Multi-electrode Array Devices Research Articles

Human neural network activity reacts to gravity changes in vitro.

During spaceflight, humans experience a variety of physiological changes due to deviations from familiar earth conditions. Specifically, the lack of gravity is responsible for many effects observed in returning astronauts. These impairments can include structural as well as functional changes of the brain and a decline in cognitive performance. However, the underlying physiological mechanisms remain elusive. Alterations in neuronal activity play a central role in mental disorders and altered neuronal transmission may also lead to diminished human performance in space. Thus, understanding the influence of altered gravity at the cellular and network level is of high importance. Previous electrophysiological experiments using patch clamp techniques and calcium indicators have shown that neuronal activity is influenced by altered gravity. By using multi-electrode array (MEA) technology, we advanced the electrophysiological investigation covering single-cell to network level responses during exposure to decreased (micro-) or increased (hyper-) gravity conditions. We continuously recorded in real-time the spontaneous activity of human induced pluripotent stem cell (hiPSC)-derived neural networks in vitro. The MEA device was integrated into a custom-built environmental chamber to expose the system with neuronal cultures to up to 6 g of hypergravity on the Short-Arm Human Centrifuge at the DLR Cologne, Germany. The flexibility of the experimental hardware set-up facilitated additional MEA electrophysiology experiments under 4.7 s of high-quality microgravity (10-6 to 10-5 g) in the Bremen drop tower, Germany. Hypergravity led to significant changes in activity. During the microgravity phase, the mean action potential frequency across the neural networks was significantly enhanced, whereas different subgroups of neurons showed distinct behaviors, such as increased or decreased firing activity. Our data clearly demonstrate that gravity as an environmental stimulus triggers changes in neuronal activity. Neuronal networks especially reacted to acute changes in mechanical loading (hypergravity) or de-loading (microgravity). The current study clearly shows the gravity-dependent response of neuronal networks endorsing the importance of further investigations of neuronal activity and its adaptive responses to micro- and hypergravity. Our approach provided the basis for the identification of responsible mechanisms and the development of countermeasures with potential implications on manned space missions.

Hyperammonemia Alters the Function of AMPA and NMDA Receptors in Hippocampus: Extracellular cGMP Reverses Some of These Alterations.

Chronic hyperammonemia alters membrane expression of AMPA and NMDA receptors subunits in hippocampus leading to impaired memory and learning. Increasing extracellular cGMP normalizes these alterations. However, it has not been studied whether hyperammonemia alters the function of AMPA and NMDA receptors. The aims of this work were: (1) assess if hyperammonemia alters AMPA and NMDA receptors function; (2) analyze if extracellular cGMP reverses these alterations. A multielectrode array device was used to stimulate Schäffer collaterals and record postsynaptic currents in the CA1 region in hippocampal slices from control and hyperammonemic rats and analyze different features of the excitatory postsynaptic potentials. Hyperammonemia reduces the amplitude and delays appearance of AMPA EPSPs, whereas increases amplitude, hyperpolarization, depolarization and desensitization area of the NMDA EPSPs. These alterations in AMPA and NMDA function are accentuated as the stimulation intensity increases. Adding extracellular cGMP reverses the alteration in amplitude in both, AMPA and NMDA EPSPs. In control slices extracellular cGMP decreases the AMPA and NMDA EPSPs amplitude and delays the response of neurons and the return to the resting potential at all stimulation intensities. In conclusion, hyperammonemia decreases the AMPA response, whereas increases the NMDA response and extracellular cGMP reverses these alterations.

Ultrasoft Silicone Gel as a Biomimetic Passivation Layer in Inkjet-Printed 3D MEA Devices.

