Event Abstract

Mapping neuronal network dynamics in developing cerebral organoids

  • 1 ETH Zürich, Department of Biosystems Science and Engineering, Switzerland
  • 2 Friedrich Miescher Institute for Biomedical Research, Switzerland
  • 3 ETH Zürich, Department of Biochemistry, Switzerland

Motivation Cerebral organoids represent an attractive, novel model system to study early brain development in vitro (Di Lullo and Kriegstein, 2017). Although recent evidence shows that cerebral organoids do recapitulate fundamental milestones of early brain morphogenesis (Lancaster and Knoblich, 2014), the emergence and functionality of brain-organoid neuronal connectivity has not been studied systematically yet. In this study, we apply high-density micro-electrode arrays (MEAs) to record from developing mouse cerebral organoids and characterize their spontaneous neuronal activity. Results provide first evidence on the potential of MEAs as a platform to study the role of spontaneous neuronal activity during brain organoid development and formation of functional microcircuits. Materials and Methods Mouse cerebral organoids: The protocol to generate mouse cerebral organoids was adapted from two previous studies (Eiraku et al., 2008; Lancaster et al., 2013). In this study, we use commercially available mouse embryonic stem cells (ESCs) (ES-D3, ATTC, CRL1934). For the generation of mouse cerebral organoids, ESCs were seeded in ultra-low-attachment V-bottom 96 well plates (4000 per well) in medium containing SB431542. After formation of embryoid bodies, primitive neuroepithelium was induced in neural induction medium (Lancaster et al., 2013). Six days after seeding (D6), organoids were embedded in Matrigel droplets to expand neuroepithelial tissue and cultured in maturation medium (Lancaster et al., 2013). For further maturation (D9 onwards), organoids were cultured in maturation medium, supplemented with Vitamin A, and maintained on an orbital shaker within a 37 C / 5% CO2 incubator. Electrophysiological recordings: Sections of cerebral organoids (300 μm) were obtained from 16-28 day-old cerebral organoids using a vibratome. Spontaneous neuronal activity was recorded with a high-density complementary-metal-oxide-semiconductor (CMOS) based MEA, comprising 26 400 platinum electrodes with 1024 readout channels over an area of 3.85 × 2.10 mm2 (Müller et al., 2015). The chip system allows for extra-cellular electrophysiological recordings of large sections of slices at a very high spatial and temporal resolution (17.5 um electrode pitch; 20 kHz sampling rate). MEA recordings were performed in BrainPhysTM medium (Bardy et al., 2015); a small holder was used to assure optimal slice attachment on the MEA. Pharmacological challenges were applied to investigate the mechanisms of observed neuronal dynamics. Results Using extracellular high-density MEA recordings, we have acquired spontaneous electrical activity of a large number of neurons in slices of developing mouse cerebral organoids. We found that the percentage of neurons demonstrating spiking activity increased during development, as did their average firing rate. Coherent network-level events could be observed relatively early (D17/18), resembling activity patterns previously reported for hippocampal neurons around birth (Crépel et al., 2008). In more mature organoids, network activity could be modified with the GABAA receptor antagonist bicuculline and blocked with the voltage-gated sodium channel blocker TTX. Immunohistochemistry confirmed the presence of excitatory and inhibitory neurons, as well as of astrocytes. Conclusion The present study combines brain organoid technology with CMOS-based high-density MEAs to investigate the emergence of neuronal spontaneous activity at the cellular- and network-level. MEAs provide a valuable platform for such experiments, since they allow for parallel recordings from a large number of cells at high temporal and spatial precision under controlled conditions. Having established a pipeline for organoids, grown from mouse ESCs, future work will expand on this innovative experimental platform to study cerebral organoids derived from human-induced pluripotent stem cells (hiPSCs).

Acknowledgements

This project has received funding from the European Research Council (ERC Advanced Grant 694829 “neuroXscales”).

References

Bardy, C., Van Den Hurk, M., Eames, T., Marchand, C., Hernandez, R. V., Kellogg, M., ... & Bang, A. G. (2015). Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro. Proceedings of the National Academy of Sciences, 112(20), E2725-E2734.

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Di Lullo, E., & Kriegstein, A. R. (2017). The use of brain organoids to investigate neural development and disease. Nature Reviews Neuroscience, 18(10), 573.

Eiraku, M., Watanabe, K., Matsuo-Takasaki, M., Kawada, M., Yonemura, S., Matsumura, M., ... & Sasai, Y. (2008). Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell stem cell, 3(5), 519-532.

Lancaster, M. A., & Knoblich, J. A. (2014). Organogenesis in a dish: modeling development and disease using organoid technologies. Science, 345(6194), 1247125.

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Müller, J., Ballini, M., Livi, P., Chen, Y., Radivojevic, M., Shadmani, A., ... & Stettler, A. (2015). High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels. Lab on a Chip, 15(13), 2767-2780.

Keywords: Organoid, Cerebral Cortex, Neurogenesis, spontaneous activity, connectivity mapping

Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.

Presentation Type: Poster Presentation

Topic: Neural Networks

Citation: Schröter M, Girr M, Boos J, Renner M, Gazorpak M, Gong W, Bartram J, Müller J and Hierlemann AR (2019). Mapping neuronal network dynamics in developing cerebral organoids. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00066

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Received: 26 Mar 2018; Published Online: 17 Jan 2019.

* Correspondence: Dr. Manuel Schröter, ETH Zürich, Department of Biosystems Science and Engineering, Zurich, Switzerland, manuel.schroeter@bsse.ethz.ch

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