Human neuronal signaling and communication assays to assess functional neurotoxicity

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 Ca2+ 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.

Functional alterations by a subgroup of neonicotinoid pesticides in human dopaminergic neurons

“…To control for the α7 specificity of PNU in the LUHMES system, we tested, whether it would also enhance signaling of other receptors. In control experiments, cells were stimulated with 1 µM α,β-meATP (P2X receptor agonist (Bianchi et al 1999 ; Khakh and North 2012 ; Loser et al 2021 )) and different concentrations of ABT 594 (non-α7 nAChR agonist). In both cases, no differences between recordings with and without PNU were detected (Fig.…”

“…This might occur by, e.g., affecting intracellular processes related to Ca 2+ -signaling. We tested the impact of neonicotinoid pretreatment on signaling via the ionotropic purinergic receptor subtype P2X3, which has been shown to be functionally expressed in LUHMES (Loser et al 2021 ). The response evoked by the agonist α,β-meATP (Bianchi et al 1999 ; Khakh and North 2012 ; Loser et al 2021 ) was not affected by the pre-applied neonicotinoids or nicotine (Fig.…”

“…It remains unclear, why the otherwise quite homogeneous LUHMES cultures show response heterogeneity at the single cell level. Since LUHMES form an interactive network, different cells in the network differ by their interconnection, their neighborhood and their firing history, and all this may contribute to differential responsiveness to nicotine or neonicotinoids (Loser et al 2021). -imaging recordings of six cells (marked in a) with and without response to the application of 100 µM Aceta.…”

“…The neuronal precursor cell line LUHMES is a widely used model to study biochemical and pharmacological effects on the human nervous system, as well as various types of toxicities (Krug et al 2013 , 2014 ; Zhang et al 2014 ; Lohren et al 2015 ; Smirnova et al 2016 ; Harris et al 2017 ; Tong et al 2017 ; Witt et al 2017 ; Delp et al 2018a , b , 2019 , 2021 ; Brüll et al 2020 ). The cells can be differentiated into post-mitotic dopaminergic neurons (Scholz et al 2011 ), and we have recently shown their usefulness for functional neurotoxicity assessment, including the measurement of effects on ionotropic (purinergic) receptors (Loser et al 2021 ). The SH-SY5Y neuroblastoma cells are another human model, which has been used for studies on Ca 2+ signaling in neuronal function such as mAChR and voltage-dependent Ca 2+ channel activation (Gustafsson et al 2010 ).…”

Acute effects of the imidacloprid metabolite desnitro-imidacloprid on human nACh receptors relevant for neuronal signaling

“…The activation of ionotropic receptors like nAChRs on neurons leads to a depolarization of the cell membrane, and thereby, activates voltage-dependent Ca 2+ channels. The transient influx of Ca 2+ into the cell increases the intracellular free Ca 2+ concentration ([Ca 2+ ] i ), which can be measured by Ca 2+ -imaging in neuronal cell cultures (Leist and Nicotera 1998 ; Sirenko et al 2019 ; Grunwald et al 2019 ; Loser et al 2021b ). This method is based on the quantifications of fluorescence signals of calcium-sensitive dyes introduced into the cells, and it is amenable to high-throughput formats (Sirenko et al 2019 ; Karreman et al 2020 ; Brüll et al 2020 ; Loser et al 2021b ).…”