA can be analyzed to assess overall cellular excitability and network activity, through a variety of parameters. Overall firing rates across the culture provide information on cellular and network excitability, while the frequency of synchronized bursts across multiple electrodes indicates the range of synaptically active networks of neurons. Multiple repeated spikes occurring across spatially distinct electrodes over a short period of time (network bursting) can serve as a biological correlate to seizure-like activity, although assembloids on MEAs remain limited compared to a whole organism that can have bona fide seizures. Figure 2C illustrates the network bursting patterns of assembloids cultured in commercially available neurophysiological basal media on Days 84 and 91, with increased bursting duration at the later timepoint likely due to functional maturation of the neurons. In this case, cellular and network activity gradually rises from the day when assembloids are plated, usually peaking on Day 92, and almost fading away on Day 125 (Figure 2D). The increased maturation at the later stage likely contributes to the extended synchronized burst duration at the later recording time point.
Bicuculline, a GABAA receptor antagonist, enhances the synchronized network activity of the assembloids, as shown by significantly higher firing rates and (network) burst frequencies after adding the drug on Day 108 (Figure 2E). This is similar to what has been previously demonstrated in single-cell electrophysiology recordings21. A power analysis for the pharmacological assay was conducted using G*Power3 base on an effect size of 0.94, and an alpha of 0.05. A total sample of 15 assembloids with three equal-sized groups of n = 5 was required to achieve a power of 0.8 (Figure 2E). A vehicle control is included to verify that the response is a true signaling event and not merely a change in signal intensity that may arise from the addition of a small amount of media (Figure 2E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67396/67396fig01.jpg" /> 
Figure 1: Overview of the study. Protocol for the generation and plating of dual labeled assembloids on MEA timeline of the differentiation protocol detailing critical stages and corresponding small-molecule supplements (for additional information on the composition, see Table 1 and Table of Materials). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67396/67396fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67396/67396fig02.jpg" /> 
Figure 2: MEA recording of assembloids. (A) Heat map of mean firing activity from a single MEA plate (n = 6 wells) at Day 82, i.e., post-plating day 10. All wells represent cultures of the same condition and genotype. (B) Representative phase-contrast and confocal images of the assembloid, with dorsal assembloid labeled with red and ventral with green. White arrowheads indicate migrated GABAergic interneurons from the ventral to the dorsal side. Scale bar = 500 &#181;m. (C) Representative raster plots over 120 s of recording time from a single well. Network bursts are highlighted with a purple box. Two separate timepoints, Day 78 (1 week after plating, starting day of recording) and Day 92 (3 weeks after plating, also when the activity peaks), are demonstrated. (D) Quantification of mean firing rates, burst frequency, and network bursting frequency from day 78 to day 127 (n = 3 differentiations, 3 assembloids/ differentiation). Each curve indicates one individual assembloid, compiled from 3 differentiations (color-coded). The red curve demonstrates the mean value. (E) Quantification of bicuculline assay results (n = 6 assembloids/ condition). The bar graph was displayed as mean &#177; SEM. Each dot represents one assembloid, compiled from 3 batches of differentiation (color-coded). A nonparametric Friedman test was performed. **p &#60; 0.01; ns, not significant. MEA, multi-electrode arrays. Heat map and raster plots are generated using the analytic software Neural Metric Tool. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67396/67396fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Components of media at various differentiation stages. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67396/67396_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>

Author Spotlight: Advancing Genetic Epilepsy Studies with Multi-Electrode Array-Based Long-Term Electrophysiological Monitoring of Human Brain Assembloids

This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal ...

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Universidad San Sebastian

Research

JoVE Journal

Neuroscience

1.1K Views.  Michigan Medicine, Ann Arbor. This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

Video: Author Spotlight: Advancing Genetic Epilepsy Studies with Multi-Electrode Array-Based Long-Term Electrophysiological Monitoring of Human Brain Assembloids

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

Developmental Biology

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Medicine

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...

Multimodal Large-Scale Neural Recordings and Analysis on HD-MEA 

Recording and Analyzing Multimodal Large-Scale Neuronal Ensemble Dynamics on CMOS-Integrated High-Density Microelectrode Array

Large-scale neuronal networks and their complex distributed microcircuits are essential to generate perception, cognition, and behavior that emerge from patterns of spatiotemporal neuronal activity. These dynamic patterns emerging from functional groups of interconnected neuronal ensembles facilitate precise computations for processing and coding multiscale neural information, thereby driving higher brain functions. To probe the computational principles of neural dynamics underlying this complexity and investigate the multiscale impact of biological processes in health and disease, large-scale simultaneous recordings have become instrumental. Here, a high-density microelectrode array (HD-MEA) is employed to study two modalities of neural dynamics - hippocampal and olfactory bulb circuits from ex-vivo mouse brain slices and neuronal networks from in-vitro cell cultures of human induced pluripotent stem cells (iPSCs). The HD-MEA platform, with 4096 microelectrodes, enables non-invasive, multi-site, label-free recordings of extracellular firing patterns from thousands of neuronal ensembles simultaneously at high spatiotemporal resolution. This approach allows the characterization of several electrophysiological network-wide features, including single/-multi-unit spiking activity patterns and local field potential oscillations. To scrutinize these multidimensional neural data, we have developed several computational tools incorporating machine learning algorithms, automatic event detection and classification, graph theory, and other advanced analyses. By supplementing these computational pipelines with this platform, we provide a methodology for studying the large, multiscale, and multimodal dynamics from cell assemblies to networks. This can potentially advance our understanding of complex brain functions and cognitive processes in health and disease. Commitment to open science and insights into large-scale computational neural dynamics could enhance brain-inspired modeling, neuromorphic computing, and neural learning algorithms. Furthermore, understanding the underlying mechanisms of impaired large-scale neural computations and their interconnected microcircuit dynamics could lead to the identification of specific biomarkers, paving the way for more accurate diagnostic tools and targeted therapies for neurological disorders.

