We have previously demonstrated presynaptic kainate receptors in the spinal cord of rats using immunostaining with a GluR 5/6/7 antibody and weak aldehyde fixation (Hwang et al., 2001). To further characterize primary afferents that express presynaptic kainate receptors, we studied with confocal microscopy double and triple immunofluorescent staining for GluR 5/6/7 and markers for glutamatergic and GABA-ergic terminals, and for various classes of nociceptive endings of primary afferent fibers. Throughout the L4-L5 segments of the spinal cord, puncta immunofluorescent for GluR 5/6/7 were densest in the superficial laminae of the dorsal horn and co-stained extensively with presynaptic markers synaptophysin and vesicular glutamate transporter (VGluT1), but not with a GABA-synthesizing enzyme (GAD-65), indicating that presynaptic kainate receptors are expressed predominantly by terminals of primary afferents (for EM evidence, see SFN abstract by Lu et al., 2004). Furthermore, GluR 5/6/7 colocalized in the inne...