Novel carbon film induces precocious calcium oscillation to promote neuronal cell maturation

International audience; Different types of carbon materials are biocompatible with neural cells and can promote maturation. The mechanism of this effect is not clear. Here we have tested the capacity of a carbon material composed of amorphous sp 3 carbon backbone, embedded with a percolating network of sp 2 carbon domains to sustain neuronal cultures. We found that cortical neurons survive and develop faster on this novel carbon material. After 3 days in culture, there is a precocious increase in the frequency of neuronal activity and in the expression of maturation marker KCC2 on carbon films as compared to a commonly used glass surface. Accelerated development is accompanied by a dramatic increase in neuronal dendrite arborization. The mechanism for the precocious maturation involves the activation of intracellular calcium oscillations by the carbon material already after 1 day in culture. Carbon-induced oscillations are independent of network activity and reflect intrinsic spontaneous activation of developing neurons. Thus, these results reveal a novel mechanism for carbon material-induced neuronal survival and maturation. Brain trauma as well as neurodegenerative diseases are the leading cause of irreversible disability and low quality of life in the elderly population 1. A way to combat neurodegeneration is to promote reparation of neuronal networks, rewiring of neuronal connections, and eventual restoration or substitution of the lost functionality 2. One putative therapeutic avenue is providing scaffolds-special materials that support targeted differentiation of neuronal stem cells and neurite outgrowth of regenerating neurons. Carbon-derived materials possess numerous properties that make them usable as scaffolds 3. Carbon nanotubes (CNTs) and graphene are among the most studied carbon materials for biological applications. These materials enhance neuronal stem cell differentiation 4-6 , as well as promote neuronal survival, neuronal activity, and neuronal process outgrowth 7-13. Neurons cultured on CNTs have increased levels of neuronal K +-Cl − cotransporter KCC2, a key component in the functional maturation of inhibitory synaptic 14 and glutamatergic 15-17 transmission. Downregulation of this protein is also implicated in reactive plasticity following brain trauma 18. CNTs have been suggested to improve the electrical responsiveness of neurons by facilitating local electronic shortcuts between somas and dendrites 19. The ability of CNTs to form tight contacts with neurons is beneficial for neuron-electrode interfaces 20-24. 3D gra-phene substrates support growth and differentiation of neurons 25-27 that in combination with anti-inflammatory properties 28,29 makes graphene a next-generation neuronal tissue scaffold. Despite the importance of novel carbon materials for future engineering, we do not fully understand the mechanisms underlining the trophic action of carbon scaffolds. In this work, we propose a novel mechanism by which a new type of sputtered carbon material accelerates neuronal maturation. The carbon film material consists of conducting nanoscale sp 2 carbon islands embedded in a diamond-like sp 3 carbon matrix. We demonstrate OPEN