Feasibility Assessment of Micro-Electrode Chip Assay as a Method of Detecting Neurotoxicity in vitro

Abstract

Detection and characterization of chemically induced toxic effects in the nervous system represent a challenge for the hazard assessment of chemicals. In vivo, neurotoxicological assessments exploit the fact that the activity of neurons in the central and peripheral nervous system has functional consequences. And so far, no in vitro method for evaluating the neurotoxic hazard has yet been validated and accepted for regulatory purpose. The micro-electrode array (MEA) assay consists of a culture chamber into which an integrated array of micro-electrodes is capable of measuring extracellular electrophysiology (spikes and bursts) from electro-active tissues. A wide variety of electrically excitable biological tissues may be placed onto the chips including primary cultures of nervous system tissue. Recordings from this type of in vitro cultured system are non-invasive, give label free evaluations and provide a higher throughput than conventional electrophysiological techniques. In this paper, 20 substances were tested in a blinded study for their toxicity and dose-response curves were obtained from fetal rat cortical neuronal networks coupled to MEAs. The experimental procedure consisted of evaluating the firing activity (spiking rate) and modification/reduction in response to chemical administration. Native/reference activity, 30 min of activity recording per dilution, plus the recovery points (after 24 h) were recorded. The preliminary data, using a set of chemicals with different mode-of-actions (13 known to be neurotoxic, 2 non-neuroactive and not toxic, and 5 non-neuroactive but toxic) show good predictivity (sensitivity: 0.77; specificity: 0.86; accuracy: 0.85). Thus, the MEA with a neuronal network has the potency to become an effective tool to evaluate the neurotoxicity of substances in vitro.

Keywords: chemical test; in vitro assay; micro-electrode array; neuronal networks; neurotoxicity.

Figure 1

Electrical activity effects on neuronal cultures after administration of not neuroactive and not toxic substances. Electrical activity following administration of substances classified in the not neuroactive and not toxic group (at the concentration indicated under the bars) has been recorded and normalized in respect to native activity (percent of control, indicated as 100 in the ordinate axis). All data are means of at least three independent experiments ± SEM. Student’s one tail paired t-test was performed to assess differences between basal spontaneous activity and activity after chemical administration. Statistical significance was indicated by * for P < 0.05.

Figure 2

Electrical activity effects on neuronal cultures after administration of toxic but not neuroactive substances. Electrical activity following administration of substances classified in the toxic but not neuroactive group (at the concentration indicated under the bars) has been recorded and normalized in respect to native activity (percent of control, indicated as 100 in the ordinate axis). All data are means of at least three independent experiments ± SEM. Student’s one tail paired t-test was performed to assess differences between basal spontaneous activity and activity after chemical administration. Statistical significance was indicated by * for P < 0.05.

Figure 3

Electrical activity effects on neuronal cultures after administration of neuroactive substances. Electrical activity following administration of substances classified in the neuroactive group (at the concentration indicated under the bars) has been recorded and normalized in respect to native activity (percent of control, indicated as 100 in the ordinate axis). All data are means of at least three independent experiments ± SEM. Student’s one tail paired t-test was performed to assess differences between basal spontaneous activity and activity after chemical administration. Statistical significance was indicated by * for P < 0.05.