Human neuronal signaling and communication assays to assess functional neurotoxicity
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ORGAN TOXICITY AND MECHANISMS
Human neuronal signaling and communication assays to assess functional neurotoxicity Dominik Loser1,2,4 · Jasmin Schaefer1,2 · Timm Danker2 · Clemens Möller4 · Markus Brüll3 · Ilinca Suciu3 · Anna‑Katharina Ückert3 · Stefanie Klima3 · Marcel Leist3 · Udo Kraushaar1 Received: 15 September 2020 / Accepted: 16 November 2020 © The Author(s) 2020
Abstract Prediction of drug toxicity on the human nervous system still relies mainly on animal experiments. Here, we developed an alternative system allowing assessment of complex signaling in both individual human neurons and on the network level. The LUHMES cultures used for our approach can be cultured in 384-well plates with high reproducibility. We established here high-throughput quantification of free intracellular C a2+ concentrations [ Ca2+]i as broadly applicable surrogate of neuronal activity and verified the main processes by patch clamp recordings. Initially, we characterized the expression pattern of many neuronal signaling components and selected the purinergic receptors to demonstrate the applicability of the [Ca2+]i signals for quantitative characterization of agonist and antagonist responses on classical ionotropic neurotransmitter receptors. This included receptor sub-typing and the characterization of the anti-parasitic drug suramin as modulator of the cellular response to ATP. To exemplify potential studies on ion channels, we characterized voltage-gated sodium channels and their inhibition by tetrodotoxin, saxitoxin and lidocaine, as well as their opening by the plant alkaloid veratridine and the food-relevant marine biotoxin ciguatoxin. Even broader applicability of [ Ca2+]i quantification as an end point was demonstrated by measurements of dopamine transporter activity based on the membrane potential-changing activity of this neurotransmitter carrier. The substrates dopamine or amphetamine triggered [ Ca2+]i oscillations that were synchronized over the entire culture dish. We identified compounds that modified these oscillations by interfering with various ion channels. Thus, this new test system allows multiple types of neuronal signaling, within and between cells, to be assessed, quantified and characterized for their potential disturbance. Keywords Channel toxins · Neuronal network · Purinoceptor · Dopamine transporter · Network oscillations
Marcel Leist and Udo Kraushaar contributed equally. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00204-020-02956-3) contains supplementary material, which is available to authorized users. * Marcel Leist marcel.leist@uni‑konstanz.de 1
NMI Natural and Medical Sciences Institute at the University of Tuebingen, 72770 Reutlingen, Germany
2
NMI TT GmbH, 72770 Reutlingen, Germany
3
In Vitro Toxicology and Biomedicine, Department Inaugurated by the Doerenkamp‑Zbinden Foundation, University of Konstanz, Universitaetsstr. 10, 78457 Constance, Germany
4
Life Sciences Faculty, Albstadt-Sigmaringen University, 72488 Si
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