Graphene has the potential to make a very significant impact onsociety, with important applications in the biomedicalfield. The possibility toengineer graphene-based medical devices at the neuronal interface is of particularinterest, making it imperative to determine the biocompatibility of graphene materialswith neuronal cells. Here we conducted a comprehensive analysis of the effects ofchronic and acute exposure of rat primary cortical neurons to few-layer pristinegraphene (GR) and monolayer graphene oxide (GO)flakes. By combining a range ofcell biology, microscopy, electrophysiology, and“omics”approaches we characterizedthe graphene−neuron interaction from thefirst steps of membrane contact andinternalization to the long-term effects on cell viability, synaptic transmission, and cellmetabolism. GR/GOflakes are found in contact with the neuronal membrane, free inthe cytoplasm, and internalized through the endolysosomal pathway, with nosignificant impact on neuron viability. However, GO exposure selectively caused theinhibition of excitatory transmission, paralleled by a reduction in the number ofexcitatory synaptic contacts, and a concomitant enhancement of the inhibitory activity. This was accompanied by inductionof autophagy, altered Ca2+dynamics, and a downregulation of some of the main players in the regulation of Ca2+homeostasis in both excitatory and inhibitory neurons. Our results show that, although graphene exposure does not impactneuron viability, it does nevertheless have important effects on neuronal transmission and network functionality, thuswarranting caution when planning to employ this material for neurobiological applications.
