Interactions between separate synaptic inputs converging on the same target appear to contribute to the fine-tuning of information processing in the central nervous system. Intersynaptic crosstalk is made possible by transmitter spillover from the synaptic cleft and its diffusion over a distance to neighboring synapses. This is the case for glutamate, which inhibits gamma-aminobutyric acid (GABA)ergic transmission in several brain regions through the activation of presynaptic receptors. Such heterosynaptic modulation depends on factors that influence diffusion in the extracellular space (ECS). Because glial cells represent a physical barrier to diffusion and, in addition, are essential for glutamate uptake, we investigated the physiological contribution of the astrocytic environment of neurons to glutamate-mediated intersynaptic communication in the brain. Here we show that the reduced astrocytic coverage of magnocellular neurons occurring in the supraoptic nucleus of lactating rats facilitates diffusion in the ECS, as revealed by tortuosity and volume fraction measurements. Under these conditions, glutamate spillover, monitored through metabotropic glutamate receptor-mediated depression of GABAergic transmission, is greatly enhanced. Conversely, impeding diffusion with dextran largely prevents crosstalk between glutamatergic and GABAergic afferent inputs. Astrocytes, therefore, by hindering diffusion in the ECS, regulate intersynaptic communication between neighboring synapses and, probably, overall volume transmission in the brain.