IDENTIFICATION AND LOCALIZATION OF AN IMMUNOREACTIVE AMPA-TYPE GLUTAMATE-RECEPTOR SUBUNIT (GLUR4) WITH RESPECT TO IDENTIFIED PHOTORECEPTOR SYNAPSES IN THE OUTER PLEXIFORM LAYER OF GOLDFISH RETINA

L-glutamate, the main excitatory synaptic transmitter in the retina, i s released from photoreceptors and evokes responses in second-order re tinal neurons (horizontal, bipolar cells) which utilize both ionotropi c and metabotropic types of glutamate receptors. in the present study, to elucidate the functional roles of glutamate receptors in synaptic transmission, we have identified a specific ionotropic receptor subuni t (GluR4) and determined its localization with respect to photorecepto r cells in the outer plexiform layer of the goldfish retina by light a nd pre-embedding electron-microscopical immunocytochemistry. We screen ed antisera to mammalian AMPA hydroxy-5-methyl-4-isoxazolepropionate)- preferring ionotropic glutamate receptors (GluR 1-4) of goldfish retin a by light-and electron-microscopical immunocytochemistry. Only immuno reactive (IR) GluR4 was found in discrete clusters in the outer plexif orm layer. The cones contacted in this manner were identified as long- wavelength (''red'') and intermediate-wavelength (''green'') cones, wh ich were strongly immunoreactive to monoclonal antibody FRet 43 and an tisera to goldfish red and green-cone opsins; and short-wavelength ('' blue'') cones, which were weakly immunoreactive to FRet 43 but strongl y immunoreactive with antiserum to blue-cone opsin. Immunoblots of gol dfish retinal homogenate with anti-GluR4 revealed a single protein at M-r = 110 kDa. Preadsorption of GluR4 antiserum with either the immuni zing rat peptide, or its goldfish homolog, reduced or abolished staini ng in retinal sections and blots. Therefore, we have detected and loca lized genuine goldfish GluR4 in the outer plexiform layer of the goldf ish retina. We characterized contacts between photoreceptor cells and GluR4-IR second-order neurons in the electron microscope. IR-GluR4 was localized to invaginating central dendrites of triads in ribbon synap ses of red cones, semi-invaginating dendrites in other cones and rods, and dendrites making wide-cleft basal junctions in rods and cones; th e GluR4-IR structures are best identified as dendrites of OFF-bipolar cells. The results of our studies indicate that in goldfish retina Glu R4-expressing neurons are postsynaptic to all types of photoreceptors and that transmission from photoreceptors to OFF-bipolars is mediated at least in part by AMPA-sensitive receptors containing GluR4 subunits .