Little is known about the functional connectivity between astrocytes in the CNS. To explore this issue we photo-released glutamate onto a single astrocyte in murine hippocampal slices and imaged calcium responses. Photo-release of glutamate causes a metabotropic glutamate receptor (mGluR)-dependent increase in internal calcium in the stimulated astrocyte and delayed calcium elevations in neighboring cells. The delayed elevation in calcium was not caused by either neuronal activity following synaptic transmission or by glutamate released from astrocytes. However, it was reduced by flufenamic acid (FFA), which is consistent with a role for adenosine triphosphate (ATP) release from astrocytes as an intercellular messenger. Exogenous ligands such as ATP (1 µM) increased the number of astrocytes that were recruited into coupled astrocytic networks, indicating that extracellular accumulation of neurotransmitters modulates neuronal excitability, synaptic transmission and functional coupling between astrocytes.