Microelectrode Array Recordings Research Articles

Ensemble learning and ground-truth validation of synaptic connectivity inferred from spike trains.

Probing the architecture of neuronal circuits and the principles that underlie their functional organization remains an important challenge of modern neurosciences. This holds true, in particular, for the inference of neuronal connectivity from large-scale extracellular recordings. Despite the popularity of this approach and a number of elaborate methods to reconstruct networks, the degree to which synaptic connections can be reconstructed from spike-train recordings alone remains controversial. Here, we provide a framework to probe and compare connectivity inference algorithms, using a combination of synthetic ground-truth and in vitro data sets, where the connectivity labels were obtained from simultaneous high-density microelectrode array (HD-MEA) and patch-clamp recordings. We find that reconstruction performance critically depends on the regularity of the recorded spontaneous activity, i.e., their dynamical regime, the type of connectivity, and the amount of available spike-train data. We therefore introduce an ensemble artificial neural network (eANN) to improve connectivity inference. We train the eANN on the validated outputs of six established inference algorithms and show how it improves network reconstruction accuracy and robustness. Overall, the eANN demonstrated strong performance across different dynamical regimes, worked well on smaller datasets, and improved the detection of synaptic connectivity, especially inhibitory connections. Results indicated that the eANN also improved the topological characterization of neuronal networks. The presented methodology contributes to advancing the performance of inference algorithms and facilitates our understanding of how neuronal activity relates to synaptic connectivity.

Simple dynamic cell culture system reduces recording noise in microelectrode array recordings

AbstractMicroelectrode arrays (MEAs) have applications in drug discovery, toxicology, and basic research. They measure the electrophysiological response of tissue cultures to quantify changes upon exposure to biochemical stimuli. Unfortunately, manual addition of chemicals introduces significant noise in the recordings. Here, we report a simple-to-fabricate fluidic system that addresses this issue. We show that cell cultures can be successfully established in the fluidic compartment under continuous flow conditions and that the addition of chemicals introduces minimal noise in the recordings. This dynamic cell culture system represents an improvement over traditional tissue culture wells used in MEAs, facilitating electrophysiology measurements. Graphical abstract

Effects of acute insecticide exposure on neuronal activity in vitro in rat cortical cultures

Exposure to pesticides, such as carbamates, organophosphates, organochlorines and pyrethroids, has been linked to various health problems, including neurotoxicity. Although most in vivo studies use only male rodents, some studies have shown in vivo sex-specific effects after acute exposure. Since in vivo studies are costly and require a large number of animals, in vitro assays that take sex-specific effects into account are urgently needed. We therefore assessed the acute effects of exposure to different carbamates (methomyl, aldicarb and carbaryl), organophosphates (chlorpyrifos (CPF), chlorpyrifos-oxon (CPO) and 3,5,6-trichloropyridinol), organochlorines (endosulfan, dieldrin and lindane) and pyrethroids (permethrin, alpha-cypermethrin and 3-phenoxy-benzoic acid (3-PBA)) on neuronal network function in sex-separated rat primary cortical cultures using micro-electrode array (MEA) recordings. Our results indicate that exposure to the carbamate carbaryl and the organophosphates CPF and CPO decreased neuronal activity, with CPO being the most potent. Notably, (network) burst patterns differed between CPF and CPO, with CPO inducing fewer, but more intense (network) bursts. Exposure to low micromolar levels of endosulfan induced a hyperexcitation, most likely due to the antagonistic effects on GABA receptors. Interestingly, females were more sensitive to endosulfan than males. Exposure to dieldrin and lindane also increased neuronal activity, albeit less than endosulfan and without sex-specific effects. Exposure to type I pyrethroid permethrin increased neuronal activity, while exposure to type II pyrethroid alpha-cypermethrin strongly decreased neuronal activity. The increase seen after permethrin exposure was more pronounced in males than in females. Together, these results show that acute exposure to different classes of pesticides exerts differential effects on neuronal activity. Moreover, it shows that MEA recordings are suited to detect sex-specific neurotoxic effects in vitro.

