2D cultures are useful platforms allowing studies of the fundamental mechanisms governing neuron and synapse functions. Yet, such models are limited when exploring changes in network dynamics due to 3D‐space topologies. 3D platforms fill this gap and favor investigating topologies closer to the real brain organization. Graphene, an atom‐thick layer of carbon, possesses remarkable properties and since its discovery is considered a highly promising material in neuroscience developments. Here, elastomeric 3D platforms endowed with graphene cues are exploited to modulate neuronal circuits when interfaced to graphene in 3D topology. Ex vivo neuronal networks are successfully reconstructed within 3D scaffolds, with and without graphene, characterized by comparable size and morphology. By confocal microscopy and live imaging, the 3D architecture of synaptic networks is documented to sustain a high rate of bursting in 3D scaffolds, an activity further increased by graphene interfacing. Changes are reported in the excitation/inhibition ratio, potentially following 3D‐graphene interfacing. A hypothesis is thus proposed, where the combination of synapse formation under 3D architecture and graphene interfaces affects the maturation of GABAergic inhibition. This will tune the balance between hyperpolarizing and depolarizing responses, potentially contributing to network synchronization in the absence of changes in GABAergic phenotype expression.
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