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.

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.

Objective.Extraction of temporal features of neuronal activity from electrophysiological data can be used for accurate classification of neural networks in healthy and pathologically perturbed conditions. In this study, we provide an extensive approach for the classification of humanin vitroneural networks with and without an underlying pathology, from electrophysiological recordings obtained using a microelectrode array (MEA) platform.Approach.We developed a Dirichlet mixture (DM) Point Process statistical model able to extract temporal features related to neurons. We then applied a machine learning algorithm to discriminate between healthy control and pathologically perturbedin vitroneural networks.Main Results.We found a high degree of separability between the classes using DM point process features (p-value <0.001 for all the features, paired t-test), which reaches 93.10 of accuracy (92.37 of ROC AUC) with the Random Forest classifier. In particular, results show a higher latency in firing for pathologically perturbed neurons (43 ± 16 ms versus 67 ± 31 ms,μIGfeature distribution).Significance.Our approach has been successful in extracting temporal features related to the neurons\' behaviour, as well as distinguishing healthy from pathologically perturbed networks, including classification of responses to a transient induced perturbation.

High resolution electrical stimulation of neural tissue is a fundamental challenge in applications such as deep brain stimulation and artificial vision. In artificial vision, achieving and validating local selective epi-retinal stimulation of different layers in the retina is particularly challenging owing to plurality of retinal cell types and delocalized wiring.
Strong selectivity and non-localized responses to epi-retinal stimulation, over a wide range of realistic stimulation parameters, was achieved and validated using asymmetric pulses.
The reported method consists of multi electrode array (MEA) stimulation and recording from a developing chick retina combined with calcium imaging. Data show direct and indirect neuronal activation in the chick retina model. In particular, axonal activation, orientation and conduction velocity are derived, and the non-local nature of the responses to direct axonal stimulation is demonstrated.
Some of the previous research with mammalian retinas demonstrated local responses around the stimulating electrode, revealing little as to axonal activation. Recent studies showed activation along the nerve fibers and studied the effect of pulse duration to improve stimulation localization (Twyford and Fried, 2016; Weitz et al., 2015). The chick retina offers a straight forward mapping of axonal activation. Here we demonstrate that the chick retina, combined with MEA recording and stimulation along with calcium imaging is a powerful tool to study retinal activation and in particular the effect of asymmetry on axonal activation.
MEA recording and stimulation from the chick retina is exceptionally powerful in distinguishing between direct and indirect responses. This method facilitates comparison between different stimulation strategies. We show that asymmetric electrical stimulations allow control over the intensity of direct activation.

The present study assesses acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), with the aim to obtain in vitro biomarkers that can be used as readouts to predict in vivo cardiotoxicity. Possible acute toxicity was investigated by assessing effects on the beating rate and the field potential duration (FPD) of doxorubicin-exposed cardiomyocytes by measuring electrical activity using multi-electrode array (MEA) analyses. No effects on the beating rate and FPD were found at concentrations up to 6μM, whereas at 12μM no electrical activity was recorded, indicating that the cardiomyocytes stopped beating. Acute and chronic effects of doxorubicin on mitochondria, which have been reported to be affected in doxorubicin-induced cardiotoxicity, were assessed using high content imaging techniques. To this end hiPSC-CMs were exposed to 150 or 300nM doxorubicin using both single dosing (3h and 2days) and repetitive dosing (3 times, of 2days each), including washout studies to assess delayed effects (assessment at day 14) and effects on cell number, mitochondrial density, mitochondrial membrane potential, mitochondrial superoxide levels and mitochondrial calcium levels were assessed. No effects of doxorubicin were found on mitochondrial density and mitochondrial superoxide levels, whereas doxorubicin reduced cell survival and slightly altered mitochondrial membrane potential and mitochondrial calcium levels, which was most profound in the washout studies. Altogether, the results of the present study show that concentrations of doxorubicin in the micromolar range were required to affect electrical activity of hiPSC-CMs, whereas nanomolar concentrations already affected cell viability and caused mitochondrial disturbances. Integration of these data with other in vitro data may enable the selection of a series of in vitro biomarkers that can be used as readouts to screen chemicals for possible cardiotoxicity.