Coherent olfactory bulb gamma oscillations arise from coupling independent columnar oscillators.

Spike timing-based representations of sensory information depend on embedded dynamical frameworks within neuronal networks that establish the rules of local computation and interareal communication. Here, we investigated the dynamical properties of olfactory bulb circuitry in mice of both sexes using microelectrode array recordings from slice and in vivo preparations. Neurochemical activation or optogenetic stimulation of sensory afferents evoked persistent gamma oscillations in the local field potential. These oscillations arose from slower, GABA(A) receptor-independent intracolumnar oscillators coupled by GABA(A)-ergic synapses into a faster, broadly coherent network oscillation. Consistent with the theoretical properties of coupled-oscillator networks, the spatial extent of zero-phase coherence was bounded in slices by the reduced density of lateral interactions. The intact in vivo network, however, exhibited long-range lateral interactions that suffice in simulation to enable zero-phase gamma coherence across the olfactory bulb. The timing of action potentials in a subset of principal neurons was phase-constrained with respect to evoked gamma oscillations. Coupled-oscillator dynamics in olfactory bulb thereby enable a common clock, robust to biological heterogeneities, that is capable of supporting gamma-band spike synchronization and phase coding across the ensemble of activated principal neurons.NEW & NOTEWORTHY Odor stimulation evokes rhythmic gamma oscillations in the field potential of the olfactory bulb, but the dynamical mechanisms governing these oscillations have remained unclear. Establishing these mechanisms is important as they determine the biophysical capacities of the bulbar circuit to, for example, maintain zero-phase coherence across a spatially extended network, or coordinate the timing of action potentials in principal neurons. These properties in turn constrain and suggest hypotheses of sensory coding.

Neuroactivity screening of botanical extracts using microelectrode array (MEA) recordings

Toxicity testing of botanicals is challenging because of their chemical complexity and variability. Since botanicals may affect many different modes of action involved in neuronal function, we used microelectrode array (MEA) recordings of primary rat cortical cultures to screen 16 different botanical extracts for their effects on cell viability and neuronal network function in vitro. Our results demonstrate that extract materials (50 μg/mL) derived from goldenseal, milk thistle, tripterygium, and yohimbe decrease mitochondrial activity following 7 days exposure, indicative of cytotoxicity. Importantly, most botanical extracts alter neuronal network function following acute exposure. Extract materials (50 μg/mL) derived from aristolochia, ephedra, green tea, milk thistle, tripterygium, and usnea inhibit neuronal activity. Extracts of kava, kratom and yohimbe are particularly potent and induce a profound inhibition of neuronal activity at the low dose of 5 μg/mL. Extracts of blue cohosh, goldenseal and oleander cause intensification of the bursts. Aconite extract (5 μg/mL) evokes a clear hyperexcitation with a marked increase in the number of spikes and (network) bursts. The distinct activity patterns suggest that botanical extracts have diverse modes of action. Our combined data also highlight the applicability of MEA recordings for hazard identification and potency ranking of botanicals.

Improvements for recording retinal function with Microelectrode Arrays

A microelectrode array (MEA) is a configuration of multiple electrodes that enables the concurrent targeting of multiple sites for extracellular recording and stimulation. By utilizing light pulses or electrical stimulations, MEA recordings unveil the complex patterns of electrical activities that arise from the signaling processes within the retinal network. Here, we present a stepwise approach for using microelectrode arrays (MEAs) for recording action potentials from the mouse retina in response to electrical and light stimuli. We provide detailed techniques accompanied by description of a custom optical system, example recordings, troubleshooting guidelines, and data processing methods including spike sorting and code resources for analyzing light and electrical responses. The comprehensive nature of this paper aims to guide researchers in utilizing MEAs effectively for investigating retinal functionality. In particular, it can be easy to have a MEA experiment fail, but hard to identify the source of the failure. This paper is meant to demystify that process. It includes:•A description of MEA setup, recording, and spike data validation.•A troubleshooting guide for common failure modes in MEA recordings from mouse retina.•Spike detection and sorting to precisely extract distinctive action potential.