Multielectrode arrays (MEAs) are versatile tools that are used for chronic recording and stimulation of neural cells and tissues. Driven by the recent progress in understanding of how neuronal growth and function respond to scaffold stiffness, development of MEAs with a soft cell-to-device interface has gained importance not only for in vivo but also for in vitro applications. However, the passivation layer, which constitutes the majority of the cell-device interface, is typically prepared with stiff materials. Herein, a fabrication of an MEA device with an ultrasoft passivation layer is described, which takes advantage of inkjet printing and a polydimethylsiloxane (PDMS) gel with a stiffness comparable to that of the brain. The major challenge in using the PDMS gel is that it cannot be patterned to expose the sensing area of the electrode. This issue is resolved by printing 3D micropillars at the electrode tip. Primary cortical neurons are grown on the fabricated device, and effective stimulation of the culture confirms functional cell-device coupling. The 3D MEA device with an ultrasoft interface provides a novel platform for investigating evoked activity and drug responses of living neuronal networks cultured in a biomimetic environment for both fundamental research and pharmaceutical applications.

Cost-effective and multifunctional acquisition system for in vitro electrophysiological investigations with multi-electrode arrays.

In vitro multi-electrode array (MEA) technology is nowadays involved in a wide range of applications beyond neuroscience, such as cardiac electrophysiology and bio-interface studies. However, the cost of commercially available acquisition systems severely limits its adoption outside specialized laboratories with high budget capabilities. Thus, the availability of low-cost methods to acquire signals from MEAs is important to allow research labs worldwide to exploit this technology for an ever-expanding pool of experiments independently from their economic possibilities. Here, we provide a comprehensive toolset to assemble a multifunctional in vitro MEA acquisition system with a total cost 80% lower than standard commercial solutions. We demonstrate the capabilities of this acquisition system by employing it to i) characterize commercial MEA devices by means of electrical impedance measurements ii) record activity from cultures of HL-1 cells extracellularly, and iii) electroporate HL-1 cells through nanostructured MEAs and record intracellular signals.

Fabrication and Characterization of 3D Multi-Electrode Array on Flexible Substrate for In Vivo EMG Recording from Expiratory Muscle of Songbird.

This work presents fabrication and characterization of flexible three-dimensional (3D) multi-electrode arrays (MEAs) capable of high signal-to-noise (SNR) electromyogram (EMG) recordings from the expiratory muscle of a songbird. The fabrication utilizes a photoresist reflow process to obtain 3D structures to serve as the electrodes. A polyimide base with a PDMS top insulation was utilized to ensure flexibility and biocompatibility of the fabricated 3D MEA devices. SNR measurements from the fabricated 3D electrode show up to a 7x improvement as compared to the 2D MEAs.

Reversal of dopamine-mediated firing inhibition through activation of the dopamine transporter in substantia nigra pars compacta neurons.

One of the hallmarks of ventral midbrain dopamine-releasing neurons is membrane hyperpolarization in response to stimulation of somato-dendritic D2 receptors. At early postnatal age, under sustained dopamine, this inhibitory response is followed by a slow recovery, resulting in dopamine inhibition reversal (DIR). In the present investigation, we aimed to get a better insight into the cellular mechanisms underlying DIR. We performed single-unit extracellular recordings with a multi-electrode array device and conventional patch-clamp recordings on midbrain mouse slices. While continuous dopamine (100μM) perfusion gave rise to firing inhibition that recovered in 10 to 15min, the same effect was not obtained with the D2 receptor agonist quinpirole (100nM). Moreover, firing inhibition caused by the GABAB receptor agonist baclofen (300nM) was reversed by dopamine (100μM), albeit D2 receptors had been blocked by sulpiride (10μM). Conversely, the block of the dopamine transporter (DAT) with cocaine (30μM) prevented firing recovery by dopamine under GABAB receptor stimulation. Accordingly, in whole-cell recordings from single cells, the baclofen-induced outward current was counteracted by dopamine (100μM) in the presence of sulpiride (10μM), and this effect was prevented by the DAT antagonists cocaine (30μM) and GBR12909 (2μM). Our results indicate that the DAT plays a major role in DIR, mediating it under conditions of sustained dopamine exposure, and point to DAT as an important target for pharmacological therapies leading to prolonged enhancement of the dopaminergic signal.

An electrically resistive sheet of glial cells for amplifying signals of neuronal extracellular recordings.

