Alexandra Gramowski

Microelectrode arrays: A physiologically based neurotoxicity testing platform for the 21st century §

Microelectrode arrays (MEAs) have been in use over the past decade and a half to study multiple aspects of electrically excitable cells. In particular, MEAs have been applied to explore the pharmacological and toxicological effects of numerous compounds on spontaneous activity of neuronal and cardiac cell networks. The MEA system enables simultaneous extracellular recordings from multiple sites in the network in real time, increasing spatial resolution and thereby providing a robust measure of network activity. The simultaneous gathering of action potential and field potential data over long periods of time allows the monitoring of network functions that arise from the interaction of all cellular mechanisms responsible for spatio-temporal pattern generation. In these functional, dynamic systems, physical, chemical, and pharmacological perturbations are holistically reflected by the tissue responses. Such features make MEA technology well suited for the screening of compounds of interest, and also allow scaling to high throughput systems that can record from multiple, separate cell networks simultaneously in multi-well chips or plates. This article is designed to be useful to newcomers to this technology as well as those who are currently using MEAs in their research. It explains how MEA systems operate, summarizes what systems are available, and provides a discussion of emerging mathematical schemes that can be used for a rapid classification of drug or chemical effects. Current efforts that will expand this technology to an influential, high throughput, electrophysiological approach for reliable determinations of compound toxicity are also described and a comprehensive review of toxicological publications using MEAs is provided as an appendix to this publication. Overall, this article highlights the benefits and promise of MEA technology as a high throughput, rapid screening method for toxicity testing.

Development of micro-electrode array based tests for neurotoxicity: assessment of interlaboratory reproducibility with neuroactive chemicals

Neuronal assemblies within the nervous system produce electrical activity that can be recorded in terms of action potential patterns. Such patterns provide a sensitive endpoint to detect effects of a variety of chemical and physical perturbations. They are a function of synaptic changes and do not necessarily involve structural alterations. In vitro neuronal networks (NNs) grown on micro-electrode arrays (MEAs) respond to neuroactive substances as well as the in vivo brain. As such, they constitute a valuable tool for investigating changes in the electrophysiological activity of the neurons in response to chemical exposures. However, the reproducibility of NN responses to chemical exposure has not been systematically documented. To this purpose six independent laboratories (in Europe and in USA) evaluated the response to the same pharmacological compounds (Fluoxetine, Muscimol, and Verapamil) in primary neuronal cultures. Common standardization principles and acceptance criteria for the quality of the cultures have been established to compare the obtained results. These studies involved more than 100 experiments before the final conclusions have been drawn that MEA technology has a potential for standard in vitro neurotoxicity/neuropharmacology evaluation. The obtained results show good intra- and inter-laboratory reproducibility of the responses. The consistent inhibitory effects of the compounds were observed in all the laboratories with the 50% Inhibiting Concentrations (IC50s) ranging from: (mean ± SEM, in μM) 1.53 ± 0.17 to 5.4 ± 0.7 (n = 35) for Fluoxetine, 0.16 ± 0.03 to 0.38 ± 0.16 μM (n = 35) for Muscimol, and 2.68 ± 0.32 to 5.23 ± 1.7 (n = 32) for Verapamil. The outcome of this study indicates that the MEA approach is a robust tool leading to reproducible results. The future direction will be to extend the set of testing compounds and to propose the MEA approach as a standard screen for identification and prioritization of chemicals with neurotoxicity potential.