Functional engineering of human iPSC-derived parasympathetic neurons enhances responsiveness to gastrointestinal hormones.

Food-derived biological signals are transmitted to the brain via peripheral nerves through the paracrine activity of gastrointestinal (GI) hormones. The signal transduction circuit of the brain-gut axis has been analyzed in animals; however, species-related differences and animal welfare concerns necessitate investigation using in vitro human experimental models. Here, we focused on the receptors of five GI hormones (CCK, GLP1, GLP2, PYY, and serotonin (5-HT)), and established human induced pluripotent stem cell (iPSC) lines that functionally expressed each receptor. Compared to the original iPSCs, iPSCs expressing one of the receptors did not show any differences in global mRNA expression, genomic stability, or differentiation capacities of the three germ layers. We induced parasympathetic neurons from these established iPSC lines to assess vagus nerve activity. We generated GI hormone receptor-expressing neurons (CCKAR, GLP1R, and NPY2R-neuron) and tested their responsiveness to each ligand using Ca2+ imaging and microelectrode array recording. GI hormone receptor-expressing neurons (GLP2R and HTR3A) were generated directly by gene induction into iPSC-derived peripheral nerve progenitors. These receptor-expressing neurons promise to contribute to a better understanding of how the body responds to GI hormones via the brain-gut axis, aid in drug development, and offer an alternative to animal studies.

Screening Alzheimer’s genetic risk factors against amyloid‐βeta synaptotoxicity

AbstractBackgroundSynapse degeneration is one of the earliest events in Alzheimer’s disease (AD) and toxic oligomers of amyloid‐beta (Aβ) peptide, cleavage product of APP, plays a major role in this process. Genome‐wide association studies recently reported 75 genomic risk loci for developing AD, but how the potential risk genes contribute to AD remains unknown. This project aims to identify AD genetic risk factors that mediate Aβ‐induced synaptotoxicity.MethodWe conducted a high‐content shRNA screening to assess the impact of ca. 200 AD risk genes on synaptic density and connectivity. Twenty‐four genes expressed in neurons with highest impact on synapses when silenced were shortlisted for a medium‐throughput screening to assess their capacity to block Aβ‐induced synaptotoxicity. Synaptotoxicity was modelled in microfluidic devices that allow primary postnatal rat neurons to be co‐cultured with Chinese hamster ovary cells, that overexpress wild‐type APP or APP with V717I (or London) mutation with increased secretion of Aβ1‐42 oligomers. Microfluidic devices allow the fluidic isolation of neurites from neuronal cell bodies and therefore provide exclusive access to pre‐ and post‐synaptic neurons. Target genes expression will be modulated either in pre‐ or post‐synaptic neurons and its impact on synaptic connectivity will be assessed.ResultBased on the shRNA screening, we shortlisted 17 detrimental genes, for which we obtained overexpression vectors packed in lentiviruses, and 6 protective genes. We have developed a medium‐throughput co‐culture device that permits screening twelve genes per imaging session using the INCell 6000 automated microscope, as well as an automated image analysis workflow for detecting and assigning presynaptic to postsynaptic puncta based on their relative proximity. Gene expression modulation is validated and the screen is on‐going.ConclusionWe have developed a microfluidic co‐culture model to rapidly and systematically interrogate genetic risk factors for their impact on synaptic connectivity. This study will help us identify AD genetic risk factors with a potential to block Aβ‐induced synaptotoxicity in vitro. Hit genes will be functionally validated via microelectrode array recordings.

Data Compression Versus Signal Fidelity Tradeoff in Wired-OR Analog-to-Digital Compressive Arrays for Neural Recording.