Author Spotlight: Advancing Large-Scale Neural Dynamics Through HD-MEA Technology

Here, we employ HD-MEA to delve into computational dynamics of large-scale neuronal ensembles, particularly in hippocampal, olfactory bulb circuits, and human neuronal networks. Capturing spatiotemporal activity, combined with computational tools, provides insights into neuronal ensemble complexity. The method enhances understanding of brain functions, potentially identifying biomarkers and treatments for neurological disorders.

Large-scale neuronal networks and their complex distributed microcircuits are essential to generate perception, cognition, and behavior that emerge from ...

Consistent Induction and Recording of Seizure-Like Activity Using CMOS-HD-MEAs

High-Quality Seizure-Like Activity from Acute Brain Slices Using a Complementary Metal-Oxide-Semiconductor High-Density Microelectrode Array System

Complementary metal-oxide-semiconductor high-density microelectrode array (CMOS-HD-MEA) systems can record neurophysiological activity from cell cultures and ex vivo brain slices in unprecedented electrophysiological detail. CMOS-HD-MEAs were first optimized to record high-quality neuronal unit activity from cell cultures but have also been shown to produce quality data from acute retinal and cerebellar slices. Researchers have recently used CMOS-HD-MEAs to record local field potentials (LFPs) from acute, cortical rodent brain slices. One LFP of interest is seizure-like activity. While many users have produced brief, spontaneous epileptiform discharges using CMOS-HD-MEAs, few users reliably produce quality seizure-like activity. Many factors may contribute to this difficulty, including electrical noise, the inconsistent nature of producing seizure-like activity when using submerged recording chambers, and the limitation that 2D CMOS-MEA chips only record from the surface of the brain slice. The techniques detailed in this protocol should enable users to consistently induce and record high-quality seizure-like activity from acute brain slices with a CMOS-HD-MEA system. In addition, this protocol outlines the proper treatment of CMOS-HD-MEA chips, the management of solutions and brain slices during experimentation, and equipment maintenance.

Author Spotlight: Unraveling Seizure Dynamics and Novel Therapeutics for Status Epilepticus Using CMOS High-Density Microelectrode Array Systems

Here, we outline a protocol for using complementary metal-oxide-semiconductor high-density microelectrode array systems (CMOS-HD-MEAs)&#160;to record seizure-like activity from ex vivo brain slices.

Complementary metal-oxide-semiconductor high-density microelectrode array (CMOS-HD-MEA) systems can record neurophysiological activity from cell cultures ...

Measuring the Electrophysiological Activity of Neuronal Networks Using Micro-Electrode Arrays

Source: Frega, M. et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54900">Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays.</a> J. Vis. Exp. (2017).
The video demonstrates how to use a microelectrode array (MEA) to record the electrophysiological activity of neurons derived from human induced pluripotent stem cells (hiPSCs). Electrophysiological activity from a multiwell MEA containing a neuron-astrocyte co-culture is recorded to detect action potentials from multiple neurons in the network, confirming the successful differentiation of hiPSCs into neurons.

Pomiar aktywności elektrofizjologicznej sieci neuronalnych za pomocą układów mikroelektrod

1. Differentiation of&#160;rtTA/Ngn2-positive hiPSCs to Neurons on 6-well MEAs and Glass Coverslips
NOTE:&#160;In this protocol, the details are provided ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Differentiating Human Induced Pluripotent Stem Cells into Neurons on Micro-Electrode Arrays

Source: Frega, M., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54900">Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays.</a> J. Vis. Exp. (2017)
The video demonstrates a method for generating excitatory cortical neurons from human-induced pluripotent stem cells (hiPSCs). Upon doxycycline induction, the hiPSCs overexpress neurogenin-2, a neural-specific transcription factor. By using specific media and co-culturing with astrocytes, this process facilitates the hiPSCs&#39; differentiation into neurons with synaptic connections.

Różnicowanie ludzkich indukowanych pluripotencjalnych komórek macierzystych w neurony na układach mikroelektrod

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Differentiation of ...

Neuronal Culture Techniques

Primary Neuronal Culture

Utilizing Multielectrode Arrays to Measure Synaptic Physiology in 3D Coculture Spheres

Source: Cvetkovic, C., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018)
This video demonstrates the procedure of using the multielectrode array (MEA) to measure synaptic physiology in 3D coculture spheres. This method allows to understand the neural network dynamics and synaptic network burst activity within the 3D spheres.

Wykorzystanie matryc wieloelektrodowych do pomiaru fizjologii synaptycznej w sferach kokultury 3D

Measurement of Live Synaptic Physiology with Multielectrode Arrays (MEAs)
1. Prepare MEAs for cell culture.

	Clean the surface of each MEA with 1 mL of ...