Astrocytes are responsible for maintaining homoeostasis and cognitive functions through calcium signalling, a process that is altered in brain diseases. Current bioelectronic tools are designed to study neurons and are not suitable for controlling calcium signals in astrocytes. Here, we show that electrical stimulation of astrocytes using electrodes coated with graphene oxide and reduced graphene oxide induces respectively a slow response to calcium, mediated by external calcium influx, and a sharp one, exclusively due to calcium release from intracellular stores. Our results suggest that the different conductivities of the substrate influence the electric field at the cell-electrolyte or cell-material interfaces, favouring different signalling events in vitro and ex vivo. Patch-clamp, voltage-sensitive dye and calcium imaging data support the proposed model. In summary, we provide evidence of a simple tool to selectively control distinct calcium signals in brain astrocytes for straightforward investigations in neuroscience and bioelectronic medicine.

2nd Faculty of Medicine Charles University Prague Czech Republic

Consiglio Nazionale delle Ricerche Istituto per la Sintesi Organica e la Fotoreattività Bologna Italy

Consiglio Nazionale delle Ricerche Istituto per lo Studio dei Materiali Nanostrutturati Bologna Italy

Department of Cellular Neurophysiology Institute of Experimental Medicine CAS Prague Czech Republic

Department of Pharmacy and Biotechnology University of Bologna Bologna Italy

Dipartimento di Ingegneria dell'Energia Elettrica e dell'Informazione 'Guglielmo Marconi' University of Bologna Cesena Italy

Erratum In

Nat Nanotechnol. 2024 Sep;19(9):1420. doi: 10.1038/s41565-024-01797-w. PubMed

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Verkhratsky, A. & Nedergaard, M. Physiology of astroglia. Physiol. Rev.98, 239–389 (2018). PubMed PMC

Goenaga, J., Araque, A., Kofuji, P. & Herrera Moro Chao, D. Calcium signalling in astrocytes and gliotransmitter release. Front. Synaptic Neurosci.15, 1138577 (2023). PubMed PMC

Bazargani, N. & Attwell, D. Astrocyte calcium signalling: the third wave. Nat. Neurosci.19, 182–189 (2016). PubMed

de Ceglia, R. et al. Specialized astrocytes mediate glutamatergic gliotransmission in the CNS. Nature622, 120–129 (2023). PubMed PMC

Lia, A. et al. Calcium signals in astrocyte microdomains, a decade of great advances. Front. Cell Neurosci.15, 673433 (2021). PubMed PMC

Scemes, E. & Giaume, C. Astrocyte calcium waves: what they are and what they do. Glia54, 716–725 (2006). PubMed PMC

Scemes, E., Suadicani, S. O., Dahl, G. & Spray, D. C. Connexin and pannexin mediated cell–cell communication. Neuron Glia Biol.3, 199–208 (2007). PubMed PMC

Halassa, M. M., Fellin, T. & Haydon, P. G. The tripartite synapse: roles for gliotransmission in health and disease. Trends Mol. Med.13, 54–63 (2007). PubMed

Araque, A., Carmignoto, G. & Haydon, P. G. Dynamic signalling between astrocytes and neurons. Annu. Rev. Physiol.63, 795–813 (2001). PubMed

Filosa, J. A. & Iddings, J. A. Astrocyte regulation of cerebral vascular tone. Am. J. Physiol. Heart Circ. Physiol.305, H609–H619 (2013). PubMed PMC

Dunn, K. M., Hill-Eubanks, D. C., Liedtke, W. B. & Nelson, M. T. TRPV4 channels stimulate Ca2+-induced Ca2+ release in astrocytic endfeet and amplify neurovascular coupling responses. Proc. Natl Acad. Sci. USA110, 6157–6162 (2013). PubMed PMC

Santello, M., Toni, N. & Volterra, A. Astrocyte function from information processing to cognition and cognitive impairment. Nat. Neurosci.22, 154–166 (2019). PubMed

Mederos, S. et al. GABAergic signalling to astrocytes in the prefrontal cortex sustains goal-directed behaviors. Nat. Neurosci.24, 82–92 (2021). PubMed

Dossi, E., Vasile, F. & Rouach, N. Human astrocytes in the diseased brain. Brain Res. Bull.136, 139–156 (2018). PubMed PMC

Kimelberg, H. K. & Nedergaard, M. Functions of astrocytes and their potential as therapeutic targets. Neurotherapeutics7, 338–353 (2010). PubMed PMC

Seifert, G. & Steinhäuser, C. Neuron–astrocyte signalling and epilepsy. Exp. Neurol.244, 4–10 (2013). PubMed

Butenko, O. et al. The increased activity of TRPV4 channel in the astrocytes of the adult rat hippocampus after cerebral hypoxia/ischemia. PLoS ONE7, e39959 (2012). PubMed PMC

Shigetomi, E., Tong, X., Kwan, K. Y., Corey, D. P. & Khakh, B. S. TRPA1 channels regulate astrocyte resting calcium levels and inhibitory synapse efficacy via GAT-3. Nat. Neurosci.15, 70–80 (2011). PubMed PMC

