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Organophosphorus nerve agents (OPNA) are hazardous environmental exposures to the civilian population and have been historically weaponized as chemical warfare agents (CWA). OPNA exposure can lead to several neurological, sensory, and motor symptoms that can manifest into chronic neurological illnesses later in life. There is still a large need for technological advancement to better understand changes in brain function following OPNA exposure. The human-relevant in vitro multi-electrode array (MEA) system, which combines the MEA technology with human stem cell technology, has the potential to monitor the acute, sub-chronic, and chronic consequences of OPNA exposure on brain activity. However, the application of this system to assess OPNA hazards and risks to human brain function remains to be investigated. In a concentration-response study, we have employed a human-relevant MEA system to monitor and detect changes in the electrical activity of engineered neural networks to increasing concentrations of the sarin surrogate 4-nitrophenyl isopropyl methylphosphonate (NIMP). We report a biphasic response in the spiking (but not bursting) activity of neurons exposed to low (i.e., 0.4 and 4 µM) versus high concentrations (i.e., 40 and 100 µM) of NIMP, which was monitored during the exposure period and up to 6 days post-exposure. Regardless of the NIMP concentration, at a network level, communication or coordination of neuronal activity decreased as early as 60 min and persisted at 24 h of NIMP exposure. Once NIMP was removed, coordinated activity was no different than control (0 µM of NIMP). Interestingly, only in the high concentration of NIMP did coordination of activity at a network level begin to decrease again at 2 days post-exposure and persisted on day 6 post-exposure. Notably, cell viability was not affected during or after NIMP exposure. Also, while the catalytic activity of AChE decreased during NIMP exposure, its activity recovered once NIMP was removed. Gene expression analysis suggests that human iPSC-derived neurons and primary human astrocytes resulted in altered genes related to the cell's interaction with the extracellular environment, its intracellular calcium signaling pathways, and inflammation, which could have contributed to how neurons communicated at a network level.

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Sharp wave-ripple complexes (SPW-Rs) are spontaneous oscillatory events that characterize hippocampal activity during resting periods and slow-wave sleep. SPW-Rs are related to memory consolidation - the process during which newly acquired memories are transformed into long-lasting memory traces. To test the involvement of SPW-Rs in this process, it is crucial to understand how SPW-Rs originate and propagate throughout the hippocampus. SPW-Rs can originate in CA3, and they typically spread from CA3 to CA1, but little is known about their formation within CA3. To investigate the generation and propagation of SPW-Rs in CA3, we recorded from mouse hippocampal slices using multi-electrode arrays and patch-clamp electrodes. We characterized extracellular and intracellular correlates of SPW-Rs and quantified their propagation along the pyramidal cell layer of CA3. We found that a hippocampal slice can be described by a speed and a direction of propagation of SPW-Rs. The preferred propagation direction was from CA3c (the subfield closer to the dentate gyrus) toward CA3a (the subfield at the boundary to CA2). In patch-clamp recordings from CA3 pyramidal neurons, propagation was estimated separately for excitatory and inhibitory currents associated with SPW-Rs. We found that propagation speed and direction of excitatory and inhibitory currents were correlated. The magnitude of the speed of propagation of SPW-Rs within CA3 was consistent with the speed of propagation of action potentials in axons of CA3 principal cells. KEY POINTS: Hippocampal sharp waves are considered important for memory consolidation; therefore, it is of interest to understand the mechanisms of their generation and propagation. Here, we used two different approaches to study the propagation of sharp waves in mouse CA3 in vitro: multi-electrode arrays and multiple single-cell recordings. We find a preferred direction of propagation of sharp waves from CA3c toward CA3a - both in the local field potential and in sharp wave-associated excitatory and inhibitory synaptic activity. The speed of sharp wave propagation is consistent with the speed of action potential propagation along the axons of CA3 pyramidal neurons. These new insights into the dynamics of sharp waves in the CA3 network will inform future experiments and theoretical models of sharp-wave generation mechanisms.

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Objective.The visual perception provided by retinal prostheses is limited by the overlapping current spread of adjacent electrodes. This reduces the spatial resolution attainable with unipolar stimulation. Conversely, simultaneous multipolar stimulation guided by the measured neural responses-neural activity shaping (NAS)-can attenuate excessive spread of excitation allowing for more precise control over the pattern of neural activation. However, defining effective multipolar stimulus patterns is a challenging task. Previous attempts focused on analytical solutions based on an assumed linear nonlinear model of retinal response; an analytical model inversion (AMI) approach. Here, we propose a model-free solution for NAS, using artificial neural networks (ANNs) that could be trained with data acquired from the implant.Approach.Our method consists of two ANNs trained sequentially. The measurement predictor network (MPN) is trained on data from the implant and is used to predict how the retina responds to multipolar stimulation. The stimulus generator network is trained on a large dataset of natural images and uses the trained MPN to determine efficient multipolar stimulus patterns by learning its inverse model. We validate our methodin silicousing a realistic model of retinal response to multipolar stimulation.Main results.We show that our ANN-based NAS approach produces sharper retinal activations than the conventional unipolar stimulation strategy. As a theoretical bench-mark of optimal NAS results, we implemented AMI stimulation by inverting the model used to simulate the retina. Our ANN strategy produced equivalent results to AMI, while not being restricted to any specific type of retina model and being three orders of magnitude more computationally efficient.Significance.Our novel protocol provides a method for efficient and personalized retinal stimulation, which may improve the visual experience and quality of life of retinal prosthesis users.

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During the last decade brain organoids have emerged as an attractive model system, allowing stem cells to be differentiated into complex 3D models, recapitulating many aspects of human brain development. Whilst many studies have analysed anatomical and cytoarchitectural characteristics of organoids, their functional characterisation has been limited, and highly variable between studies. Standardised, consistent methods for recording functional activity are critical to providing a functional understanding of neuronal networks at the synaptic and network level that can yield useful information about functional network phenotypes in disease and healthy states. In this study we outline a detailed methodology for calcium imaging and Multi-Electrode Array (MEA) recordings in brain organoids. To illustrate the utility of these functional interrogation techniques in uncovering induced differences in neural network activity we applied various stimulating media protocols. We demonstrate overlapping information from the two modalities, with comparable numbers of active cells in the four treatment groups and an increase in synchronous behaviour in BrainPhys treated groups. Further development of analysis pipelines to reveal network level changes in brain organoids will enrich our understanding of network formation and perturbation in these structures, and aid in the future development of drugs that target neurological disorders at the network level.

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The mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway is a ubiquitous cellular pathway. mTORopathies, a group of disorders characterized by hyperactivity of the mTORC1 pathway, illustrate the prominent role of the mTOR pathway in disease pathology, often profoundly affecting the central nervous system. One of the most debilitating symptoms of mTORopathies is drug-resistant epilepsy, emphasizing the urgent need for a deeper understanding of disease mechanisms to develop novel anti-epileptic drugs. In this study, we explored the multiwell Multi-electrode array (MEA) system as a tool to identify robust network activity parameters in an approach to model mTORopathy-related epilepsy in vitro. To this extent, we cultured mouse primary hippocampal neurons on the multiwell MEA to identify robust network activity phenotypes in mTORC1-hyperactive neuronal networks. mTOR-hyperactivity was induced either through deletion of Tsc1 or overexpression of a constitutively active RHEB variant identified in patients, RHEBp.P37L. mTORC1 dependency of the phenotypes was assessed using rapamycin, and vigabatrin was applied to treat epilepsy-like phenotypes. We show that hyperactivity of the mTORC1 pathway leads to aberrant network activity. In both the Tsc1-KO and RHEB-p.P37L models, we identified changes in network synchronicity, rhythmicity, and burst characteristics. The presence of these phenotypes is prevented upon early treatment with the mTORC1-inhibitor rapamycin. Application of rapamycin in mature neuronal cultures could only partially rescue the network activity phenotypes. Additionally, treatment with the anti-epileptic drug vigabatrin reduced network activity and restored burst characteristics. Taken together, we showed that mTORC1-hyperactive neuronal cultures on the multiwell MEA system present reliable network activity phenotypes that can be used as an assay to explore the potency of new drug treatments targeting epilepsy in mTORopathy patients and may give more insights into the pathophysiological mechanisms underlying epilepsy in these patients.

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Repeated and uncontrolled seizures in epilepsy result in brain cell loss and neural inflammation. Current anticonvulsants primarily target ion channels and receptors implicated in seizure activity. Identification of neurotherapeutics that can inhibit epileptiform activity and reduce inflammation in the brain may offer significant benefits in the long-term management of epilepsy. Fenamates are unique because they are both non-steroidal anti-inflammatory drugs (NSAIDs) and highly subunit selective modulators of GABAA receptors. In the current study we have investigated the hypothesis that fenamates have antiseizure properties using mature human stem cell-derived neuro-glia cell cultures, maintained in long-term culture, and previously shown to be sensitive to first, second and third generation antiepileptics. Mefenamic acid, flufenamic acid, meclofenamic acid, niflumic acid, and tolfenamic acid (each tested at 10-100 µM) attenuated 4-aminopyridine (4-AP, 100 µM) evoked epileptiform activity in a dose-dependent fashion. These actions were as effective diazepam (3-30 µM) and up to 200 times more potent than phenobarbital (300-1,000 µM). The low (micromolar) concentrations of fenamates that inhibited 4-AP evoked epileptiform activity correspond to those reported to potentiate GABAA receptor function. In contrast, the fenamates had no effect on neural spike amplitudes, indicating that their antiseizure actions did not result from inhibition of sodium-channels. The antiseizure actions of fenamates were also not replicated by either of the two non-fenamate NSAIDs, ibuprofen (10-100 µM) or indomethacin (10-100 µM), indicating that inhibition of cyclooxygenases is not the mechanism through which fenamates have anticonvulsant properties. This study therefore shows for the first time, using functionally mature human stem cell-derived neuroglial circuits, that fenamate NSAIDs have powerful antiseizure actions independent of, and in addition to their well-established anti-inflammatory properties, suggesting these drugs may provide a novel insight and new approach to the treatment of epilepsy in the future.

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Objective.Detection of the epileptogenic zone is critical, especially for patients with drug-resistant epilepsy. Accurately mapping cortical regions exhibiting high activity during spontaneous seizure events while detecting neural activity up to 500 Hz can assist clinicians' surgical decisions and improve patient outcomes.Approach.We designed, fabricated, and tested a novel hybrid, multi-scale micro-electrocorticography (micro-ECoG) array with a unique embedded configuration. This array was compared to a commercially available microelectrode array (Neuronexus) for recording neural activity in rodent sensory cortex elicited by somatosensory evoked potentials and pilocarpine-induced seizures.Main results.Evoked potentials and spatial maps recorded by the multi-scale array ('micros', 'mesos', and 'macros' refering to the relative electrode sizes, 40 micron, 1 mm, and 4 mm respectively) were comparable to the Neuronexus array. The SSEPs recorded with the micros had higher peak amplitudes and greater signal power than those recorded by the larger mesos and macro. Seizure onset events and high-frequency oscillations (∼450 Hz) were detected on the multi-scale, similar to the commercially available array. The micros had greater SNR than the mesos and macro over the 5-1000 Hz frequency range during seizure monitoring. During cortical stimulation experimentation, the mesos successfully elicited motor effects.Significance.Previous studies have compared macro- and microelectrodes for localizing seizure activity in adjacent regions. The multi-scale design validated here is the first to simultaneously measure macro- and microelectrode signals from the same overlapping cortical area. This enables direct comparison of microelectrode recordings to the macroelectrode recordings used in standard neurosurgical practice. Previous studies have also shown that cortical regions generating high-frequency oscillations are at an increased risk for becoming epileptogenic zones. More accurate mapping of these micro seizures may improve surgical outcomes for epilepsy patients.

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BACKGROUND: The retinotopic map property of the superior colliculus (SC) is a reliable indicator of visual functional changes in rodents. Electrophysiological mapping of the SC using a single electrode has been employed for measuring visual function in rat and mouse disease models. Single electrode mapping is highly laborious requiring long-term exposure to the SC surface and prolonged anesthetic conditions that can adversely affect the mapping data. NEW METHOD: To avoid the above-mentioned issues, we fabricated a fifty-six (56) electrode multi-electrode array (MEA) for rapid and reliable visual functional mapping of the SC. Since SC is a dome-shaped structure, the array was made of electrodes with dissimilar tip lengths to enable simultaneous and uniform penetration of the SC. RESULTS: SC mapping using the new MEA was conducted in retinal degenerate (RD) Royal College of Surgeons (RCS) rats and rats with focal retinal damage induced by green diode laser. For SC mapping, the MEA was advanced into the SC surface and the visual activities were recorded during full-filed light stimulation of the eye. Based on the morphological examination, the MEA electrodes covered most of the exposed SC area and penetrated the SC surface at a relatively uniform depth. MEA mapping in RCS rats (n=9) demonstrated progressive development of a scotoma in the SC that corresponded to the degree of photoreceptor loss. MEA mapping in the laser damaged rats demonstrated the presence of a scotoma in the SC area that corresponded to the location of retinal laser injury. COMPARISON WITH EXISTING METHODS AND CONCLUSIONS: The use of MEA for SC mapping is advantageous over single electrode recording by enabling faster recordings and reducing anesthesia time. This study establishes the feasibility of the MEA technique for rapid and efficient SC mapping, particularly advantageous for evaluating therapeutic effects in retinal degenerate rat disease models.

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The investigation of the human brain at cellular and microcircuit level remains challenging due to the fragile viability of neuronal tissue, inter- and intra-variability of the samples and limited availability of human brain material. Especially brain slices have proven to be an excellent source to investigate brain physiology and disease at cellular and small network level, overcoming the temporal limits of acute slices. Here we provide a revised, detailed protocol of the production and in-depth knowledge on long-term culturing of such human organotypic brain slice cultures for research purposes. We highlight the critical pitfalls of the culturing process of the human brain tissue and present exemplary results on viral expression, single-cell Patch-Clamp recordings, as well as multi-electrode array recordings as readouts for culture viability, enabling the use of organotypic brain slice cultures of these valuable tissue samples for basic neuroscience and disease modeling (Fig. 1).

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In clinical rhythmology, intracardiac bipolar electrograms (EGMs) play a critical role in investigating the triggers and substrates inducing and perpetuating atrial fibrillation (AF). However, the interpretation of bipolar EGMs is ambiguous due to several aspects of electrodes, mapping algorithms and wave propagation dynamics, so it requires several variables to describe the effects of these uncertainties on EGM analysis. In this narrative review, we critically evaluate the potential impact of such uncertainties on the design of cardiac mapping tools on AF-related substrate characterization. Literature suggest uncertainties are due to several variables, including the wave propagation vector, the wave's incidence angle, inter-electrode spacing, electrode size and shape, and tissue contact. The preprocessing of the EGM signals and mapping density will impact the electro-anatomical representation and the features extracted from the local electrical activities. The superposition of multiple waves further complicates EGM interpretation. The inclusion of these uncertainties is a nontrivial problem but their consideration will yield a better interpretation of the intra-atrial dynamics in local activation patterns. From a translational perspective, this review provides a concise but complete overview of the critical variables for developing more precise cardiac mapping tools.

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MicroGraphited-Diamond-Multi Electrode Arrays (µG-D-MEAs) can be successfully used to reveal, in real time, quantal exocytotic events occurring from many individual neurosecretory cells and/or from many neurons within a network. As µG-D-MEAs arrays are patterned with up to 16 sensing microelectrodes, each of them recording large amounts of data revealing the exocytotic activity, the aim of this work was to support an adequate analysis code to speed up the signal detection. The cutting-edge technology of microGraphited-Diamond-Multi Electrode Arrays (µG-D-MEAs) has been implemented with an automated analysis code (APE, Amperometric Peak Analysis) developed using Matlab R2022a software to provide easy and accurate detection of amperometric spike parameters, including the analysis of the pre-spike foot that sometimes precedes the complete fusion pore dilatation. Data have been acquired from cultured PC12 cells, either collecting events during spontaneous exocytosis or after L-DOPA incubation. Validation of the APE code was performed by comparing the acquired spike parameters with those obtained using Quanta Analysis (Igor macro) by Mosharov et al.

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[This corrects the article DOI: 10.3389/fncel.2023.1287089.].

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Whereas studies of the V1 cortex have focused mainly on neural line orientation preference, color inputs are also known to have a strong presence among these neurons. Individual neurons typically respond to multiple colors and nearby neurons have different combinations of preferred color inputs. However, the computations performed by V1 neurons on such color inputs have not been extensively studied. Here we aimed to address this issue by studying how different V1 neurons encode different combinations of inputs composed of four basic colors. We quantified the decoding accuracy of individual neurons from multi-electrode array recordings, comparing multiple individual neurons located within 2 mm along the vertical axis of the V1 cortex of the anesthetized rat. We found essentially all V1 neurons to be good at decoding spatiotemporal patterns of color inputs and they did so by encoding them in different ways. Quantitative analysis showed that even adjacent neurons encoded the specific input patterns differently, suggesting a local cortical circuitry organization which tends to diversify rather than unify the neuronal responses to each given input. Using different pairs of monocolor inputs, we also found that V1 neocortical neurons had a diversified and rich color opponency across the four colors, which was somewhat surprising given the fact that rodent retina express only two different types of opsins. We propose that the processing of color inputs in V1 cortex is extensively composed of multiple independent circuitry components that reflect abstract functionalities resident in the internal cortical processing rather than the raw sensory information per se.

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While there is a growing appreciation of three-dimensional (3D) neural tissues (i.e., hydrogel-based, organoids, and spheroids), shown to improve cellular health and network activity to mirror brain-like activity in vivo, functional assessment using current electrophysiology techniques (e.g., planar multi-electrode arrays or patch clamp) has been technically challenging and limited to surface measurements at the bottom or top of the 3D tissue. As next-generation MEAs, specifically 3D MEAs, are being developed to increase the spatial precision across all three dimensions (X, Y, Z), development of improved computational analytical tools to discern region-specific changes within the Z dimension of the 3D tissue is needed. In the present study, we introduce a novel computational analytical pipeline to analyze 3D neural network activity recorded from a "bottom-up" 3D MEA integrated with a 3D hydrogel-based tissue containing human iPSC-derived neurons and primary astrocytes. Over a period of ~6.5 weeks, we describe the development and maturation of 3D neural activity (i.e., features of spiking and bursting activity) within cross sections of the 3D tissue, based on the vertical position of the electrode on the 3D MEA probe, in addition to network activity (identified using synchrony analysis) within and between cross sections. Then, using the sequential addition of postsynaptic receptor antagonists, bicuculline (BIC), 2-amino-5-phosphonovaleric acid (AP-5), and 6-cyano-5-nitroquinoxaline-2,3-dione (CNQX), we demonstrate that networks within and between cross sections of the 3D hydrogel-based tissue show a preference for GABA and/or glutamate synaptic transmission, suggesting differences in the network composition throughout the neural tissue. The ability to monitor the functional dynamics of the entire 3D reconstructed neural tissue is a critical bottleneck; here we demonstrate a computational pipeline that can be implemented in studies to better interpret network activity within an engineered 3D neural tissue and have a better understanding of the modeled organ tissue.

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This study aims to investigate the efficacy of electrical stimulation by comparing network-mediated RGC responses in normal and degenerate retinas using a N-methyl-N-nitrosourea (MNU)-induced non-human primate (NHPs) retinitis pigmentosa (RP) model. Adult cynomolgus monkeys were used for normal and outer retinal degeneration (RD) induced by MNU. The network-mediated RGC responses were recorded from the peripheral retina mounted on an 8 × 8 multielectrode array (MEA). The amplitude and duration of biphasic current pulses were modulated from 1 to 50 µA and 500 to 4000 µs, respectively. The threshold charge density for eliciting a network-mediated RGC response was higher in the RD monkeys than in the normal monkeys (1.47 ± 0.13 mC/cm2 vs. 1.06 ± 0.09 mC/cm2, p < 0.05) at a 500 µs pulse duration. The monkeys required a higher charge density than rodents among the RD models (monkeys; 1.47 ± 0.13 mC/cm2, mouse; 1.04 ± 0.09 mC/cm2, and rat; 1.16 ± 0.16 mC/cm2, p < 0.01). Increasing the pulse amplitude and pulse duration elicited more RGC spikes in the normal primate retinas. However, only pulse amplitude variation elicited more RGC spikes in degenerate primate retinas. Therefore, the pulse strategy for primate RD retinas should be optimized, eventually contributing to retinal prosthetics. Given that RD NHP RGCs are not sensitive to pulse duration, using shorter pulses may potentially be a more charge-effective approach for retinal prosthetics.

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Loss of photoreceptors in retinal degenerative diseases also impacts the inner retina: bipolar cell dendrites retract, neurons rewire, and protein expression changes. ON-bipolar cells (OBCs) represent an attractive target for optogenetic vision restoration. However, the above-described maladaptations may negatively impact the quality of restored vision. To investigate this question, we employed human post-mortem retinas and transgenic rd1_Opto-mGluR6 mice expressing the optogenetic construct Opto-mGluR6 in OBCs and carrying the retinal degeneration rd1 mutation. We found significant changes in delayed rectifier potassium channel expression in OBCs of degenerative retinas. In particular, we found an increase in Kv1.3 expression already in early stages of degeneration. Immunohistochemistry localized Kv1.3 channels specifically to OBC axons. In whole-cell patch-clamp experiments, OBCs in the degenerated murine retina were less responsive, which could be reversed by application of the specific Kv1.3 antagonist Psora-4. Notably, Kv1.3 block significantly increased the amplitude and kinetics of Opto-mGluR6-mediated light responses in OBCs of the blind retina and increased the signal-to-noise ratio of light-triggered responses in retinal ganglion cells. We propose that reduction in Kv1.3 activity in the degenerated retina, either by pharmacological block or by KCNA3 gene silencing, could improve the quality of restored vision.