Electrical signals of neuronal cells can be recorded non-invasively and with a high degree of temporal resolution using multielectrode arrays (MEAs). However, signals that are recorded with these devices are small, usually 0.01%-0.1% of intracellular recordings. Here, we show that the amplitude of neuronal signals recorded with MEA devices can be amplified by covering neuronal networks with an electrically resistive sheet. The resistive sheet used in this study is a monolayer of glial cells, supportive cells in the brain. The glial cells were grown on a collagen-gel film that is permeable to oxygen and other nutrients. The impedance of the glial sheet was measured by electrochemical impedance spectroscopy, and equivalent circuit simulations were performed to theoretically investigate the effect of covering the neurons with such a resistive sheet. Finally, the effect of the resistive glial sheet was confirmed experimentally, showing a 6-fold increase in neuronal signals. This technique feasibly amplifies signals of MEA recordings.

Astrocyte-Dependent Vulnerability to Excitotoxicity in Spermine Oxidase-Overexpressing Mouse.

Transgenic mice overexpressing spermine oxidase (SMO) in the cerebral cortex (Dach-SMO mice) showed increased vulnerability to excitotoxic brain injury and kainate-induced epileptic seizures. To investigate the mechanisms by which SMO overexpression leads to increased susceptibility to kainate excitotoxicity and seizure, in the cerebral cortex of Dach-SMO and control mice we assessed markers for astrocyte proliferation and neuron loss, and the ability of kainate to evoke glutamate release from nerve terminals and astrocyte processes. Moreover, we assessed a possible role of astrocytes in an in vitro model of epileptic-like activity in combined cortico-hippocampal slices recorded with a multi-electrode array device. In parallel, as the brain is a major metabolizer of oxygen and yet has relatively feeble protective antioxidant mechanisms, we analyzed the oxidative status of the cerebral cortex of both SMO-overexpressing and control mice by evaluating enzymatic and non-enzymatic scavengers such as metallothioneins. The main findings in the cerebral cortex of Dach-SMO mice as compared to controls are the following: astrocyte activation and neuron loss; increased oxidative stress and activation of defense mechanisms involving both neurons and astrocytes; increased susceptibility to kainate-evoked cortical epileptogenic activity, dependent on astrocyte function; appearance of a glutamate-releasing response to kainate from astrocyte processes due to activation of Ca(2+)-permeable AMPA receptors in Dach-SMO mice. We conclude that reactive astrocytosis and activation of glutamate release from astrocyte processes might contribute, together with increased reactive oxygen species production, to the vulnerability to kainate excitotoxicity in Dach-SMO mice. This mouse model with a deregulated polyamine metabolism would shed light on roles for astrocytes in increasing vulnerability to excitotoxic neuron injury.

Neurointerfaces: Review and development

Electrical activity seems to be the key issue for disclosing information processing mechanisms in neuronal networks; however, the related phenomena such as long-term memory, learning behavior and synaptic plasticity have not been adequately understood yet on cellular level. A great challenge in the fundamental research and practical implementation of those phenomena is to build up multi-electrode array (MEA) devices for simultaneous neuronal recordings and stimulation. We outline the state-of-the-art MEA designs, development trends, and benefits and shortcomings of the MEA concept. As a rule, the general task in neurointerface designing is to achieve biocompatible, low-invasive interface performance and the spatio-temporal resolution. In this paper, we propose and describe two innovative neurointerface designs. One of these designs is introduced as a conceptual device based on a dense array of vertically standing semiconductor microtubes, which can be implemented in next-generation in vivo neuronal interfaces. Another design is a pilot in vitro MEA device included 60 planar patch-clamp electrode sites array. Here, we report results of measurements performed with the help of patch-clamp electrodes, each of the electrodes is a kind of a 2 μm diameter pipette that can be used for making contacts to the cell membrane. An advantageous feature of the method consists in that the treated cells have no immediate contact with the metal as they contact with the microchannel conducting liquid, or ionic conductor; this approach ensures more adequate measurements and, simultaneously, it improves the cell survivability during experiments.

Redox cycling in nanoporous electrochemical devices

Nanoscale redox cycling is a powerful technique for detecting electrochemically active molecules, based on fast repetitive oxidation and reduction reactions. An ideal implementation of redox cycling sensors can be realized by nanoporous dual-electrode systems in easily accessible and scalable geometries. Here, we introduce a multi-electrode array device with highly efficient nanoporous redox cycling sensors. Each of the sensors holds up to 209,000 well defined nanopores with minimal pore radii of less than 40 nm and an electrode separation of ~100 nm. We demonstrate the efficiency of the nanopore array by screening a large concentration range over three orders of magnitude with area-specific sensitivities of up to 81.0 mA (cm(-2) mM(-1)) for the redox-active probe ferrocene dimethanol. Furthermore, due to the specific geometry of the material, reaction kinetics has a unique potential-dependent impact on the signal characteristics. As a result, redox cycling experiments in the nanoporous structure allow studies on heterogeneous electron transfer reactions revealing a surprisingly asymmetric transfer coefficient.

Neurons on nanometric topographies: insights into neuronal behaviors in vitro.

Topography, the physical characteristics of an environment, is one of the most prominent stimuli neurons can encounter in the body. Many aspects of neurons and neuronal behavior are affected by the size, shape, and pattern of the physical features of the environment. A recent increase in the use of nanometric topographies, due to improved fabrication techniques, has resulted in new findings on neuronal behavior and development. Factors such as neuron adhesion, neurite alignment, and even the rate of neurite formation have all been highlighted through nanotopographies as complex phenomena that are driven by intricate intracellular mechanisms. Nanotopographies are suitable platforms, not only for fundamental studies on neuronal development, but also in practical applications, including multielectrode array devices and neuro-regenerative medicine. We reviewed recent publications that address the effects of nanotopography on neurons and categorized the observed behaviors as adherence, directional guidance, or accelerated outgrowth. We also discussed possible biological mechanisms of the molecular and cellular responses to topography, and suggested future perspectives for this field.

Hippocampus versus entorhinal cortex decoupling by an NR2 subunit–specific block of NMDA receptors in a rat in vitro model of temporal lobe epilepsy

The role of N-methyl-D-aspartate receptors (NMDARs) in the generation and maintenance of epileptic seizures has been widely investigated, however, little is known of possible separate roles played by NMDARs that contain different NR2 subunits. A better comprehension of how distinct NMDARs subtypes participate in seizure generation and/or diffusion may lead to the development of more targeted pharmacologic strategies to treat epilepsy. Therefore, we have performed an electrophysiologic investigation using a multielectrode array device, on slices comprising entorhinal cortex (EC) and hippocampus, continuously perfused in a Mg(2+) -free medium, with added 4-aminopiridine (4AP; 10-15 μm). Two separate rhythmic patterns of interictal-like activity were generated in EC and hippocampus, with EC seizures entrained to those in CA3, so that a significant degree of cross-correlation occurred. Perfusion with the NR2A-containing NMDAR antagonist [(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077; 50 nm) or Zn(2+) (200 nm), did not affect the rate of interictal-like events in EC and hippocampus; however, it significantly reduced their cross-correlation, causing a substantial decoupling of the two rhythm generators. The same effect was observed with (αR,βS)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidinepropanol maleate (Ro25-6981; 1 μm), when coapplied with a subthreshold dose of NVP-AAM077. Our results suggest that NR2 subunits may be crucial in entraining cortical networks, leading to recruitment of wider range oscillations during epilepsy. Therefore, a pharmacologic strategy directed onto NR2 subunits may help to limit seizure diffusion and recruitment of potentially entrained oscillatory networks.