Future high-density and high channel count neural interfaces that enable simultaneous recording of tens of thousands of neurons will provide a gateway to study, restore and augment neural functions. However, building such technology within the bit-rate limit and power budget of a fully implantable device is challenging. The wired-OR compressive readout architecture addresses the data deluge challenge of a high channel count neural interface using lossy compression at the analog-to-digital interface. In this article, we assess the suitability of wired-OR for several steps that are important for neuroengineering, including spike detection, spike assignment and waveform estimation. For various wiring configurations of wired-OR and assumptions about the quality of the underlying signal, we characterize the trade-off between compression ratio and task-specific signal fidelity metrics. Using data from 18 large-scale microelectrode array recordings in macaque retina ex vivo, we find that for an event SNR of 7-10, wired-OR correctly detects and assigns at least 80% of the spikes with at least 50× compression. The wired-OR approach also robustly encodes action potential waveform information, enabling downstream processing such as cell-type classification. Finally, we show that by applying an LZ77-based lossless compressor (gzip) to the output of the wired-OR architecture, 1000× compression can be achieved over the baseline recordings.

Human acute microelectrode array recordings with broad cortical access, single-unit resolution, and parallel behavioral monitoring

There are vast gaps in our understanding of the organization and operation of the human nervous system at the level of individual neurons and their networks. Here, we report reliable and robust acute multichannel recordings using planar microelectrode arrays (MEAs) implanted intracortically in awake brain surgery with open craniotomies that grant access to large parts of the cortical hemisphere. We obtained high-quality extracellular neuronal activity at the microcircuit, local field potential level and at the cellular, single-unit level. Recording from the parietal association cortex, a region rarely explored in human single-unit studies, we demonstrate applications on these complementary spatial scales and describe traveling waves of oscillatory activity as well as single-neuron and neuronal population responses during numerical cognition, including operations with uniquely human number symbols. Intraoperative MEA recordings are practicable and can be scaled up to explore cellular and microcircuit mechanisms of a wide range of human brain functions.

Spatiotemporal spike-centered averaging reveals symmetry of temporal and spatial components of the spike-LFP relationship during human focal seizures

The electrographic manifestation of neural activity can reflect the relationship between the faster action potentials of individual neurons and the slower fluctuations of the local field potential (LFP). This relationship is typically examined in the temporal domain using the spike-triggered average. In this study, we add a spatial component to this relationship. Here we first derive a theoretical model of the spike-LFP relationship across a macroelectrode. This mathematical derivation showed a special symmetry in the spike-LFP relationship wherein a sinc function in the temporal domain predicts a sinc function in the spatial domain. We show that this theoretical result is observed in a real-world system by characterizing the spike-LFP relationship using microelectrode array (MEA) recordings of human focal seizures. To do this, we present a approach, termed the spatiotemporal spike-centered average (st-SCA), that allows for visualization of the spike-LFP relationship in both the temporal and spatial domains. We applied this method to 25 MEA recordings obtained from seven patients with pharmacoresistant focal epilepsy. Of the five patients with MEAs implanted in recruited territory, three exhibited spatiotemporal patterns consistent with a sinc function, and two exhibited spatiotemporal patterns resembling deep wells of excitation. These results suggest that in some cases characterization of the spike-LFP relationship in the temporal domain is sufficient to predict the underlying spatial pattern. Finally, we discuss the biological interpretation of these findings and propose that the sinc function may reflect the role of mid-range excitatory connections during seizure activity.

