We investigated the use of flavoprotein autofluorescence (FA) as a tool to map long-range neural connections and combined FA with laser-uncaging of glutamate to facilitate rapid long-range mapping in vitro. Using the somatosensory thalamocortical slice, we determined that the spatial resolution of FA is >or=100-200 microm and that the sensitivity for detecting thalamocortical synaptic activity approximates that of whole cell recording. Blockade of ionotropic glutamate receptors with DNQX and AP5 abolished cortical responses to electrical thalamic stimulation. The combination of FA with photostimulation using caged glutamate revealed robust long-distance connectivity patterns that could be readily assessed in slices from the somatosensory, auditory, and visual systems that contained thalamocortical, corticothalamic, or corticocortical connections. We mapped the projection from the ventral posterior nucleus of thalamus (VPM) to the primary somatosensory cortex-barrel field and confirmed topography that had been previously described using more laborious methods. We also produced a novel map of the projections from the VPM to the thalamic reticular nucleus, showing precise topography along the dorsoventral axis. Importantly, only about 30 s were needed to generate the connectivity map (six stimulus locations). These data suggest that FA is a sensitive tool for exploring and measuring connectivity and, when coupled with glutamate photostimulation, can rapidly map long-range projections in a single animal.