Shibasaki, K., Ikenaka, K., Tamalu, F., Tominaga, M. & Ishizaki, Y. A novel subtype of astrocytes expressing TRPV4 (transient receptor potential vanilloid 4) regulates neuronal excitability via release of gliotransmitters. J. Biol. Chem.289, 14470–14480 (2014). PubMed PMC

Stobart, J. L. et al. Cortical circuit activity evokes rapid astrocyte calcium signals on a similar timescale to neurons. Neuron98, 726–735.e4 (2018). PubMed

Srinivasan, R. et al. Ca2+ signalling in astrocytes from Ip3r2−/− mice in brain slices and during startle responses in vivo. Nat. Neurosci.18, 708–717 (2015). PubMed PMC

Rungta, R. L. et al. Ca2+ transients in astrocyte fine processes occur via Ca2+ influx in the adult mouse hippocampus. Glia64, 2093–2103 (2016). PubMed

Benfenati, V. et al. Expression and functional characterization of transient receptor potential vanilloid-related channel 4 (TRPV4) in rat cortical astrocytes. Neuroscience148, 876–892 (2007). PubMed

Benfenati, V. et al. An aquaporin-4/transient receptor potential vanilloid 4 (AQP4/TRPV4) complex is essential for cell-volume control in astrocytes. Proc. Natl Acad. Sci. USA108, 2563–2568 (2011). PubMed PMC

Shigetomi, E., Jackson-Weaver, O., Huckstepp, R. T., O’Dell, T. J. & Khakh, B. S. TRPA1 channels are regulators of astrocyte basal calcium levels and long-term potentiation via constitutive d-serine release. J. Neurosci.33, 10143–10153 (2013). PubMed PMC

Cheli, V. T. et al. L-type voltage-operated calcium channels contribute to astrocyte activation in vitro. Glia64, 1396–1415 (2016). PubMed PMC

Letellier, M. et al. Astrocytes regulate heterogeneity of presynaptic strengths in hippocampal networks. Proc. Natl Acad. Sci. USA113, E2685–E2694 (2016). PubMed PMC

Wang, F., Du, T., Liang, C., Verkhratsky, A. & Peng, L. Ammonium increases Ca2+ signalling and upregulates expression of Cav1.2 gene in astrocytes in primary cultures and in the in vivo brain. Acta Physiol.214, 261–274 (2015). PubMed

De Pittà, M., Goldberg, M., Volman, V., Berry, H. & Ben-Jacob, E. Glutamate regulation of calcium and IP3 oscillating and pulsating dynamics in astrocytes. J. Biol. Phys.35, 383–411 (2009). PubMed PMC

Kofuji, P. & Araque, A. G-protein-coupled receptors in astrocyte–neuron communication. Neuroscience456, 71–84 (2021). PubMed PMC

Durkee, C. A. et al. Gi/o protein-coupled receptors inhibit neurons but activate astrocytes and stimulate gliotransmission. Glia67, 1076–1093 (2019). PubMed PMC

Matyash, M., Matyash, V., Nolte, C., Sorrentino, V. & Kettenmann, H. Requirement of functional ryanodine receptor type 3 for astrocyte migration. FASEB J.16, 84–86 (2002). PubMed

Petravicz, J., Boyt, K. M. & McCarthy, K. D. Astrocyte IP3R2-dependent Ca2+ signalling is not a major modulator of neuronal pathways governing behavior. Front. Behav. Neurosci.8, 384 (2014). PubMed PMC

Gu, X. et al. Synchronized astrocytic Ca2+ responses in neurovascular coupling during somatosensory stimulation and for the resting state. Cell Rep.23, 3878–3890 (2018). PubMed PMC

Maiolo, L. et al. Glial interfaces: advanced materials and devices to uncover the role of astroglial cells in brain function and dysfunction. Adv. Health. Mater.10, e2001268 (2021). PubMed

Shigetomi, E. et al. Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses. J. Gen. Physiol.141, 633–647 (2013). PubMed PMC

Srinivasan, R. et al. New transgenic mouse lines for selectively targeting astrocytes and studying calcium signals in astrocyte processes in situ and in vivo. Neuron92, 1181–1195 (2016). PubMed PMC

Fabbri, R. et al. Graphene glial-interfaces: challenges and perspectives. Nanoscale13, 4390–4407 (2021). PubMed

Kostarelos, K., Vincent, M., Hebert, C. & Garrido, J. A. Graphene in the design and engineering of next-generation neural interfaces. Adv. Mater.29, 1700909 (2017). PubMed

Capasso, A. et al. Interactions between primary neurons and graphene films with different structure and electrical conductivity. Adv. Funct. Mater.31, 2005300 (2021).

Viana, D. et al. Nanoporous graphene-based thin-film microelectrodes for in vivo high-resolution neural recording and stimulation. Nat. Nanotechnol.19, 514–523 (2024). PubMed PMC

Bramini, M. et al. Interfacing graphene-based materials with neural cells. Front. Syst. Neurosci. 12, 12 (2018). PubMed PMC

Kovtun, A. et al. Accurate chemical analysis of oxygenated graphene-based materials using X-ray photoelectron spectroscopy. Carbon143, 268–275 (2019).

