Dieter G. Weiss

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.

Mutational analysis of the N-ras,p53,p16INK4a,CDK4, and MC1R genes in human congenital melanocytic naevi

Eighteen human congenital melanocytic naevi (CMN) from 17 patients were screened for activating point mutations in the oncogenesN-ras andCDK4 and for sequence variants in the MC1R gene by combined RFLP-PCR/SSCP analysis. In addition, all lesions were screened for deletions and point mutations in the tumour suppressor genesp53 andp16INK4a (CDKN2A) by combined multiplex PCR/SSCP analysis. Positive screening data were specified by sequencing of the corresponding PCR product. Activating point mutations in theN-ras gene (nine CAA (Gln) to AAA (Lys) transversions and one CAA (Gln) to CGA (Arg) transition at codon 61) were detected at high frequency (56%). Furthermore, three missense mutations (V92M) and two silent mutations (CGA (Arg) to CGG (Arg), codon 213, exon 6) were found in theMC1R andp53 genes, respectively. No mutations were found in p16 orCDK4. The activatedN-ras oncogene, which is also found in human cutaneous melanomas, may constitute a potential risk factor for melanoma formation within CMN.

Substance identification by quantitative characterization of oscillatory activity in murine spinal cord networks on microelectrode arrays

This paper presents a novel and comprehensive method to identify substances on the basis of electrical activity and is a substantial improvement for drug screening. The spontaneous activity of primary neuronal networks is influenced by neurotransmitters, ligands, and other substances in a similar fashion as known from in vivo pharmacology. However, quantitative methods for the identification of substances through their characteristic effects on network activity states have not yet been reported. We approached this problem by creating a database including native activity and five drug‐induced oscillatory activity states from extracellular multisite recordings from microelectrode arrays. The response profiles consisted of 30 activity features derived from the temporal distribution of action potentials, integrated burst properties, calculated coefficients of variation, and features of Gabor fits to autocorrelograms. The different oscillatory states were induced by blocking neurotransmitter receptors for: (i) GABAA; (ii) glycine; (iii) GABAA and glycine; (iv) all major synaptic types except AMPA, and (v) all major synapses except NMDA. To test the identification capability of the six substance‐specific response profiles, five blind experiments were performed. The response features from the unknown substances were compared to the database using proximity measures using the normalized Euclidian distance to each activity state. This process created six identification coefficients where the smallest correctly identified the unknown substances. Such activity profiles are expected to become substance‐specific ‘finger prints’ that classify unique responses to known and unknown substances. It is anticipated that this kind of approach will help to quantify pharmacological responses of networks used as biosensors.

The Keratocyte Network of Human Cornea: A Three-dimensional Study Using Confocal Laser Scanning Fluorescence Microscopy

Purpose. Keratocytes of the living human cornea were examined to compare quantitatively spatial arrangement and cell volume of the stromal layers. This knowledge is required for further studies toward a quantitative understanding of cellular alterations in corneal pathology. Methods. Three human corneas were stained with calcein AM and ethidium homodimer (Live/Dead Kit) directly after enucleation. The fluorescent cells were examined with confocal laser scanning fluorescence microscopy. High-resolution three-dimensional (3-D) volumes of ≤270 &mgr;m in the z-axis were reconstructed. Cell density and volume density were determined by computer-aided morphometry. Results. Three keratocyte subpopulations were distinguished. Their spatial arrangement was visualized by 3-D reconstructions of the scanned volumes. Whereas cell density decreased progressively from the anterior (100%) to posterior (53.7%) stroma, volume density was highest in the posterior stroma (17.03 ± 5.05%) and lowest in the central stroma (9.31 ± 1.09%). In the anterior stroma, volume density was found to be 10.19 ± 4.37%. Conclusion. Confocal laser scanning fluorescence microscopy allowed quantitative analysis and the visualization of the spatial arrangement of the keratocyte network in the living human corneal tissue for the first time. The results provide a basis for further studies of alterations of the normal cellular arrangements in corneal disease.

Oxidative stress-induced cytotoxic and genotoxic effects of nano-sized titanium dioxide particles in human HaCaT keratinocytes

Since nano-sized particles (NPs) are increasingly used in various fields of innovative biomedicine and industrial technologies, it is of importance to identify their potential human health risk. We investigated whether ROS-induced mitochondrial DNA damage is the mode of action of titanium dioxide-NPs (TiO2-NPs; ≤20 nm) to induce cytotoxic and genotoxic effects in human HaCaT keratinocytes in vitro. We showed that TiO2-NPs accumulate at the cell surface and are taken up by endocytosis. Micronucleus (MN) formation was found to be significantly maximal increased 24 h after treatment with 10 μg/ml and 48 h after treatment with 5 μg/ml TiO2-NPs about 1.8-fold respectively 2.2-fold of control. Mitochondrial DNA damage measured as common deletion was observed to be significantly 14-fold increased 72 h after treatment with 10 μg/ml TiO2-NPs when compared to control. Four hours after treatment with 5 and 50 μg/ml TiO2-NPs the level of ROS in HaCaT cells was found to be significantly increased about 7.5-fold respectively 16.7-fold of control. In conclusion, for the first time we demonstrate the induction of the mitochondrial common deletion in HaCaT cells following exposure to TiO2-NPs, which strongly suggests a ROS-mediated cytotoxic and genotoxic potential of NPs. However, the effects of the modification of TiO2-NPs, such as agglomeration, size distribution pattern and exposure time have to be further critically examined.

