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. 2024 Nov 17;15(1):433.
doi: 10.1186/s13287-024-04018-2.

In vitro electrophysiological drug testing on neuronal networks derived from human induced pluripotent stem cells

Giulia Parodi  1 , Giorgia Zanini  2 , Linda Collo  2 , Roberta Impollonia  2 , Chiara Cervetto  3   4 , Monica Frega  5 , Michela Chiappalone  2   4 , Sergio Martinoia  6   7
Affiliations

In vitro electrophysiological drug testing on neuronal networks derived from human induced pluripotent stem cells

Giulia Parodi et al. Stem Cell Res Ther. .

Abstract

Background: In vitro models for drug testing constitute a valuable and simplified in-vivo-like assay to better comprehend the biological drugs effect. In particular, the combination of neuronal cultures with Micro-Electrode Arrays (MEAs) constitutes a reliable system to investigate the effect of drugs aimed at manipulating the neural activity and causing controlled changes in the electrophysiology. While chemical modulation in rodents' models has been extensively studied in the literature, electrophysiological variations caused by chemical modulation on neuronal networks derived from human induced pluripotent stem cells (hiPSCs) still lack a thorough characterization.

Methods: In this work, we created three different configurations of hiPSCs-derived neuronal networks composed of fully glutamatergic neurons (100E), 75% of glutamatergic and 25% of GABAergic neurons (75E25I) and fully GABAergic neurons (100I). We focused on the effects caused by antagonists of three of the most relevant ionotropic receptors of the human brain, i.e., 2-amino-5-phosphonovaleric (APV, NMDA receptors antagonist), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, AMPA receptors antagonist), and bicuculline, picrotoxin and pentylenetetrazole (BIC, PTX, and PTZ, respectively, GABAA receptors antagonists).

Results: We found that APV and CNQX completely abolished the network bursting activity and caused major changes in the functional connectivity. On the other hand, the effect of BIC, PTX and PTZ mostly affected configurations in which the inhibitory component was present by increasing the firing and network bursting activity as well as the functional connectivity.

Conclusions: Our work revealed that hiPSCs-derived neuronal networks are very sensitive to pharmacological manipulation of the excitatory ionotropic glutamatergic and inhibitory ionotropic GABAergic transmission, representing a preliminary and necessary step forward in the field of drug testing that can rely on pathological networks of human origin.

Keywords: Drug testing; Electrophysiology; Human induced pluripotent stem cells; Micro-Electrode Arrays.

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Conflict of interest statement

