The present study was performed as part of a systematic examination of glycine's coexistence with other classical transmitters and neuropeptides in neuronal populations of the larval tiger salamander retina. Substance P immunocytochemistry was combined with either glycine immunocytochemistry or autoradiography of glycine high-affinity uptake to examine whether tiger salamander substance P-amacrine cells express these glycine markers. Double-label analyses revealed two populations of substance P-amacrine cells that express glycine immunoreactivity and glycine high-affinity uptake. The large majority of double-labeled cells were situated in the innermost cell row of the inner nuclear layer, while a smaller number were located in the inner nuclear layer in the second cell row distal to the inner plexiform layer. Double-label immunocytochemistry revealed that these double-labeled cells accounted for 91.7% of substance P-immunoreactive amacrine cells. A slightly lower percentage (90.1%) of substance P-amacrine cells were found to exhibit a glycine high-affinity uptake mechanism. Substance P-amacrine cells that did not co-label for markers of glycine activity were situated in the innermost cell row of the inner nuclear layer. Substance P-immunoreactive displaced amacrine cells were not observed to co-label for either glycine immunoreactivity or glycine high-affinity uptake.

The present study reveals that the large majority of substance P-amacrine cells in the larval tiger salamander retina co-express markers of glycine activity. This finding suggests a functional diversity in the population of tiger salamander substance P-amacrine cells relative to their coexisting relationship with a major inhibitory neurotransmitter.