Extracellular spread of synaptically released glutamate is tightly controlled to guarantee a high fidelity of point-to-point excitatory synaptic connections. Reduced glutamate uptake increases the likelihood of activation of extrasynaptic receptors and the extent of synaptic crosstalk. Astrocytes mediate the bulk of glutamate uptake suggesting that perturbations of astrocyte function and morphology can rapidly affect glutamate uptake. Our recent work along these lines uncovered that induction of epileptiform activity in vitro and status epilepticus in vivo leads to rapid astrocyte morphology changes over tens of minutes in the hippocampal CA1 region. We have also discovered that the action range of glutamate is significantly higher than expected in healthy tissue and, importantly, that it increases after induction of epilepsy. Together these novel findings indicate that epileptic activity or, more general, ongoing neuronal activity can rapidly affect extracellular signalling of the N-methyl-D-aspartate receptor (NMDAR) ligand glutamate by inducing structural changes of astrocytes. We predict that extracellular signalling of the NMDAR ligands D-serine and glycine, the receptor’s co-agonists, is also affected because their extracellular concentration and diffusion are controlled by astrocytes. Therefore, the aim of the current project is to reveal the relationship between neuronal network activity, astrocyte morphology and extracellular dynamics of NMDAR ligands in vitro and in vivo. Our work will shed light on a new mechanism that, by modifying NMDAR ligand signaling, could shape synaptic processing and contribute to the development of epilepsy.