Immunocytochemical localization of the NMDA-R2A receptor subunit in the cat retina.

Immunocytochemical studies were performed to determine the distribution and cellular localization of the NMDA-R2A receptor subunit (R2A) in the cat retina. R2A-immunoreactivity (R2A-IR) was noted in all layers of the retina, with specific localizations in the outer segments of red/green and blue cone photoreceptors, B-type horizontal cells, several types of amacrine cells, Müller cells and the majority of cells in the ganglion cell layer. In the inner nuclear layer, 48% of all cells residing in the amacrine cell layer were R2A-IR including a cell resembling the GABAergic A17 amacrine cell. Interestingly, the AII rod amacrine cell was devoid of R2A-IR. Although the localization of the R2A subunit was anticipated in ganglion cells, amacrines and Müller cells, the presence of this receptor subunit to the cells in the outer retina was not expected. Here, both the R2A and the R2B subunits were found to be present in the outer segments of cone photoreceptors and to the tips of rod outer segments. Although the function of these receptor subunits in rod and cone photoreceptors remains to be determined, the fact that both R2A and R2B receptor subunits are localized to cone outer segments suggests a possible alternative pathway for calcium entry into a region where this cation plays such a crucial role in the process of phototransduction. To further classify the cells that display NR2A-IR, we performed dual labeling experiments showing the relationship between R2A-labeled cells with GABA. Results showed that all GABAergic-amacrines and displaced amacrines express the R2A-subunit protein. In addition, approximately 11% of the NR2A-labeled amacrines, did not stain for GABA. These findings support pharmacological data showing that NMDA directly facilitates GABA release in retina and retinal cultures [I.L. Ferreira, C.B. Duarte, P.F. Santos, C.M. Carvalho, A.P. Carvalho, Release of [3H]GABA evoked by glutamate receptor agonist in cultured chick retinal cells: effect of Ca2+, Brain Res. 664 (1994) 252-256; G.D. Zeevalk, W.J. Nicklas, Action of the anti-ischemic agent ifenprodil on N-methyl-d-aspartate and kainate-mediated excitotoxicity, Brain Res. 522 (1990) 135-139; R. Huba, H.D. Hofmann, Transmitter-gated currents of GABAergic amacrine-like cells in chick retinal cultures, Vis. Neurosci. 6 (1991) 303-314; M. Yamashita, R. Huba, H.D. Hofmann, Early in vitro development of voltage- and transmitter-gated currents in GABAergic amacrine cells, Dev. Brain Res. 82 (1994) 95-102; R. Ientile, S. Pedale, V. Picciurro, V. Macaione, C. Fabiano, S. Macaione, Nitric oxide mediates NMDA-evoked [3H]GABA release from chick retina cells, FEBS Lett. 417 (1997) 345-348; R.C. Kubrusly, M.C. deMello, F.G. deMello, Aspartate as a selective NMDA agonist in cultured cells from the avian retina, Neurochem. Intl. 32 (1998) 47-52] or reduction of GABA in vivo [N.N. Osborn, A.J. Herrera, The effect of experimental ischaemia and excitatory amino acid agonist on the GABA and serotonin immunoreactivities in the rabbit retina, Neurosci. 59 (1994) 1071-1081]. Since the majority of GABAergic synapses in the inner retina are onto both rod and cone bipolar axon terminals [R.G. Pourcho, M.T. Owzcarzak, Distribution of GABA immunoreactivity in the cat retina: A light and electron-microscopic study, Vis. Neurosci. 2 (1989) 425-435], we hypothesize that the NMDA-receptor plays a crucial role in providing feedback inhibition onto rod and cone bipolar cells.

Glutamate immunoreactivity in the cat retina: a quantitative study.

Immunocytochemical methods were used to visualize glutamate immunoreactivity in the cat retina and to compare its localization with that of aspartate, GABA, and glycine. The cellular and subcellular distribution of glutamate was analyzed at the light-microscopic level by optical densitometry and at the electron-microscopic level by immunogold quantification. The findings were consistent with the proposed role for glutamate as the neurotransmitter of photoreceptors and bipolar cells as particularly high concentrations of staining were found in synaptic terminals of these cells. Ganglion cells were also consistently stained. Aspartate was totally colocalized with glutamate in neuronal cell bodies but the synaptic levels of aspartate were much lower than for glutamate. In addition to the staining of photoreceptor, bipolar, and ganglion cells, glutamate immunoreactivity was also observed in approximately 60% of the amacrine cells. These cells exhibited colocalization with either GABA or glycine. The elevated levels of Glu in amacrine cells may reflect its role as a transmitter precursor in GABAergic cells and as an energy source for mitochondria in glycinergic cells.

Immunocytochemical evidence for the involvement of glycine in sensory centers of the rat brain.

Glycine-like immunoreactivity was localized to a number of sites in the rat brain which are involved in processing sensory information. In the auditory and vestibular systems, glycine immunoreactivity was seen in dorsal and ventral cochlear nuclei, superior olive, trapezoid body, medial and lateral vestibular nuclei, and inferior colliculus. Staining in the visual system was seen in retina, dorsal lateral geniculate nucleus, and superior colliculus. The olfactory system exhibited staining in the olfactory bulb and accessory olfactory formation. Somatosensory centers with glycine immunoreactivity included the dorsal column nuclei, spinal trigeminal nucleus, principal sensory nucleus of V, reticular formation, and periaqueductal gray. Glycine-immunoreactive neurons were also seen in cerebellar cortex, deep cerebellar nuclei, hippocampus, cerebral cortex, and striatum. The distribution of staining indicates that glycine plays a major role in sensory centers with actions at both strychnine-sensitive and strychnine-insensitive receptors.