Comprehensive Collection of Current Methods for Contractility and Electrophysiology Recordings in Human Cardiac Cells

ARTICLES DISCUSSED: Gerges, N. A. et al. Measurement of heart contractility in isolated adult human primary cardiomyocytes. Journal of Visualized Experiments. (186), e64394 (2022). Lickiss, B. et al. Hybrid cell analysis system to assess structural and contractile changes of human iPSC-derived cardiomyocytes for preclinical cardiac risk evaluation. Journal of Visualized Experiments. (188), e64283 (2022). Feaster, T. K., Casciola, M., Narkar, A., Blinova, K. Evaluation of cardiac contractility modulation therapy in 2D human stem cell-derived cardiomyocytes. Journal of Visualized Experiments. (190), e64848 (2022). Schaefer, J., Danker, T., Gebhardt, K., Kraushaar, U. Laser-induced action potential-like measurements of cardiomyocytes on microelectrode arrays for increased predictivity of safety pharmacology. Journal of Visualized Experiments. (187), e64355 (2022). Berry, B. J. et al. Preclinical drug testing in scalable 3D engineered muscle tissues. Journal of Visualized Experiments. e64399 (2022). Zhao, S. R., Mondéjar-Parreño, G., Li, D., Shen, M., Wu, J. C. Technical applications of microelectrode array and patch clamp recordings on human induced pluripotent stem cell-derived cardiomyocytes. Journal of Visualized Experiments. (186), e64265 (2022).

An Innovative Toolkit to Investigate the Complex Mechanisms of Cardiac Arrhythmias

ARTICLES DISCUSSED: Tomsits, P., Schuttler, D., Kaab, S., Clauss, S., Voigt, N. Isolation of high quality murine atrial and ventricular myocytes for simultaneous measurements of Ca2+ transients and L-Type calcium current. Journal of Visualized Experiments. (165), 10.3791/61964 (2020). Seibertz, F., Reynolds, M., Voigt, N. Single-Cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes. Journal of Visualized Experiments. (166), 10.3791/61890 (2020). Xia, R. et al. Isolation and culture of resident cardiac macrophages from the murine sinoatrial and atrioventricular node. Journal of Visualized Experiments. (171), 10.3791/62236 (2021). Xia, R. et al. Whole-Mount immunofluorescence staining, confocal imaging and 3D reconstruction of the sinoatrial and atrioventricular node in the mouse. Journal of Visualized Experiments. (166), 10.3791/62058 (2020). Kumar, P., Si, M., Paulhus, K., Glasscock, E. Microelectrode array recording of sinoatrial node firing rate to identify intrinsic cardiac pacemaking defects in mice. Journal of Visualized Experiments. (173), 10.3791/62735 (2021). Scherschel, K. et al. Location, dissection, and analysis of the murine stellate ganglion. Journal of Visualized Experiments. (166), 10.3791/62026 (2020). Shimura, D., Hunter, J., Katsumata, M., Shaw, R. M. Removal of an internal translational start site from mRNA while retaining expression of the full-length protein. Journal of Visualized Experiments. (181), 10.3791/63405 (2022). Rotzer, R. D. et al. Implantation of combined telemetric ECG and blood pressure transmitters to determine spontaneous baroreflex sensitivity in conscious mice. Journal of Visualized Experiments. (168), 10.3791/62101 (2021). Tomsits, P. et al. Analyzing long-term electrocardiography recordings to detect arrhythmias in mice. Journal of Visualized Experiments. (171), 10.3791/62386 (2021). Grab, M. et al. Development and evaluation of 3D-printed cardiovascular phantoms for interventional planning and training. Journal of Visualized Experiments. (167), 10.3791/62063 (2021).

Organophosphate insecticides disturb neuronal network development and function via non-AChE mediated mechanisms