Liscio, A. et al. Charge transport in graphene–polythiophene blends as studied by Kelvin probe force microscopy and transistor characterization. J. Mater. Chem.21, 2924–2931 (2011).

Durso, M. et al. Biomimetic graphene for enhanced interaction with the external membrane of astrocytes. J. Mater. Chem. B6, 5335–5342 (2018). PubMed

Borrachero-Conejo, A. I. et al. Electrical stimulation by an organic transistor architecture induces calcium signalling in nonexcitable brain cells. Adv. Healthc. Mater.8, 1801139 (2019). PubMed

Chiacchiaretta, M. et al. Graphene oxide upregulates the homeostatic functions of primary astrocytes and modulates astrocyte-to-neuron communication. Nano Lett.18, 5827–5838 (2018). PubMed

Bramini, M. et al. An increase in membrane cholesterol by graphene oxide disrupts calcium homeostasis in primary astrocytes. Small15, 1900147 (2019). PubMed

Musto, M. et al. Shedding plasma membrane vesicles induced by graphene oxide nanoflakes in brain cultured astrocytes. Carbon176, 458–469 (2021).

OʼShea, T. M. et al. Foreign body responses in mouse central nervous system mimic natural wound responses and alter biomaterial functions. Nat. Commun.11, 6203 (2020). PubMed PMC

Salatino, J. W., Ludwig, K. A., Kozai, T. D. Y. & Purcell, E. K. Glial responses to implanted electrodes in the brain. Nat. Biomed. Eng.1, 862–877 (2017). PubMed PMC

Schulte, A. et al. Homeostatic calcium fluxes, ER calcium release, SOCE, and calcium oscillations in cultured astrocytes are interlinked by a small calcium toolkit. Cell Calcium101, 102515 (2022). PubMed

Muschol, M., Dasgupta, B. R. & Salzberg, B. M. Caffeine interaction with fluorescent calcium indicator dyes. Biophys. J.77, 577–586 (1999). PubMed PMC

Trefalt, G., Behrens, S. H. & Borkovec, M. Charge regulation in the electrical double layer: ion adsorption and surface interactions. Langmuir32, 380–400 (2016). PubMed

Gurney, R.W. Ionic Processes In Solution (McGraw-Hill, 1953).

Wan, X. et al. Bimodal voltage dependence of TRPA1: mutations of a key pore helix residue reveal strong intrinsic voltage-dependent inactivation. Pflug. Arch.466, 1273–1287 (2014). PubMed PMC

Garcia-Elias, A., Lorenzo, I. M., Vicente, R. & Valverde, M. A. IP3 receptor binds to and sensitizes TRPV4 channel to osmotic stimuli via a calmodulin-binding site. J. Biol. Chem.283, 31284–31288 (2008). PubMed

Lee, K. S., Ladinsky, H., Choi, S. J. & Kasuya, Y. Studies on the in vitro interaction of electrical stimulation and Ca++ movement in sarcoplasmic reticulum. J. Gen. Physiol.49, 689–715 (1966). PubMed PMC

Bedut, S., Kettenhofen, R. & D’Angelo, J.-M. Voltage-sensing optical recording: a method of choice for high-throughput assessment of cardiotropic effects. J. Pharmacol. Toxicol. Methods105, 106888 (2020). PubMed

Pampaloni, N. P. et al. Single-layer graphene modulates neuronal communication and augments membrane ion currents. Nat. Nanotechnol.13, 755–764 (2018). PubMed

Vaidyanathan, T. V., Collard, M., Yokoyama, S., Reitman, M. E. & Poskanzer, K. E. Cortical astrocytes independently regulate sleep depth and duration via separate GPCR pathways. eLife10, e63329 (2021). PubMed PMC

Giacomello, M. et al. Stimulation of Ca2+ signals in neurons by electrically coupled electrolyte–oxide–semiconductor capacitors. J. Neurosci. Methods198, 1–7 (2011). PubMed

Monai, H. et al. Calcium imaging reveals glial involvement in transcranial direct current stimulation-induced plasticity in mouse brain. Nat. Commun.7, 11100 (2016). PubMed PMC

Zhao, S. et al. Full activation pattern mapping by simultaneous deep brain stimulation and fMRI with graphene fiber electrodes. Nat. Commun.11, 1788 (2020). PubMed PMC

Mola, M. G. et al. The speed of swelling kinetics modulates cell volume regulation and calcium signaling in astrocytes: a different point of view on the role of aquaporins. Glia64, 139–154 (2016). PubMed PMC

Ferroni, S., Marchini, C., Schubert, P. & Rapisarda, C. Two distinct inwardly rectifying conductances are expressed in long term dibutyryl-cyclic-AMP treated rat cultured cortical astrocytes. FEBS Lett.367, 319–325 (1995). PubMed

Benfenati, V. et al. A transparent organic transistor structure for bidirectional stimulation and recording of primary neurons. Nat. Mater.12, 672–680 (2013). PubMed

Fromherz, P. Three levels of neuroelectronic interfacing. Ann. N. Y. Acad. Sci.1093, 143–160 (2006). PubMed