title: Neurotoxicity screening of (illicit) drugs using novel methods for analysis of microelectrode array (MEA) recordings.
abstract: Annual prevalence of the use of common illicit drugs and new psychoactive substances (NPS) is high, despite the often limited knowledge on the health risks of these substances. Recently, cortical cultures grown on multi-well microelectrode arrays (mwMEAs) have been used for neurotoxicity screening of chemicals, pharmaceuticals, and toxins with a high sensitivity and specificity. However, the use of mwMEAs to investigate the effects of illicit drugs on neuronal activity is largely unexplored. We therefore first characterised the cortical cultures using immunocytochemistry and show the presence of astrocytes, glutamatergic and GABAergic neurons. Neuronal activity is concentration-dependently affected following exposure to six neurotransmitters (glutamate, GABA, serotonin, dopamine, acetylcholine and nicotine). Most neurotransmitters inhibit neuronal activity, although glutamate and acetylcholine transiently increase activity at specific concentrations. These transient effects are not detected when activity is determined during the entire 30min exposure window, potentially resulting in false-negative results. As expected, exposure to the GABAA-receptor antagonist bicuculline increases neuronal activity. Exposure to a positive allosteric modulator of the GABAA-receptor (diazepam) or to glutamate receptor antagonists (CNQX and MK-801) reduces neuronal activity. Further, we demonstrate that exposure to common drugs (3,4-methylenedioxymethamphetamine (MDMA) and amphetamine) and NPS (1-(3-chlorophenyl)piperazine (mCPP), 4-fluoroamphetamine (4-FA) and methoxetamine (MXE)) decreases neuronal activity. MXE most potently inhibits neuronal activity with an IC50 of 0.5μM, whereas 4-FA is least potent with an IC50 of 113μM. Our data demonstrate the importance of analysing neuronal activity within different time windows during exposure to prevent false-negative results. We also show that cortical cultures grown on mwMEAs can successfully be applied to investigate the effects of different (illicit) drugs on neuronal activity. Compared to investigating multiple single endpoints for neurotoxicity or neuromodulation, such as receptor activation or calcium channel function, mwMEAs can provide information on integrated aspects of drug-induced neurotoxicity more rapidly. Therefore, this approach could contribute to a faster insight in possible health risks and shorten the regulation process.
authors.affiliation: National Poisons Information Center (NVIC), University Medical Center Utrecht, P.O. Box 85500, NL-3508 GA Utrecht, The Netherlands. Electronic address: L.Hondebrink@umcutrecht.nl.
authors.forename: L
authors.initials: L
authors.lastname: Hondebrink
authors.affiliation: Neurotoxicology Research Group, Division Toxicology, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.177, NL-3508 Utrecht, The Netherlands.
authors.forename: A H A
authors.initials: AHA
authors.lastname: Verboven
authors.affiliation: Neurotoxicology Research Group, Division Toxicology, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.177, NL-3508 Utrecht, The Netherlands.
authors.forename: W S
authors.initials: WS
authors.lastname: Drega
authors.affiliation: Neurotoxicology Research Group, Division Toxicology, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.177, NL-3508 Utrecht, The Netherlands.
authors.forename: S
authors.initials: S
authors.lastname: Schmeink
authors.affiliation: Neurotoxicology Research Group, Division Toxicology, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.177, NL-3508 Utrecht, The Netherlands.
authors.forename: M W G D M
authors.initials: MWGDM
authors.lastname: de Groot
authors.affiliation: Neurotoxicology Research Group, Division Toxicology, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.177, NL-3508 Utrecht, The Netherlands.
authors.forename: R G D M
authors.initials: RGDM
authors.lastname: van Kleef
authors.affiliation: Neurotoxicology Research Group, Division Toxicology, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.177, NL-3508 Utrecht, The Netherlands.
authors.forename: F M J
authors.initials: FMJ
authors.lastname: Wijnolts
authors.affiliation: Neurotoxicology Research Group, Division Toxicology, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.177, NL-3508 Utrecht, The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: de Groot
authors.affiliation: National Poisons Information Center (NVIC), University Medical Center Utrecht, P.O. Box 85500, NL-3508 GA Utrecht, The Netherlands; Neurotoxicology Research Group, Division Toxicology, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.177, NL-3508 Utrecht, The Netherlands; Department of Intensive Care Medicine, University Medical Center Utrecht, P.O. Box 85500, NL-3508 GA, Utrecht, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Meulenbelt
authors.affiliation: Neurotoxicology Research Group, Division Toxicology, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.177, NL-3508 Utrecht, The Netherlands.
authors.forename: R H S
authors.initials: RHS
authors.lastname: Westerink
mesh_terms: D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000200:Action Potentials / Q000187:drug effects*; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects; D002540:Cerebral Cortex / Q000166:cytology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018682:GABA Agents / Q000494:pharmacology; D050485:GABA Plasma Membrane Transport Proteins / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D013287:Illicit Drugs / Q000633:toxicity*; D008839:Microelectrodes; D009474:Neurons / Q000187:drug effects*; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D050598:Vesicular Glutamate Transport Protein 1 / Q000378:metabolism
chemical_list: D018691:Excitatory Amino Acid Antagonists; D018682:GABA Agents; D050485:GABA Plasma Membrane Transport Proteins; D005904:Glial Fibrillary Acidic Protein; D013287:Illicit Drugs; D050598:Vesicular Glutamate Transport Protein 1; D016291:Dizocilpine Maleate; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D014446:Tyrosine 3-Monooxygenase
keywords: Analysis strategy; Characterisation; Illicit drugs; Integrated testing strategy; Neurotoxicity screening