Exposure to organophosphate (OP) insecticides has been related to several adverse health effects, including neurotoxicity. The primary insecticidal mode of action of OP insecticides relies on (irreversible) binding to acetylcholine esterase (AChE), with –oxon metabolites having a much higher potency for AChE inhibition than the parent compounds. However, OP insecticides can also have non-AChE-mediated effects, including changes in gene expression, neuroendocrine effects, disruption of neurite outgrowth and disturbance of the intracellular calcium (Ca2+) homeostasis. Since Ca2+ is involved in neurotransmission and neuronal development, our research aimed to assess the effects of two widely used OP insecticides, chlorpyrifos (CPF) and diazinon (DZ) and their respective -oxon metabolites, on intracellular Ca2+ homeostasis in human SH-SY5Y cells and rat primary cortical cultures. Furthermore, we assessed the acute and chronic effects of exposure to these compounds on neuronal network maturation and function in rat primary cortical cultures using microelectrode array (MEA) recordings. While inhibition of AChE appears to be the primary mode of action of oxon-metabolites, our data indicate that both parent OP insecticides (CPF and DZ) inhibit depolarization-evoked Ca2+ influx and neuronal activity at concentrations far below their sensitivity for AChE inhibition, indicating that inhibition of voltage-gated calcium channels is a common mode of action of OP insecticides. Notably, parent compounds were more potent than their oxon metabolites, with exposure to diazinon-oxon (DZO) having no effect on both neuronal activity and Ca2+ influx. Human SH-SY5Y cells were more sensitive to OP-induced inhibition of depolarization-evoked Ca2+ influx than rat cortical cells. Acute exposure to OP insecticides had more potent effects on neuronal activity than on Ca2+ influx, suggesting that neuronal activity parameters are especially sensitive to OP exposure. Interestingly, the effects of DZ and chlorpyrifos-oxon (CPO) on neuronal activity lessened after 48 h of exposure, while the potency of CPF did not differ over time. This suggests that neurotoxicity after exposure to different OPs has different effects over time and occurs at levels that are close to human exposure levels. In line with these results, chronic exposure to CPF during 10 days impaired neuronal network development, illustrating the need to investigate possible links between early-life OP exposure and neurodevelopmental disorders in children and highlighting the importance of non-AChE mediated mechanisms of neurotoxicity after OP exposure.

Acute, sub-chronic and chronic exposures to TiO2 and Ag nanoparticles differentially affects neuronal function in vitro

In vivo toxicokinetic studies provide evidence for the translocation and accumulation of nanoparticles (NP) in the brain, thereby causing concern for adverse health effects, particularly for effects following chronic exposure. To date, only few studies investigated the effects of NP exposure on neuronal function in vitro, primarily focusing on short-term effects. The aim of this study was therefore to investigate the effects of two common types of NP, titanium dioxide NP (TiO2NP) and silver NP (AgNP), on neuronal function following acute (0.5 h), sub-chronic (24 h and 48 h) and chronic (14 days) exposure in vitro. Effects of NP exposure on intracellular calcium homeostasis, spontaneous neuronal (network) activity and neuronal network morphology were investigated in rat primary cortical cells using respectively, single-cell microscopy calcium imaging, micro-electrode array (MEA) recordings and immunohistochemistry. Our data demonstrate that high doses of AgNP (≥ 30 µg/mL) decrease calcium influx after 24 h exposure, although neuronal activity is not affected following acute and sub-chronic exposure. However, chronic exposure to non-cytotoxic doses of AgNP (1–10 µg/mL) potently decreases spontaneous neuronal (network) activity, without affecting network morphology and viability. Exposure to higher doses (≥ 30 µg/mL) affects network morphology and is also associated with cytotoxicity. In contrast, acute and sub-chronic exposure to TiO2NP is without effects, whereas chronic exposure only modestly reduces neuronal function without affecting morphology. Our combined findings indicate that TiO2NP exposure is of limited hazard for neuronal function whereas AgNP, in particularly during chronic exposure, has profound effects on neuronal (network) function and morphology.

Neuronal avalanche dynamics and functional connectivity elucidate information propagation in vitro.

Cascading activity is commonly observed in complex dynamical systems, including networks of biological neurons, and how these cascades spread through the system is reliant on how the elements of the system are connected and organized. In this work, we studied networks of neurons as they matured over 50 days in vitro and evaluated both their dynamics and their functional connectivity structures by observing their electrophysiological activity using microelectrode array recordings. Correlations were obtained between features of their activity propagation and functional connectivity characteristics to elucidate the interplay between dynamics and structure. The results indicate that in vitro networks maintain a slightly subcritical state by striking a balance between integration and segregation. Our work demonstrates the complementarity of these two approaches—functional connectivity and avalanche dynamics—in studying information propagation in neurons in vitro, which can in turn inform the design and optimization of engineered computational substrates.