Declarations Ethics approval and consent to participate The experimental protocol was approved by the European Animal Care Legislation (2010/63/EU), by the Italian Ministry of Health in accordance with the D.L. 116/1992 and by the guidelines of the University of Genova (Prot. 75F11.N.6JI, 08/08/2018). We received the Ngn2-positive and Ascl1-positive hiPSCs lines in frozen vials, kindly provided by Prof. Nadif Kasri (Radboud University Medical Centre, the Netherlands). The genetically modified organism (GMO) approval under which the lines have been used is IG22-071. The two lines provided by our collaborators were previously characterized [21]. The lines were infected, according to a previously published protocol [10], with lentiviral constructs encoding rtTA combined with Ngn2 (Control line 1) or Ascl1 (Control line 2) to generate doxycycline-inducible excitatory or inhibitory neurons arrays [21, 22]. Both lines were generated from reprogrammed fibroblasts. Control line 1 (C1, healthy 30-years-old female, Ngn2) was reprogrammed via episomal reprogramming (Coriell Institute for medical research, GM25256). Control line 2 (C2, healthy 51-years-old male, Ascl1) was reprogrammed via a non-integrating Sendai virus (KULSTEM iPSC core facility Leuven, Belgium, KSF-16-025). Karyotypes of hiPSCs lines were verified, and hiPSCs lines were tested for pluripotency and genomic integrity based on single nucleotide polymorphism arrays [21, 22]. We declare that the research was conducted in accordance with the principles embodied in the Declaration of Helsinki and in accordance with local statutory requirements. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Experimental protocol. a Schematic representation of the cell culture protocol. Starting from hiPSCs cultures, excitatory (E, red) and inhibitory (I, blue) iNeurons were obtained. b Sketch of the three configurations obtained by varying the ratio between glutamatergic (red) and GABAergic (blue) neurons in the neuronal cultures. Neurons were co-plated with rat astrocytes (green) in the active area of the MEAs, characterized by 60 electrodes. c Sketch of the experimental protocol performed. The spontaneous activity was recorded for 10 min. Subsequently, one of the five drugs (i.e., APV, CNQX, BIC, PTX, or PTZ) was added to the neuronal culture medium and the electrophysiological activity was recorded for 40 min. d Table representing the number of the recorded neuronal cultures for each configuration and for each drug
Fig. 2
Fig. 2
Spiking activity characterization during the chemical modulation. a–i Normalized Instantaneous Firing Rate (IFR) of 100E (red), 75E25I (orange), and 100I (blue) configurations when APV (first row), CNQX (second row), or BIC (third row) were used. The timepoint “Spont” represents the normalized firing rate computed over the 10-min recording of the spontaneous activity. Data are represented with the mean (dot) and the standard error of the mean (whiskers) (∗ refers to p < 0.05)
Fig. 3
Fig. 3
a Representative raster plots and respective cumulative instantaneous firing rate profiles (bin = 10 ms, overlapped) of the electrophysiological activity of the 100E (red) and 75E25I (orange) configurations in spontaneous conditions (first column) and after APV (second column), CNQX (third column), and BIC (fourth column) administration. A black dot represents a detected spike, while a dense black band indicates a network burst event. b-g Network bursting activity characterization during the chemical modulation. Normalized Network Bursting Rate (NBR) of 100E (red) and 75E25I (orange) configurations when APV (b, e), CNQX (c, f), or BIC (d, g) were used. The timepoint “Spont” represents the normalized NBR computed over the 10-min recording of the spontaneous activity. Data are represented with the mean (dot) and the standard error of the mean (whiskers) (∗ refers to p < 0.05 and ∗ ∗ to p < 0.01)
Fig. 4
Fig. 4
Drugs' effect in the 10–15 min after the administration. a Plot of the normalized number of network bursting (NB) units for 100E (red) and 75E25I (orange) configurations. b Box plots of the normalized Network Bursting Rate (NBR) for each configuration. c, d Average network burst shapes (Spike Time Histogram, STH) for the spontaneous (grey) and the BIC-modulated activity for the 100E (c, red) and 75E25I (d, orange) configurations. Inset: box plots of the Network Burst Duration (NBD) for each configuration in spontaneous (grey) and BIC modulated (red or orange) conditions. Scatter plots are represented with the mean (dot) and the standard error of the mean (whiskers). Box plots are represented with the percentile 25–75 (box), the standard deviation (whiskers), the median (line), the mean (square), and the minimum and maximum (crosses) values (∗ refers to p < 0.05)
Fig. 5
Fig. 5
Functional connectivity maps and graphs. Representative connectivity maps and connectivity graphs of 100E (red) and 75E25I (orange) configurations. In each pair, the functional connectivity map/graph of the spontaneous activity (on the left) is compared with the one after the drug treatment (on the right). In the connectivity maps, the connection between two units is represented by a pixel which colour represents its strength. In the connectivity graphs, each node is represented by dots and functional connections are represented with edges
Fig. 6
Fig. 6
Characterization of the functional connectivity. a–f Pie charts of the number of edges and number of links new, unchanged, and extinguished with respect to the spontaneous activity for each configuration and for each administrated drug. g–h Box plots of the Cpeak values of the 100E g and 75E25I h configurations. Box plots are represented with the percentile 25–75 (box), the standard deviation (whiskers), the median (line), the mean (square), and the minimum and maximum (crosses) values (∗ refers to p < 0.05)
Fig. 7
Fig. 7
Characterization of PTX e PTZ effects. a–c Normalized Instantaneous Firing Rate (IFR) over time of the 100E (red), 75E25I (orange), and 100I (blue) configurations when PTX was used. d–f Normalized IFR over time of the 100E (red), 75E25I (orange), and 100I (blue) configurations when PTZ was used. g–h Normalized Network Bursting Rate (NBR) over time of the 100E (red) and 75E25I (orange) configurations in the presence of PTX. Insets: Normalized number of network bursting units of the configurations in the spontaneous and PTX evoked conditions. i Normalized Network Burst Duration (NBD) of the 100E (left) and 75E25I (right) configurations in spontaneous (grey) and PTX evoked conditions (red and orange, respectively). j–k Normalized Network Bursting Rate (NBR) over time of the 100E (red) and 75E25I (orange) configurations in the presence of PTZ. Insets: Normalized number of network bursting units of the configurations in the spontaneous and PTZ evoked conditions. l Normalized Network Burst Duration (NBD) of the 100E (left) and 75E25I (right) configurations in spontaneous (grey) and PTZ evoked conditions (red and orange, respectively). Scatter plots are represented with the mean (dot) and the standard error of the mean (whiskers). Box plots are represented with the percentile 25–75 (box), the standard deviation (whiskers), the median (line), the mean (square), and the minimum and maximum (crosses) values (∗ refers to p < 0.05)
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