Modeling large-scale neural network culture interface on very-high density multi-electrode arrays

The use of multi-electrode arrays (MEA) technology is developing in neuroscience fields like neuro-pharmacology [1,2], network plasticity investigation [3-5] or neurological diseases [6] and disorders [7]. Dissociated cultures or slices are now often employed on 60–100 multi-site arrays. Recently, matrixes of several thousands of microelectrodes have been developed in order to gain higher spatial resolution from the cell scale up to large network scale. With the framework of a European Consortium (IDEA Project) we developed a 4096 electrode MEA using the Active Pixel Sensor APS technology as well as a computer model of cortical dissociated cultures grown on this device including the neuron-to-electrode interface. Our goal is to better understand the network mechanisms responsible for recorded activity, and to provide integrated software for Computer Aided Design (CAD) of neural engineering devices. Since it was computationally too heavy to work with thousands of interconnected Hodgkin-Huxley cell models, we chose to implement the Izhikevich model which is known as a good compromise between realistic cellular properties and computation time [8]. Indeed, the classical standard leaky integrate-and-fire cellular model can hardly mimic the rich repertoire of intrinsic cellular properties that can be found in biological substrates. We present here the first recordings of high-density MEAs together with dedicated software which can simulate the complete system composed of the electrode matrix and the biological network grown on top. The first results of these large-scale interconnected networks simulations (size and number of cells similar to those recorded in vitro) are consistent with the first recorded data using our prototype of high-density MEAs: (i) the bursts initiation location varies randomly from one place to another, (ii) their propagation varies with the connectivity and the level of presynaptic firing, (iii) the average bursting frequency with no inhibitory connections is close to 1 Hz and similar activity is obtained with bicucculin treated dissociated culture activity. Finally, (iv) the model burst propagation speed is about 100 mm/s and this value has also been computed on real cultures in our lab. This tool is currently used to optimize the design and to investigate the properties of large-scale MEA devices under development and constitutes an innovative neuro-engineering CAD environment.

Chronically recording with a multi-electrode array device in the auditory cortex of an awake ferret

It is known that anesthesia depresses neural activity and inhibits cortico-cortical interactions and cortical output. Hence, it is important to record from awake animals in order to better understand the full dynamic range of neural responses. We have developed a preparation for chronic, multi-electrode physiological recording in the cortex of the awake ferret. This paper discusses several of the advantages and disadvantages of the technique as well as procedures used to overcome potential complications associated with chronic implants in the ferret. Our solutions are well suited to the special species requirements, yet are also easily generalizable to other species.

Protective role of hydrogen peroxide in oxygen‐deprived dopaminergic neurones of the rat substantia nigra

Hydrogen peroxide (H2O2) is a reactive oxygen species, responsible for cytotoxic damage through the formation of hydroxyl radicals. Dopamine (DA) neurones of the substantia nigra pars compacta (SNc) are highly sensitive to metabolic stress, and they typically respond to energy deprivation with membrane hyperpolarization, mainly through opening of ATP-dependent K+ channels. Accordingly, H2O2 (3 mM) induced a tolbutamide-sensitive outward current in DA neurones. Conversely, in a hypoxic medium, H2O2 reverted membrane hyperpolarization, which is associated with oxygen deprivation in DA neurones, restored their action potential firing, and reduced the hypoxia-mediated outward current in a concentration-dependent manner, between 0.1 and 3 mM (IC50 0.6+/-0.1 mM). Notably, H2O2 did not counteract membrane hyperpolarization associated with hypoglycaemia, moreover, when catalase was inhibited with 3-amino-1,2,4-triazole (3-AT; 30 mM), H2O2 did not reduce hypoxia-mediated outward current. The counteracting action of H2O2 on hypoxia-mediated effects was further confirmed by single-unit extracellular recordings of presumed DA neurones in acute midbrain slices preparations, using a planar multi-electrode array device. Whilst a prolonged period of hypoxia (40 min) caused firing suppression, which did not recover after perfusion in normoxic conditions, the presence of H2O2 (3 mM) during this prolonged hypoxic period rescued most of the neurones from irreversible firing inhibition. Accordingly, morphological studies showed that H2O2 counteracts the cytochrome c release provoked by prolonged hypoxic treatment. Taken together, our data suggest that H2O2 prevents the metabolic stress of DA neurones induced by hypoxia by serving as a supplementary source of molecular oxygen, through its degradation by catalase.