Abstract
Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of forming a functional network. Starting from a stably proliferating neuroepithelial stem cell (NESC) population, we generate “mixed cortical cultures” (MCC) within 24 days. Characterization by immunocytochemistry, gene expression profiling and functional tests (multi-electrode arrays) showed that MCC contain various functional neurotransmitter receptors, and in particular, the N-methyl-d-aspartate subtype of ionotropic glutamate receptors (NMDA-R). As this important receptor is found neither on conventional neural cell lines nor on most stem cell-derived neurons, we focused here on the characterization of rapid glutamate-triggered Ca2+ signalling. Changes of the intracellular free calcium ion concentration ([Ca2+]i) were measured by fluorescent imaging as the main endpoint, and a method to evaluate and quantify signals in hundreds of cells at the same time was developed. We observed responses to glutamate in the low µM range. MCC responded to kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and a subpopulation of 50% had functional NMDA-R. The receptor was modulated by Mg2+, Zn2+ and Pb2+ in the expected ways, and various toxicologically relevant agonists (quinolinic acid, ibotenic acid, domoic acid) triggered [Ca2+]i responses in MCC. Antagonists, such as phencyclidine, ketamine and dextromethorphan, were also readily identified. Thus, the MCC developed here may fill an important gap in the panel of test systems available to characterize the effects of chemicals on neurotransmitter receptors.
Highlights
Identifying compounds that interfere with ionotropic glutamate receptor signalling is important for the safety evaluation of drugs and environmental chemicals
Several agonists, antagonist and modulators of the NMDA receptor (NMDA-R) have been developed as drug candidates or pharmacological tools: NMDA has been identified as a specific agonist for the NMDA-R; the NMDAR antagonist ketamine is used clinically as an anaesthetic (Sinner and Graf 2008); the receptor blocker memantine is an approved medication for Alzheimer’s disease (Witt et al 2004); and some NMDA-R antagonists, e.g., phencyclidine (PCP) and dextromethorphan (DXM) cause hallucinogenic and dissociative symptoms and are used as illicit recreational drugs (Williams and Lundahl 2019)
We showed that [Ca2+]i imaging can be used as a quantitative readout despite the heterogeneity of cell types present in mixed cortical cultures” (MCC)
Summary
Identifying compounds that interfere with ionotropic glutamate receptor signalling is important for the safety evaluation of drugs and environmental chemicals. Such information is of utmost toxicological relevance, as l-glutamate is the main excitatory neurotransmitter in the brain (Fukaya et al 2003; Maycox et al 1988). The many receptors of this acidic amino acid can be divided into metabotropic receptors and ionotropic receptors. The latter group can be further subdivided into N-methyl-d-aspartate (NMDA) receptors and non-NMDA receptors (kainate and AMPA subtypes) (Traynelis et al 2010). Several agonists, antagonist and modulators of the NMDA-R have been developed as drug candidates or pharmacological tools: NMDA has been identified as a specific agonist for the NMDA-R; the NMDAR antagonist ketamine is used clinically as an anaesthetic (Sinner and Graf 2008); the receptor blocker memantine is an approved medication for Alzheimer’s disease (Witt et al 2004); and some NMDA-R antagonists, e.g., phencyclidine (PCP) and dextromethorphan (DXM) cause hallucinogenic and dissociative symptoms and are used as illicit recreational drugs (Williams and Lundahl 2019)
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