Multi electrode arrays (MEAs) are increasingly used to detect external field potentials in electrically active cells. Recently, in combination with cardiomyocytes derived from human (induced) pluripotent stem cells they have started to become a preferred tool to examine newly developed drugs for potential cardiac toxicity in pre-clinical safety pharmacology. The most important risk parameter is proarrhythmic activity in cardiomyocytes which can cause sudden cardiac death. Whilst MEAs can provide medium- to high- throughput noninvasive assay platform, the translation of a field potential to cardiac action potential (normally measured by low-throughput patch clamp) is complex so that accurate assessment of drug risk to the heart is in practice still challenging. To address this, we used computational simulation to study the theoretical relationship between aspects of the field potential and the underlying cardiac action potential. We then validated the model in both primary mouse- and human pluripotent (embryonic) stem cell-derived cardiomyocytes showing that field potentials measured in MEAs could be converted to action potentials that were essentially identical to those determined directly by electrophysiological patch clamp. The method significantly increased the amount of information that could be extracted from MEA measurements and thus combined the advantages of medium/high throughput with more informative readouts. We believe that this will benefit the analysis of drug toxicity screening of cardiomyocytes using in time and accuracy.

ATP activates P2X receptors and acts as a neurotransmitter in the nervous system. We have previously reported that P2X receptors modulate the firing rate of retinal ganglion cells. Since many subtypes of P2X receptors are distributed in the mouse retina, it is likely that the modulatory effects of P2X receptor-mediated signaling can occur at multiple synaptic levels in the retina. In this study, we investigated whether P2X receptors expressed between the photoreceptor layer and the inner nuclear layer in the mouse retina were physiologically functional, by electroretinography (ERG). In the combined rod-cone ERG and the scotopic ERG, intravitreal injection of PPADS, an antagonist of P2X receptors, had no effects on the amplitude of the a-wave, but decreased the amplitude of the b-wave. In the photopic ERG, intravitreal injection of PPADS significantly decreased the amplitude of both the a-wave and the b-wave. In ex vivo recordings, a decrease in the b-wave amplitude was observed at 20μM PPADS, confirming that the inhibition of the b-wave by intravitreal injection of PPADS is due to the inhibition of P2X receptors. Our findings suggest that P2X receptor-mediated signaling has a physiological effect in both the rod and the cone pathways in postreceptoral processing.

Cultured neurons on multi electrode arrays (MEAs) have been widely used to study various aspects of neuronal (network) functioning. A possible drawback of this approach is the lack of structure in these networks. At the single cell level, several solutions have been proposed to enable directed connectivity, and promising results were obtained. At the level of connected sub-populations, a few attempts have been made with promising results. First assessment of the designs\' functionality, however, suggested room for further improvement. We designed a two chamber MEA aiming to create a unidirectional connection between the networks in both chambers (\"emitting\" and \"receiving\"). To achieve this unidirectionality, all interconnecting channels contained barbs that hindered axon growth in the opposite direction (from receiving to emitting chamber). Visual inspection showed that axons predominantly grew through the channels in the promoted direction. This observation was confirmed by spontaneous activity recordings. Cross-correlation between the signals from two electrodes inside the channels suggested signal propagation at ≈2 m/s from emitting to receiving chamber. Cross-correlation between the firing patterns in both chambers indicated that most correlated activity was initiated in the emitting chamber, which was also reflected by a significantly lower fraction of partial bursts (i.e., a one-chamber-only burst) in the emitting chamber. Finally, electrical stimulation in the emitting chamber induced a fast response in that chamber, and a slower response in the receiving chamber. Stimulation in the receiving chamber evoked a fast response in that chamber, but no response in the emitting chamber. These results confirm the predominantly unidirectional nature of the connecting channels from emitting to receiving chamber.

Gene electrotransfer (GET) enhances delivery of DNA vaccines by increasing both gene expression and immune responses. Our lab has developed the multi-electrode array (MEA) for DNA delivery to skin. The MEA was used at constant pulse duration (150 ms) and frequency (6.67 Hz). In this study, delivery parameters including applied voltage (5-45 V), amount of plasmid (100-300 μg), and number of treatments (2-3) were evaluated for delivery of a DNA vaccine. Mice were intradermally injected with plasmid expressing Bacillus anthracis protective antigen with or without GET and αPA serum titers measured. Within this experiment no significant differences were noted in antibody levels from varying dose or treatment number. However, significant differences were measured from applied voltages of 25 and 35 V. These voltages generated antibody levels between 20,000 and 25,000. Serum from animals vaccinated with these conditions also resulted in toxin neutralization in 40-60% of animals. Visual damage was noted at MEA conditions of 40 V. No damage was noted either visually or histologically from conditions of 35 V or below. These results reflect the importance of establishing appropriate electrical parameters and the potential for the MEA in non-invasive DNA vaccination against B. anthracis.

Carbon nanotube (CNT) coatings have been demonstrated over the past several years as a promising material for neuronal interfacing applications. In particular, in the realm of neuronal implants, CNTs have major advantages owing to their unique mechanical and electrical properties. Here we review recent investigations utilizing CNTs in neuro-interfacing applications. Cell adhesion, neuronal engineering and multi electrode recordings with CNTs are described. We also highlight prospective advances in this field, in particular, progress toward flexible, bio-compatible CNT-based technology.