Functional screening of traditional antidepressants with primary cortical neuronal networks grown on multielectrode neurochips

We optimized the novel technique of multielectrode neurochip recordings for the rapid and efficient screening of neuroactivity. Changes in the spontaneous activity of cultured networks of primary cortical neurons were quantified to evaluate the action of drugs on the firing dynamics of complex network activity. The multiparametric assessment of electrical activity changes caused by psychoactive herbal extracts from Hypericum, Passiflora and Valeriana, and various combinations thereof revealed a receptor‐specific and concentration‐dependent inhibition of the firing patterns. The spike and burst rates showed significant substance‐dependent effects and significant differences in potency. The effects of specific receptor blockades on the inhibitory responses provided evidence that the herbal extracts act on gamma‐amino butyric acid (GABA) and serotonin (5‐HT) receptors, which are recognized targets of pharmacological antidepressant treatment. A biphasic effect, serotonergic stimulation of activity at low concentrations that is overridden by GABAergic inhibition at higher concentrations, is apparent with Hypericum alone and the triple combination of the extracts. The more potent neuroactivity of the triple combination compared to Hypericum alone and the additive effect of Passiflora and Valeriana suggest a synergy between constituent herbal extracts. The extracts and their combinations affected the set of derived activity parameters in a concomitant manner suggesting that all three constituent extracts and their combinations have largely similar modes of action. This study also demonstrates the sensitivity, selectivity and robustness of neurochip recordings for high content screening of complex mixtures of neuroactive substances and for providing multiparametric information on neuronal activity changes to assess the therapeutic potential of psychoactive substances.

Nanoparticles Induce Changes of the Electrical Activity of Neuronal Networks on Microelectrode Array Neurochips

Background Nanomaterials are extensively used in industry and daily life, but little is known about possible health effects. An intensified research regarding toxicity of nanomaterials is urgently needed. Several studies have demonstrated that nanoparticles (NPs; diameter < 100 nm) can be transported to the central nervous system; however, interference of NPs with the electrical activity of neurons has not yet been shown. Objectives/methods We investigated the acute electrophysiological effects of carbon black (CB), hematite (Fe2O3), and titanium dioxide (TiO2) NPs in primary murine cortical networks on microelectrode array (MEA) neurochips. Uptake of NPs was studied by transmission electron microscopy (TEM), and intracellular formation of reactive oxygen species (ROS) was studied by flow cytometry. Results The multiparametric assessment of electrical activity changes caused by the NPs revealed an NP-specific and concentration-dependent inhibition of the firing patterns. The number of action potentials and the frequency of their patterns (spike and burst rates) showed a significant particle-dependent decrease and significant differences in potency. Further, we detected the uptake of CB, Fe2O3, and TiO2 into glial cells and neurons by TEM. Additionally, 24 hr exposure to TiO2 NPs caused intracellular formation of ROS in neuronal and glial cells, whereas exposure to CB and Fe2O3 NPs up to a concentration of 10 μg/cm2 did not induce significant changes in free radical levels. Conclusion NPs at low particle concentrations are able to exhibit a neurotoxic effect by disturbing the electrical activity of neuronal networks, but the underlying mechanisms depend on the particle type.

Information extraction from biphasic concentration-response curves for data obtained from neuronal activity of networks cultivated on multielectrode-array-neurochips

We aim to fit biphasic concentration-response curves to extract information about the effect of given biochemical substances to in-vitro neurons. Neurons extracted from embryonic mice are cultivated on multielectrode-array-neurochips (MEA-neurochip) [1]. The activity of single neurons in such networks is recorded especially the change of network activity caused by long-term application of neuroactive substances. This results in quasi-stable patterns of neuronal activity. Based on the data, different features [2] are calculated adapted from spikes and bursts and separately displayed in concentration-response curves [3]. These concentration-response curves can exhibit non sigmoid shape, then indicating that different mechanisms affect the neuronal activity. Hence, the concentration-response curves presumably include currently hidden and unused information.

Effect of blood plasma components from patients with hepatic encephalopathy on electrophysiological activity of primary frontal cortex networks in vitro

Hepatic encephalopathy (HE) results from impaired detoxification capacity of the liver leading to accumulation of various substances that may impair the function of the central nervous system (CNS).1—5

Concentration-dependent multi-parametric functional screening of CNS drugs with neuronal networks on microelectrode arrays

Functional screening of CNS drugs with neuronal network cultures on microelectrode arrays can elucidate different modes of action for a given substance at different concentrations. This concentration-dependent profiling of CNS drug candidates is a new feature of this technology.