Laser-Induced Action Potential-Like Measurements of Cardiomyocytes on Microelectrode Arrays for Increased Predictivity of Safety Pharmacology.

Life-threatening drug-induced cardiac arrhythmia is often preceded by prolonged cardiac action potentials (AP), commonly accompanied by small proarrhythmic membrane potential fluctuations. The shape and time course of the repolarizing fraction of the AP can be pivotal for the presence or absence of arrhythmia. Microelectrode arrays (MEA) allow easy access to cardiotoxic compound effects via extracellular field potentials (FP). Although a powerful and well-established tool in research and cardiac safety pharmacology, the FP waveform does not allow to infer the original AP shape due to the extracellular recording principle and the resulting intrinsic alternating current (AC) filtering. A novel device, described here, can repetitively open the membrane of cardiomyocytes cultivated on top of the MEA electrodes at multiple cultivation time points, using a highly focused nanosecond laser beam. The laser poration results in transforming the electrophysiological signal from FP to intracellular-like APs (laser-induced AP, liAP) and enables the recording of transcellular voltage deflections. This intracellular access allows a better description of the AP shape and a better and more sensitive classification of proarrhythmic potentials than regular MEA recordings. This system is a revolutionary extension to the existing electrophysiological methods, permitting accurate evaluation of cardiotoxic effect with all advantages of MEA-based recordings (easy, acute, and chronic experiments, signal propagation analysis, etc.).

An improved platform for cultured neuronal network electrophysiology: multichannel optogenetics integrated with MEAs.

Cultured neuronal networks (CNNs) are powerful tools for studying how neuronal representation and adaptation emerge in networks of controlled populations of neurons. To ensure the interaction of a CNN and an artificial setting, reliable operation in both open and closed loops should be provided. In this study, we integrated optogenetic stimulation with microelectrode array (MEA) recordings using a digital micromirror device and developed an improved research tool with a 64-channel interface for neuronal network control and data acquisition. We determined the ideal stimulation parameters including light intensity, frequency, and duty cycle for our configuration. This resulted in robust and reproducible neuronal responses. We also demonstrated both open and closed loop configurations in the new platform involving multiple bidirectional channels. Unlike previous approaches that combined optogenetic stimulation and MEA recordings, we did not use binary grid patterns, but assigned an adjustable-size, non-binary optical spot to each electrode. This approach allowed simultaneous use of multiple input-output channels and facilitated adaptation of the stimulation parameters. Hence, we advanced a 64-channel interface in that each channel can be controlled individually in both directions simultaneously without any interference or interrupts. The presented setup meets the requirements of research in neuronal plasticity, network encoding and representation, closed-loop control of firing rate and synchronization. Researchers who develop closed-loop control techniques and adaptive stimulation strategies for network activity will benefit much from this novel setup.

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety assessment models is a critical step during drug development. Currently, cardiac safety assessment is still highly dependent on animal studies. However, animal models are plagued by poor translational specificity to humans due to species-specific differences, particularly in terms of cardiac electrophysiological characteristics. Thus, there is an urgent need to develop a reliable, efficient, and human-based model for preclinical cardiac safety assessment. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an invaluable in vitro model for drug-induced cardiotoxicity screening and disease modeling. hiPSC-CMs can be obtained from individuals with diverse genetic backgrounds and various diseased conditions, making them an ideal surrogate to assess drug-induced cardiotoxicity individually. Therefore, methodologies to comprehensively investigate the functional characteristics of hiPSC-CMs need to be established. In this protocol, we detail various functional assays that can be assessed on hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium handling. Overall, the incorporation of hiPSC-CMs into preclinical cardiac safety assessment has the potential to revolutionize drug development.

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes