Microelectrode Arrays Research Articles

HiPSC-CM Electrophysiology: Impact of Temporal Changes and Study Parameters on Experimental Reproducibility.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are frequently used for preclinical cardiotoxicity testing and remain an important tool for confirming model-based predictions of drug effects in accordance with Comprehensive in Vitro Proarrhythmia Assay (CiPA). Despite the considerable benefits hiPSC-CMs provide, concerns surrounding experimental reproducibility have emerged. We investigated the effects of temporal changes and experimental parameters on hiPSC-CM electrophysiology. iCell cardiomyocytes2 were cultured and biosignals were acquired using a microelectrode array (MEA) system (1-14 days). Continuous recordings revealed a 22.6% increase in the beating rate and 7.7% decrease in the field potential duration (FPD) during a 20-minute equilibration period. Location specific differences across a multiwell plate were also observed, with iCell cardiomyocytes2 in the outer rows beating 8.8 beats per minute (BPM) faster than the inner rows. Cardiac endpoints were also impacted by cell culture duration; from 2-14 days the beating rate decreased (-12.7 BPM), FPD lengthened (+257 ms), and spike amplitude increased (+3.3 mV). Cell culture duration (4-10 days) also impacted cardiomyocyte drug responsiveness (E-4031, nifedipine, isoproterenol). qRT-PCR results suggest that daily variations in cardiac metrics may be linked to the continued maturation of hiPSC-CMs in culture (2-30 days). Daily experiments were also repeated using a second cell line (Cor.4U). Collectively, our study highlights multiple sources of variability to consider and address when performing hiPSC-CM MEA studies. To improve reproducibility and data interpretation, MEA-based studies should establish a standardized protocol and report key experimental conditions (e.g., cell line, culture time, equilibration time, electrical stimulation settings, raw data values).

Versatile micro-electrode array to monitor human iPSC derived 3D neural tissues at air-liquid interface

Engineered 3D neural tissues made of neurons and glial cells derived from human induced pluripotent stem cells (hiPSC) are among the most promising tools in drug discovery and neurotoxicology. They represent a cheaper, faster, and more ethical alternative to in vivo animal testing that will likely close the gap between in vitro animal models and human clinical trials. Micro-Electrode Array (MEA) technology is known to provide an assessment of compound effects on neural 2D cell cultures and acute tissue preparations by real-time, non-invasive, and long-lasting electrophysiological monitoring of spontaneous and evoked neuronal activity. Nevertheless, the use of engineered 3D neural tissues in combination with MEA biochips still involves series of constraints, such as drastically limited diffusion of oxygen and nutrients within tissues mainly due to the lack of vascularization. Therefore, 3D neural tissues are extremely sensitive to experimental conditions and require an adequately designed interface that provides optimal tissue survival conditions. A well-suited technique to overcome this issue is the combination of the Air-Liquid Interface (ALI) tissue culture method with the MEA technology. We have developed a full 3D neural tissue culture process and a data acquisition system composed of high-end electronics and novel MEA biochips based on porous, flexible, thin-film membranes integrating recording electrodes, named as “Strip-MEA,” to allow the maintenance of an ALI around the 3D neural tissues. The main motivation of the porous MEA biochips development was the possibility to monitor and to study the electrical activity of 3D neural tissues under different recording configurations, (i) the Strip-MEA can be placed below a tissue, (ii) or by taking advantage of the ALI, be directly placed on top of the tissue, or finally, (iii) it can be embedded into a larger neural tissue generated by the fusion of two (or more) tissues placed on both sides of the Strip-MEA allowing the recording from its inner part. This paper presents the recording and analyses of spontaneous activity from the three positioning configurations of the Strip-MEAs. Obtained results are discussed with the perspective of developing in vitro models of brain diseases and/or impairment of neural network functioning.

Porcine Model of Cerebral Ischemic Stroke Utilizing Intracortical Recordings for the Continuous Monitoring of the Ischemic Area

Purpose: Our aim was to use intracortical recording to enable the tracking of ischemic infarct development over the first few critical hours of ischemia with a high time resolution in pigs. We employed electrophysiological measurements to obtain quick feedback on neural function, which might be useful for screening, e.g., for the optimal dosage and timing of agents prior to further pre-clinical evaluation. Methods: Micro-electrode arrays containing 16 (animal 1) or 32 electrodes (animal 2–7) were implanted in the primary somatosensory cortex of seven female pigs, and continuous electrical stimulation was applied at 0.2 Hz to a cuff electrode implanted on the ulnar nerve. Ischemic stroke was induced after 30 min of baseline recording by injection of endothelin-1 onto the cortex adjacent to the micro-electrode array. Evoked responses were extracted over a moving window of 180 s and averaged across channels as a measure of cortical excitability. Results: Across the animals, the cortical excitability was significantly reduced in all seven 30 min segments following endothelin-1 injection, as compared to the 30 min preceding this intervention. This difference was not explained by changes in the anesthesia, ventilation, end-tidal CO2, mean blood pressure, heart rate, blood oxygenation, or core temperature, which all remained stable throughout the experiment. Conclusions: The animal model may assist in maturing neuroprotective approaches by testing them in an accessible model of resemblance to human neural and cardiovascular physiology and body size. This would constitute an intermediate step for translating positive results from rodent studies into human application, by more efficiently enabling effective optimization prior to chronic pre-clinical studies in large animals.

Highly stretchable and customizable microneedle electrode arrays for intramuscular electromyography.

Stretchable three-dimensional (3D) penetrating microelectrode arrays have potential utility in various fields, including neuroscience, tissue engineering, and wearable bioelectronics. These 3D microelectrode arrays can penetrate and conform to dynamically deforming tissues, thereby facilitating targeted sensing and stimulation of interior regions in a minimally invasive manner. However, fabricating custom stretchable 3D microelectrode arrays presents material integration and patterning challenges. In this study, we present the design, fabrication, and applications of stretchable microneedle electrode arrays (SMNEAs) for sensing local intramuscular electromyography signals ex vivo. We use a unique hybrid fabrication scheme based on laser micromachining, microfabrication, and transfer printing to enable scalable fabrication of individually addressable SMNEA with high device stretchability (60 to 90%). The electrode geometries and recording regions, impedance, array layout, and length distribution are highly customizable. We demonstrate the use of SMNEAs as bioelectronic interfaces in recording intramuscular electromyography from various muscle groups in the buccal mass of Aplysia.

Intrahippocampal Supramolecular Assemblies Directed Bioorthogonal Liberation of Neurotransmitters to Suppress Seizures in Freely Moving Mice.

The precise delivery of anti-seizure medications (ASM) to epileptic loci remains the major challenge to treat epilepsy without causing adverse drug reactions. The unprovoked nature of epileptic seizures raises the additional need to release ASMs in a spatiotemporal controlled manner. Targeting the oxidative stress in epileptic lesions, here the reactive oxygen species (ROS) induced in situ supramolecular assemblies that synergized bioorthogonal reactions to deliver inhibitory neurotransmitter (GABA) on-demand, are developed. Tetrazine-bearing assembly precursors undergo oxidation and selectively self-assemble under pathological conditions inside primary neurons and mice brains. Assemblies induce local accumulation of tetrazine in the hippocampus CA3 region, which allows the subsequent bioorthogonal release of inhibitory neurotransmitters. For induced acute seizures, the sustained release of GABA extends the suppression than the direct supply of GABA. In the model of permanent damage of CA3, bioorthogonal ligation on assemblies provides a reservoir of GABA that behaves prompt release upon 365nm irradiation. Incorporated with the state-of-the-art microelectrode arrays, it is elucidated that the bioorthogonal release of GABA shifts the neuron spike waveforms to suppress seizures at the single-neuron precision. The strategy of in situ supramolecular assemblies-directed bioorthogonal prodrug activation shall be promising for the effective delivery of ASMs to treat epilepsy.

Functional Optical Coherence Tomography of Rat Cortical Neurovascular Activation during Monopulse Electrical Stimulation with the Microelectrode Array

This paper presents a study to evoke rat cortical functional activities, including hemodynamic and neural tissue signal changes, by monopulse electrical stimulation with a microelectrode array using functional optical coherence tomography (fOCT). Based on the principal component analysis and fuzzy clustering method (PCA-FCM), the hemodynamic response of different size blood vessels in rat cortex are analyzed, showing that the hemodynamic response of the superficial large blood vessels is more concentrated. In the regions of neural tissue where blood vessels are removed, positive significant pixels (the intensity of the pixel for five consecutive frames is greater than the average value plus triple standard deviation) and negative significant pixels (the intensity of the pixel for five consecutive frames is less than the average value minus triple standard deviation) exist, and the averaged intensity signal responds rapidly with an onset time of ~20.8 ms. Furthermore, the hemodynamic response was delayed by ~3.5 s from the neural tissue response. fOCT can provide a label-free, large-scale and depth-resolved map of cortical neurovascular activation, which is a promising technology to monitor cortical small-scale neurovascular activities.

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.

Purinergic Signalling Mediates Aberrant Excitability of Developing Neuronal Circuits in the Fmr1 Knockout Mouse Model.

Neuronal hyperexcitability within developing cortical circuits is a common characteristic of several heritable neurodevelopmental disorders, including Fragile X Syndrome (FXS), intellectual disability and autism spectrum disorders (ASD). While this aberrant circuitry is typically studied from a neuron-centric perspective, glial cells secrete soluble factors that regulate both neurite extension and synaptogenesis during development. The nucleotide-mediated purinergic signalling system is particularly instrumental in facilitating these effects. We recently reported that within a FXS animal model, theFmr1 KO mouse, the purinergic signalling system is upregulated in cortical astrocytes leading to altered secretion of synaptogenic and plasticity-related proteins. In this study, we examined whether elevated astrocyte purinergic signalling also impacts neuronal morphology and connectivity of Fmr1 KO cortical neurons. Here, we found that conditioned media from primary Fmr1 KO astrocytes was sufficient to enhance neurite extension and complexity of both wildtype and Fmr1 KO neurons to a similar degree as UTP-mediated outgrowth. Significantly enhanced firing was also observed in Fmr1 KO neuron-astrocyte co-cultures grown on microelectrode arrays but was associated with large deficits in firing synchrony. The selective P2Y2 purinergic receptor antagonist AR-C 118925XX effectively normalized much of the aberrant Fmr1 KO activity, designating P2Y2 as a potential therapeutic target in FXS. These results not only demonstrate the importance of astrocyte soluble factors in the development of neural circuitry, but also show that P2Y purinergic receptors play a distinct role in pathological FXS neuronal activity.

Highly durable, stretchable multielectrode array for electro-mechanical co-stimulation of cells

Highly durable, stretchable multielectrode array for electro-mechanical co-stimulation of cells

Modeling sporadic juvenile ALS in iPSC-derived motor neurons explores the pathogenesis of FUSR503fs mutation.

Fused in sarcoma (FUS) mutations represent the most common genetic etiology of juvenile amyotrophic lateral sclerosis (JALS), for which effective treatments are lacking. In a prior report, we identified a novel FUS mutation, c.1509dupA: p. R503fs (FUSR503fs), in a sporadic JALS patient. The physicochemical properties and structure of FUSR503fs protein were analyzed by software: Multi-electrode array (MEA) assay, calcium activity imaging assay and transcriptome analysis were used to explore the pathophysiological mechanism of iPSC derived motor neurons. Structural analysis and predictions regarding physical and chemical properties of this mutation suggest that the reduction of phosphorylation and glycosylation sites, along with alterations in the amino acid sequence, may contribute to abnormal FUS accumulation within the cytoplasm and nucleus of induced pluripotent stem cell- derived motor neurons (MNs). Multi-electrode array and calcium activity imaging indicate diminished spontaneous electrical and calcium activity signals in MNs harboring the FUSR503fs mutation. Transcriptomic analysis reveals upregulation of genes associated with viral infection and downregulation of genes involved in neural function maintenance, such as the ATP6V1C2 gene. Treatment with ropinirole marginally mitigates the electrophysiological decline in FUSR503fs MNs, suggesting the utility of this cell model for mechanistic exploration and drug screening. iPSCs-derived motor neurons from JALS patients are promising tools for drug screening. The pathological changes in motor neurons of FUSR503fs may occur earlier than in other known mutation types that have been reported.

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

Multi-Sensor Origami Platform: A Customizable System for Obtaining Spatiotemporally Precise Functional Readouts in 3D Models.

