We present a method to perform simultaneous microdialysis with light reflectance imaging of neural activity in a discrete brain region of the freely behaving animal. We applied this method to the dorsal hippocampus of freely behaving cats to (1) measure extracellular glutamate and reflectance variations across a sleep-waking cycle, (2) assess spatially coherent neural activity changes accompanying local perfusion of cocaine and (3) measure local changes in cell volume induced by infusion of hyper- and hypo-osmotic solutions. Higher extracellular glutamate concentrations corresponded to higher imaged neural activity. Sequential images showed that cocaine perfusion elicited a propagating reflectance change as cocaine reached the tissue. Microperfusion of hypo-osmotic solution (-100 mOsm), which increases cell volume, decreased reflectance. Microperfusion of hyperosmotic sucrose solutions, which reduce cell volume, increased reflectance in a dose-dependent manner. The data indicate that reflectance imaging can measure changes in cell volume, and could, thus, measure neural activity through activity/cell volume corollaries, Combining microdialysis and optical imaging enables investigation of the neurochemical bases of spontaneous neural activity patterns within discrete brain nuclei.