Fig 1.
Immunohistochemical analysis of the mouse retina using an anti-GluK5 antibody.
(A) Immunofluorescence analysis showing strong, punctate anti-GluK5 staining in the outer (arrowheads) and inner (open arrowheads) plexiform layer of the wild-type mouse retina. In the OPL (A’) the horseshoe-like appearance of the photoreceptor ribbons is clearly visible (arrowheads). (B) GluK5 immunolabeling does not colocalize with the presynaptic marker synaptophysin in the outer plexiform layer. High power photographs (B’) demonstrate adjacent localization (arrowheads). (C) Fluorescence immunostaining for CtBP2 and GluK5 in the outer plexiform layer. The horseshoe-like appearance of the photoreceptor synaptic ribbons colabels for CtBP2 and GluK5 and is clearly visible at higher magnification (arrowheads in C and C’). D) Double fluorescence immunolabeling for anti-GluK5 and anti-Calbindin, a marker for horizontal cells. The high-power photograph in D’ shows only very weak colocalization (arrowhead). (E) Double fluorescence immunolabeling with anti-PKC labeling rod bipolar cells and GluK5. GluK5 expression in the OPL does not colocalize with processes and terminals of rod bipolar cells (see higher magnification in E’). Scale bars: A—E: 10 μm; A’–E’: 2 μm.
Fig 2.
Ultrastructural localization of GluK5 in the outer and inner plexiform layer of the mouse retina.
(A and B) Electron micrograph of rod photoreceptor ribbons showing the ultrastructural localization of GluK5 immunoreactivity in rod spherules. Note the distribution of GluK5 immunoreactivity at the rod photoreceptor ribbon synapse covering the entire presynaptic ribbon structure (arrowheads). (C) Electron micrograph of a cone pedicle in the OPL showing GluK5 localization (arrowheads) in processes of OFF-cone bipolar cells. (D—F) Electron microscopic analysis of GluK5 distribution in the IPL. In the outer part of the inner plexiform layer GluK5 labeling is found in processes postsynaptic to OFF-cone bipolar cells (D). In the inner part of the inner plexiform layer labeling is seen in processes postsynaptic to ON-cone bipolar cells (E) and to rod bipolar cells (F). The arrows point to the presynaptic ribbon, the presynaptic terminals are surrounded with red dotted lines; the postsynaptic GluK5 positive elements are visualized with blue dotted lines. For better visualization in the electron micrographs presynaptic elements are visualized with blue and postsynaptic elements with red dotted lines. H = horizontal cell, RS = rod spherule, CP = cone pedicle, Off-CBP = off-cone bipolar cell, On-CBP = on-cone bipolar cell, RBP = rod bipolar cell. All scale bars: 100 nm.
Fig 3.
The absence of GluK5 disrupts the normal organization of presynaptic rod photoreceptor ribbons.
The electron micrographs show sections through rod ribbon synapses in the outer plexiform layer of wild-type (A) and GluK5-knockout retinae (B-D, G). Wild-type terminals (A) show normal synaptic architecture with a synaptic ribbon anchored to the presynaptic membrane and with postsynaptic elements formed by processes of horizontal and bipolar cells. (B-D) Rod terminals in adult GluK5-/- mice showing “disintegrated” synaptic ribbons. In some cases ribbon material seems to be free-floating within the rod terminal (B and D, arrowheads). (E –F) The synaptic ribbon structure is not affected at synapses in the IPL as demonstrated for ribbons of ON-cone (E) and OFF-cone (F) bipolar cells. The lower magnification (G) shows that most of rod photoreceptor ribbons are affected in the GluK5 knockout retina. (H) Histogram showing the number of ribbon fragments at rod photoreceptor terminals in GluK5 knockout and wild type animals. n = number of presynaptic elements analysed. ***P < 0,001 Mann-Whitney-U-Test. H = horizontal cell, B = Bipolar cell, Off-CBP = off-cone bipolar cell, On-CBP = on-cone bipolar cell. All scale bars: 250 nm.
Fig 4.
Electron microscopic analysis of synapses deficient in GluK5.
(A1–A8) Electron micrographs showing a series of ultrathin sections taken through two rod photoreceptor synaptic terminals of a GluK5-/- retina. The ultrastructural abnormalities in the course of the synaptic ribbons are clearly visible. In most sections the ribbons appear disintegrated (see arrowheads in A). (B1-B7) Electron micrographs showing a series of ultrathin sections taken through a rod photoreceptor synaptic terminal of a GluK5 knockout mouse. The ribbon and its fragments are visualized in green. For a better visualization the postsynaptic elements are colored in light and dark blue and the outline of the rod terminal is labelled in red. (C) Overlay of the colored green ribbon fragments. It is possible to follow the course of the ribbon through the matrix of the rod terminal. The overlay suggests that although the ribbon course is undulated its continuity might be conserved. (D and E) Immunohistochemistry for CtBP2 in wild-type and GluK5-/- retinae. (D) Typical horseshoe shaped ribbons are observed (open arrowheads). (E) GluK5 deficient retinae reveal to a high degree punctuated immunolabeling (arrowheads). (D’ and E’) Higher magnification of tagged frames in D (D’) and E (E’). Scale bars (A—C): 500 nm; D and E: 5 μm.