Bioprinting technology offers unprecedented opportunities to construct in vitro tissue models that recapitulate the 3D morphology and functionality of native tissue. Yet, it remains difficult to obtain adequate functional readouts from such models. In particular, it is challenging to position sensors in desired locations within pre-fabricated 3D bioprinted structures. At the same time, bioprinting tissue directly onto a sensing device is not feasible due to interference with the printer head. As such, a multi-sensing platform inspired by origami that overcomes these challenges by "folding" around a separately fabricated 3D tissue structure is proposed, allowing for the insertion of electrodes into precise locations, which are custom-defined using computer-aided-design software. The multi-sensing origami platform (MSOP) can be connected to a commercial multi-electrode array (MEA) system for data-acquisition and processing. To demonstrate the platform, how integrated 3D MEA electrodes can record neuronal electrical activity in a 3D model of a neurovascular unit is shown. The MSOP also enables a microvascular endothelial network to be cultured separately and integrated with the 3D tissue structure. Accordingly, how impedance-based sensors in the platform can measure endothelial barrier function is shown. It is further demonstrated the device's versatility by using it to measure neuronal activity in brain organoids.

Denis Aristide Baylor. 30 January 1940 — 16 March 2022

Denis Baylor was one of the leading sensory neuroscientists of the latter part of the twentieth century. His research on the rod and cone photoreceptors of the retina provided a deep understanding of the electrical responses of these cells to light and also of the molecular mechanisms of photo­transduction, whereby light absorption is linked via biochemical reactions within the cell to ion channels in its membrane and thence to cellular membrane voltage changes. He also made major contributions to knowledge of the transmission of these signals to the retina's ganglion cells, which forward information to the brain. Over his career, he invented and/or exploited three important methods for electrophysiological recording: ultra-high-resistance sharp microelectrodes; the suction-pipette technique for photoreceptors; and multielectrode arrays for retina. His research findings have provided important advances in our understanding of visual neuroscience, from the detection of individual photon absorptions by rod photoreceptors, through the underlying molecular mechanisms, to the processing and transmission of these visual signals through the retina. He instilled a love for experimental neuroscience and rigorous scientific investigation into a generation of postdoctoral trainees, students and colleagues. He died of a heart attack on 16 March 2022.

Flexible high-density microelectrode arrays for closed-loop brain-machine interfaces: a review.

Flexible high-density microelectrode arrays (HDMEAs) are emerging as a key component in closed-loop brain-machine interfaces (BMIs), providing high-resolution functionality for recording, stimulation, or both. The flexibility of these arrays provides advantages over rigid ones, such as reduced mismatch between interface and tissue, resilience to micromotion, and sustained long-term performance. This review summarizes the recent developments and applications of flexible HDMEAs in closed-loop BMI systems. It delves into the various challenges encountered in the development of ideal flexible HDMEAs for closed-loop BMI systems and highlights the latest methodologies and breakthroughs to address these challenges. These insights could be instrumental in guiding the creation of future generations of flexible HDMEAs, specifically tailored for use in closed-loop BMIs. The review thoroughly explores both the current state and prospects of these advanced arrays, emphasizing their potential in enhancing BMI technology.

Phase-amplitude coupling detection and analysis of human 2-dimensional neural cultures in multi-well microelectrode array in vitro

BackgroundHuman induced pluripotent stem cell (hiPSC)- derived neurons offer the possibility of studying human-specific neuronal behaviors in physiologic and pathologic states in vitro. It is unclear whether cultured neurons can achieve the fundamental network behaviors required to process information in the brain. Investigating neuronal oscillations and their interactions, as occurs in cross-frequency coupling (CFC), addresses this question. New methodsWe examined whether networks of two-dimensional (2D) cultured hiPSC-derived cortical neurons grown with hiPSC-derived astrocytes on microelectrode array plates recapitulate the CFC that is present in vivo. We employed the modulation index method for detecting phase-amplitude coupling (PAC) and used offline spike sorting to analyze the contribution of single neuron spiking to network behavior. ResultsWe found that PAC is present, the degree of PAC is specific to network structure, and it is modulated by external stimulation with bicuculline administration. Modulation of PAC is not driven by single neurons, but by network-level interactions. Comparison with existing methodsPAC has been demonstrated in multiple regions of the human cortex as well as in organoids. This is the first report of analysis demonstrating the presence of coupling in 2D cultures. ConclusionCFC in the form of PAC analysis explores communication and integration between groups of neurons and dynamical changes across networks. In vitro PAC analysis has the potential to elucidate the underlying mechanisms as well as capture the effects of chemical, electrical, or ultrasound stimulation; providing insight into modulation of neural networks to treat nervous system disorders in vivo.

