Functional imaging signals arise from metabolic and hemodynamic activity, but how these processes are related to the synaptic and electrical activity of neurons is not well understood. To provide insight into this issue, we used in vivo imaging and simultaneous local pharmacology to study how sensory-evoked neural activity leads to intrinsic optical signals (IOS) in the well-defined circuitry of the olfactory glomerulus. Odor-evoked IOS were tightly coupled to release of glutamate and were strongly modulated by activation of presynaptic dopamine and GABA-B receptors. Surprisingly, IOS were independent of postsynaptic transmission through ionotropic or metabotropic glutamate receptors, but instead were inhibited when uptake by astrocytic glutamate transporters was blocked. These data suggest that presynaptic glutamate release and uptake by astrocytes form a critical pathway through which neural activity is linked to metabolic processing and hence to functional imaging signals.