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Modeling the complex and prolonged development of the mammalian central nervous system in vitro remains a profound challenge. Most studies of human stem cell derived neurons are conducted over days to weeks and may or may not include glia. Here we have utilized a single human pluripotent stem cell line, TERA2.cl.SP12 to derive both neurons and glial cells and determined their differentiation and functional maturation over 1 year in culture together with their ability to display epileptiform activity in response to pro-convulsant agents and to detect antiseizure drug actions. Our experiments show that these human stem cells differentiate in vitro into mature neurons and glia cells and form inhibitory and excitatory synapses and integrated neural circuits over 6-8 months, paralleling early human neurogenesis in vivo; these neuroglia cultures display complex electrochemical signaling including high frequency trains of action potentials from single neurons, neural network bursts and highly synchronized, rhythmical firing patterns. Neural activity in our 2D neuron-glia circuits is modulated by a variety of voltage-gated and ligand-gated ion channel acting drugs and these actions were consistent in both young and highly mature neuron cultures. We also show for the first time that spontaneous and epileptiform activity is modulated by first, second and third generation antiseizure agents consistent with animal and human studies. Together, our observations strongly support the value of long-term human stem cell-derived neuroglial cultures in disease modeling and neuropsychiatric drug discovery.

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Impairment of long-term potentiation (LTP) is a common feature of many preclinical models of neurological disorders. Modeling LTP on human induced pluripotent stem cells (hiPSC) enables the investigation of this critical plasticity process in disease-specific genetic backgrounds. Here, we describe a method to chemically induce LTP across entire networks of hiPSC-derived neurons on multi-electrode arrays (MEAs) and investigate effects on neuronal network activity and associated molecular changes.

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In order to perform in vitro cardiotoxicity screening of pharmacological substances, multi-electrode array systems are increasingly used to measure the extracellular field potentials of cell layers of human induced pluripotent stem cell cardiomyocytes. The analysis of the field potentials is usually performed using complex analysis software provided by the hardware manufacturers. In the case of the Cardiac Analysis Tool software from Axion Biosystems, inconsistencies were found in the results, which can significantly influence the cardiotoxicity screening results. In order to obtain more reliable results, a new algorithm was developed and implemented in an easy-to-use software tool, the INCardio Data Analysis Tool, which, due to its high degree of automation, can also be used by inexperienced users. The validation reveals differences in the results of the two tools both in depolarization spike amplitudes and in the time course of the field potential durations. The manual analysis of all signals affected by deviations shows that the results of the newly developed Data Analysis Tool are correct in all cases and can therefore be classified as more accurate and reliable than the reference analysis software.

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Background: Field potential (FP) signals from human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) spheroid which are used for drug safety tests in the preclinical stage are different from action potential (AP) signals and require working knowledge of the multi-electrode array (MEA) system. In this study, we developed in silico three-dimensional (3-D) models of hiPSC-CM spheroids for the simulation of field potential measurement. We compared our model simulation results against in vitro experimental data under the effect of drugs E-4031 and nifedipine. Methods: In silico 3-D models of hiPSC-CM spheroids were constructed in spherical and discoidal shapes. Tetrahedral meshes were generated inside the models, and the propagation of the action potential in the model was obtained by numerically solving the monodomain reaction-diffusion equation. An electrical model of electrode was constructed and FPs were calculated using the extracellular potentials from the AP propagations. The effects of drugs were simulated by matching the simulation results with in vitro experimental data. Results: The simulated FPs from the 3-D models of hiPSC-CM spheroids exhibited highly variable shapes depending on the stimulation and measurement locations. The values of the IC50 of E-4031 and nifedipine calculated by matching the simulated FP durations with in vitro experimental data were in line with the experimentally measured ones reported in the literature. Conclusion: The 3-D in silico models of hiPSC-CM spheroids generated highly variable FPs similar to those observed in in vitro experiments. The in silico model has the potential to complement the interpretation of the FP signals obtained from in vitro experiments.