Multiplicative joint coding in preparatory activity for reaching sequence in macaque motor cortex

Although the motor cortex has been found to be modulated by sensory or cognitive sequences, the linkage between multiple movement elements and sequence-related responses is not yet understood. Here, we recorded neuronal activity from the motor cortex with implanted micro-electrode arrays and single electrodes while monkeys performed a double-reach task that was instructed by simultaneously presented memorized cues. We found that there existed a substantial multiplicative component jointly tuned to impending and subsequent reaches during preparation, then the coding mechanism transferred to an additive manner during execution. This multiplicative joint coding, which also spontaneously emerged in recurrent neural networks trained for double reach, enriches neural patterns for sequential movement, and might explain the linear readout of elemental movements.

XMEA: A New Hybrid Diamond Multielectrode Array for the In Situ Assessment of the Radiation Dose Enhancement by Nanoparticles.

This work presents a novel multielectrode array (MEA) to quantitatively assess the dose enhancement factor (DEF) produced in a medium by embedded nanoparticles. The MEA has 16 nanocrystalline diamond electrodes (in a cell-culture well), and a single-crystal diamond divided into four quadrants for X-ray dosimetry. DEF was assessed in water solutions with up to a 1000 µg/mL concentration of silver, platinum, and gold nanoparticles. The X-ray detectors showed a linear response to radiation dose (r2 ≥ 0.9999). Overall, platinum and gold nanoparticles produced a dose enhancement in the medium (maximum of 1.9 and 3.1, respectively), while silver nanoparticles produced a shielding effect (maximum of 37%), lowering the dose in the medium. This work shows that the novel MEA can be a useful tool in the quantitative assessment of radiation dose enhancement due to nanoparticles. Together with its suitability for cells' exocytosis studies, it proves to be a highly versatile device for several applications.

Spindle oscillations in communicating axons within a reconstituted hippocampal formation are strongest in CA3 without thalamus

Spindle-shaped waves of oscillations emerge in EEG scalp recordings during human and rodent non-REM sleep. The association of these 10–16 Hz oscillations with events during prior wakefulness suggests a role in memory consolidation. Human and rodent depth electrodes in the brain record strong spindles throughout the cortex and hippocampus, with possible origins in the thalamus. However, the source and targets of the spindle oscillations from the hippocampus are unclear. Here, we employed an in vitro reconstruction of four subregions of the hippocampal formation with separate microfluidic tunnels for single axon communication between subregions assembled on top of a microelectrode array. We recorded spontaneous 400–1000 ms long spindle waves at 10–16 Hz in single axons passing between subregions as well as from individual neurons in those subregions. Spindles were nested within slow waves. The highest amplitudes and most frequent occurrence suggest origins in CA3 neurons that send feed-forward axons into CA1 and feedback axons into DG. Spindles had 50–70% slower conduction velocities than spikes and were not phase-locked to spikes suggesting that spindle mechanisms are independent of action potentials. Therefore, consolidation of declarative-cognitive memories in the hippocampus may be separate from the more easily accessible consolidation of memories related to thalamic motor function.

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.

Decoding Typical Flight States Based on Neural Signals from the Midbrain Motor Nuclei of Pigeons.

Exploring the neural encoding mechanism and decoding of motion state switching during flight can advance our knowledge of avian behavior control and contribute to the development of avian robots. However, limited acquisition equipment and neural signal quality have posed challenges, thus we understand little about the neural mechanisms of avian flight. We used chronically implanted micro-electrode arrays to record the local field potentials (LFPs) in the formation reticularis medialis mesencephali (FRM) of pigeons during various motion states in their natural outdoor flight. Subsequently, coherence-based functional connectivity networks under different bands were constructed and the topological features were extracted. Finally, we used a support vector machine model to decode different flight states. Our findings indicate that the gamma band (80-150 Hz) in the FRM exhibits significant power for identifying different states in pigeons. Specifically, the avian brain transmitted flight related information more efficiently during the accelerated take-off or decelerated landing states, compared with the uniform flight and baseline states. Finally, we achieved a best average accuracy of 0.86 using the connectivity features in the 80-150 Hz band and 0.89 using the fused features for state decoding. Our results open up possibilities for further research into the neural mechanism of avian flight and contribute to the understanding of flight behavior control in birds.