Application of micro-electrode arrays (MEAs) as an emerging technology for developmental neurotoxicity: Evaluation of domoic acid-induced effects in primary cultures of rat cortical neurons

Due to lack of knowledge only a few industrial chemicals have been identified as developmental neurotoxicants. Current developmental neurotoxicity (DNT) guidelines (OECD and EPA) are based entirely on in vivo studies that are both time consuming and costly. Consequently, there is a high demand to develop alternative in vitro methods for initial screening to prioritize chemicals for further DNT testing. One of the most promising tools for neurotoxicity assessment is the measurement of neuronal electrical activity using micro-electrode arrays (MEAs) that provides a functional and neuronal specific endpoint that until now has been used mainly to detect acute neurotoxicity. Here, electrical activity measurements were evaluated to be a suitable endpoint for the detection of potential developmental neurotoxicants. Initially, primary cortical neurons grown on MEA chips were characterized for different cell markers over time, using immunocytochemistry. Our results show that primary cortical neurons could be a promising in vitro model for DNT testing since some of the most critical neurodevelopment processes such as progenitor cell commitment, proliferation and differentiation of astrocytes and maturation of neurons are present. To evaluate if electrical activity could be a suitable endpoint to detect chemicals with DNT effects, our model was exposed to domoic acid (DomA), a potential developmental neurotoxicant for up to 4 weeks. Long-term exposure to a low concentration (50nM) of DomA increased the basal spontaneous electrical activity as measured by spike and burst rates. Moreover, the effect induced by the GABA(A) receptor antagonist bicuculline was significantly lower in the DomA treated cultures than in the untreated ones. The MEA measurements indicate that chronic exposure to DomA changed the spontaneous electrical activity leading to the possible neuronal mal functioning. The obtained results suggest that the MEAs could be a useful tool to identify compounds with DNT potential.

Stimulation of phagocytosis and free radical production in murine macrophages by 50 Hz electromagnetic fields.

Effects of 50 Hz electromagnetic fields on phagocytosis and free radical production were examined in mouse bone marrow-derived macrophages. Macrophages were in vitro exposed to electromagnetic fields using different magnetic field densities (0.5-1.5 mT). Short-time exposure (45 min) to electromagnetic fields resulted in significantly increased phagocytic uptake (36.3% +/- 15.1%) as quantified by measuring the internalization rate of latex beads. Stimulation with 1 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) showed the same increased phagocytic activity as 1 mT electromagnetic fields. However, co-exposure to electromagnetic fields and TPA showed no further increase of bead uptake, and therefore we concluded that because of the absence of additive effects, the electromagnetic fields-induced stimulation of mouse bone marrow-derived macrophages does not involve the protein kinase C signal transduction pathway. Furthermore, a significant increased superoxide production after exposure to electromagnetic fields was detected.

Alteration in cellular functions in mouse macrophages after exposure to 50 Hz magnetic fields

The aim of the present study is to investigate whether extremely low frequency electromagnetic fields (ELF‐EMF) affect certain cellular functions and immunologic parameters of mouse macrophages. In this study, the influence of 50 Hz magnetic fields (MF) at 1.0 mT was investigated on the phagocytic activity and on the interleukin‐1β (IL‐1β) production in differentiated macrophages. MF‐exposure led to an increased phagocytic activity after 45 min, shown as a 1.6‐fold increased uptake of latex beads in MF‐exposed cells compared to controls. We also demonstrate an increased IL‐1β release in macrophages after 24 h exposure (1.0 mT MF). Time‐dependent IL‐1β formation was significantly increased already after 4 h and reached a maximum of 12.3‐fold increase after 24 h compared to controls. Another aspect of this study was to examine the genotoxic capacity of 1.0 mT MF by analyzing the micronucleus (MN) formation in long‐term (12, 24, and 48 h) exposed macrophages. Our data show no significant differences in MN formation or irregular mitotic activities in exposed cells. Furthermore, the effects of different flux densities (ranging from 0.05 up to 1.0 mT for 45 min) of 50 Hz MF was tested on free radical formation as an endpoint of cell activation in mouse macrophage precursor cells. All tested flux densities significantly stimulated the formation of free radicals. Here, we demonstrate the capacity of ELF‐EMF to stimulate physiological cell functions in mouse macrophages shown by the significantly elevated phagocytic activity, free radical release, and IL‐1β production suggesting the cell activation capacity of ELF‐EMF in the absence of any genotoxic effects. J. Cell. Biochem.

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.