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Purpose: To compare the morphologic and functional recovery of the retina after detachment and reattachment in an animal with a cone-dominant retina, the ground squirrel., Methods: Ground squirrel (Spermophilus beecheyi) retinas were detached for 1 day and reattached for 7, 35, or 96 days (n = 2, each time point). Flicker ERGs were recorded 1 day after the detachment and at various times after reattachment. Contrast-response functions were measured for isochromatic modulation and for selective modulation of short-wavelength-sensitive (S) and middle-wavelength-sensitive (M) cones. At the end of the experiment, retinas were prepared for light microscopy or immunocytochemical staining with antibodies to rod opsin, S and M cone opsins, cytochrome oxidase, synaptophysin, glial fibrillary acidic protein (GFAP), cellular retinaldehyde-binding protein (CRALBP), interphotoreceptor-binding protein (IRBP), and peanut agglutinin lectin (PNA). Photoreceptor density maps were created from wholemount preparations labeled with biotinylated PNA and anti-S cone opsin. Cell counts of photoreceptor nuclei and cone outer segments (OS) were compared with flicker ERG data. Cell death was examined by the TUNEL method., Results: Reattachment stopped photoreceptor cell death and reversed the disruption of interphotoreceptor matrix as well as the redistribution of Müller cell proteins. It also activated some astrocytes based on anti-GFAP staining. S- and M-cone OS showed a gradual recovery in length after reattachment, and this recovery continued to the longest time points examined. ERG contrast gains also recovered after reattachment, but these reached asymptotic levels by approximately a week after reattachment. There were significant correlations between outer nuclear layer (ONL) cell counts and ERG contrast gains. No differences were noted in the indices of recovery of M and S cones., Conclusions: The ERG can be used to follow specifically the changes in the retina that occur after retinal detachment and reattachment.

Purpose: To develop methods for recording human electroretinogram (ERG) responses to stimuli that modulate different classes of cones in various ratios, to draw inferences about the combination of cone signal in early retinal processing., Methods: Subjects viewed large-field temporal modulations presented on a computer-controlled color monitor. A flicker photometric paradigm was used to equate the ERG response elicited by interleaved reference and test modulations. Test modulations were chosen to stimulate the L- and M-cones in various ratios. Results were obtained from color-normal subjects, dichromats, and an anomalous trichromat., Results: Reliable signals were obtained from all subjects to both L- and M-cone-isolating modulations and to intermediate modulations. Signals from color-defective subjects were predominantly determined by the modulation seen by only one cone type, whereas signals from color-normal subjects were sensitive to both L- and M-cone modulations. For most color-normal subjects, the recorded signal was a linear function of the contrasts seen by the L- and M-cones. There was individual variability in how strongly each cone type contributed to the overall signal., Conclusions: It is straightforward to record signals to color modulations presented on a CRT by using the flicker photometric ERG. For most observers, signals from L- and M-cones combine linearly. The relative contribution of the two cone classes varies across observers, probably because of individual differences in the relative numbers of L- and M-cones.

Purpose: Changes in retinal photopigments represent a fundamental step in the evolution of visual systems, in that addition of new pigment types or alterations in the spectral absorption properties of existing pigments modify visual capacities and thus open new visual worlds. To provide a tool that would allow direct examination of the changes caused by the presence of novel photopigments, this study was designed to determine whether a gene encoding a human cone photopigment introduced into the mouse genome would be expressed in a cone-specific manner and would support phototransduction., Methods: Mice transgenic for the human long wavelength-sensitive (L) photopigment were generated by microinjection of fertilized mouse eggs. RNA expression in different tissues was monitored by reverse transcription-polymerase chain reaction analysis. Photopigment protein was localized in retinal cross sections and wholemounts by antibody staining. Light transduction of the cone photopigments was assessed by flicker photometric electroretinography (ERG)., Results: The human transgene was expressed specifically in the mouse cones in quantities comparable to those of the mouse middle wavelength-sensitive (M) pigment gene. Immunocytochemical analysis showed that the human L pigment was abundantly synthesized in most mouse cones, was translocated to the outer segments, and caused no detectable cone degeneration. Electroretinographic spectral sensitivity analysis showed that the human L pigment was efficient in eliciting an electrical response. The degree of expression of the transgene in the two founders correlated well with the spectral responsivity of the ERG., Conclusions: The human L photopigment transduces light efficiently in mouse cones, implying that all protein domains necessary for efficient interaction with intracellular transport and signal transduction machineries in mouse cones have been conserved through evolution. The expression of the human L photopigment gene in both classes of cone of the mouse retina indicates that the transgene did not have the regulatory elements necessary for restricting its expression to mouse M cones or that such elements are not recognized in mouse UV-sensitive cones.

We identified and localized interphotoreceptor (or interstitial) retinoid-binding protein (IRBP) and cellular retinaldehyde-binding protein (CRALBP) in the cone-dominant retinas of diurnal squirrels. Western blots were prepared from sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) from whole retina, and from retina proximal and distal to the photoreceptor nuclei. Blots were incubated with purified rabbit IgG's specific for the bovine retinal antigens, and the labeled components were visualized using immunoperoxidase techniques. Anti-bovine IRBP and anti-bovine CRALBP recognized single components on gels of retinal supernatants that corresponded to the electrophoretic migration of the bovine antigens. The component recognized by anti-bovine IRBP on blots of outer retinal proteins (Mr 146,000) was absent on blots of inner retinal proteins. Twelve and 24 hr after intravitreal injection of 3H-L-fucose, electropherograms showed one major peak of radioactivity that coincided with the component recognized by anti-bovine IRBP. By immunoelectron microscopy, anti-bovine CRALBP labeling was restricted to the cytoplasm of both RPE and Muller cells, with light labeling of nuclear euchromatin in both cell types. In contrast, anti-bovine IRBP recognized antigenic sites primarily in the interphotoreceptor space (IPS). Intracellular labeling was limited to occasional granules in the photoreceptor myoids and the apical RPE cytoplasm. Extracellular labeling with anti-bovine IRBP was strongly associated with patches or small clumps of amorphous, electron opaque material distributed throughout the IPS. This material was particularly prominent near the cone outer segment plasma membranes, and was tentatively identified as the residual interphotoreceptor matrix that remained after exposure to the solvents used during tissue processing. In general, the results are consistent with those obtained in rod-dominant species. In addition, they imply that cones as well as rods are responsible for IRBP synthesis in the ground squirrel.

A comparison of electroretinogram (ERG) thresholds measured on rats reared in continuous darkness and under cyclic lighting conditions shows that by 30 days of age the dark-reared animals have achieved significantly lower thresholds than the animals reared under cyclic illumination. Ten days of continuous dark exposure produces this same increase in sensitivity in adult rats reared in cyclic lighting. These changes in sensitivity appear to reflect structural changes occurring within the rod outer segments.