Full-field electroretinograms in individuals with the Laurence-Mood-Bardet-Biedl syndrome

PURPOSE: To evaluate rod and cone function in individuals with the Laurence-Moon-Bardet-Biedl syndrome. METHODS: We obtained a full-field electroretinograms in 36 patients. If responses less than 10 microV were recorded with single white flashes a special techniques with narrow band filter and computer averaging was used. RESULTS: No rod responses to dim blue light could be obtained in any of the patients. Residual cone flicker responses were measurable in 28 of the individuals. Those with amplitudes < 0.05 microV were significantly older than those with amplitudes > 1.00 microV. The ERG pattern was consistent within affected pairs of siblings in 8 families. CONCLUSION: The retinal dystrophy in Laurence-Moon-Bardet-Biedl syndrome is primarily a rod-cone dystrophy, but even cone flicker amplitudes are severely reduced with further progression with age. There is no intrafamilial variability of the electroretinograms in affected siblings.     

Identification and distribution of photoreceptor subtypes in the neotenic tiger salamander retina

The neotenic tiger salamander retina is a major model system for the study of retinal physiology and circuitry, yet there are unresolved issues regarding the organization of the photoreceptors and the photoreceptor mosaic. The rod and cone subtypes in the salamander retina were identified using a combination of morphological and immunocytochemical markers for specific rod and cone opsin epitopes. Because the visual pigment mechanisms present in the tiger salamander retina are well characterized and the antibodies employed in these studies are specific for particular rod and cone opsin epitopes, we also were able to identify the spectral class of the various rod and cone subtypes. Two classes of rods corresponding to the "red" and "green" rods previously reported in amphibian retinas were identified. In serial semithin section analyses, rods and cones comprised 62.4+/-1.4% and 37.6+/-1.4% of all photoreceptors, respectively. One rod type comprising 98.0+/-0.7% of all rods showed the immunological and morphological characteristics of "red" rods, which are maximally sensitive to middle wavelengths. The second rod subtype comprised 2.0+/-0.7% of all rods and possessed the immunological and morphological characteristics of "green" rods, which are maximally sensitive to short wavelengths. By morphology four cone types were identified, showing three distinct immunological signatures. Most cones (84.8+/-1.5% of all cones), including most large single cones, the accessory and principal members of the double cone, and some small single cones, showed immunolabeling by antisera that recognize long wavelength-sensitive cone opsins. A subpopulation of small single cones (8.4+/-1.7% of all cones) showed immunolabeling for short wavelength-sensitive cone opsin. A separate subpopulation of single cones which included both large and small types (6.8+/-1.4% of all cones) was identified as the UV-Cone population and showed immunolabeling by antibodies that recognize rod opsin epitopes. Analysis of flatmounted retinas yielded similar results. All photoreceptor types appeared to be distributed in all retinal regions. There was no obvious crystalline organization of the various photoreceptor subtypes in the photoreceptor mosaic.     

Nerve repair using freezing and fibrin glue: immediate histologic improvement of axonal coaptation

The chicken retina has several types of cone photoreceptor cells, each of which contains a visual pigment, chicken red (iodopsin), green, blue or violet. Although biochemical and photochemical properties of these cone pigments have been well characterized, no information is available about the chicken photoreceptor G-protein, transducin, which couples with the visual pigment to convert a photon signal into a cellular response. To identify alpha-subunits of chicken rod and cone transducins (Tr alpha and Tc alpha, respectively), we produced two site-directed antibodies which discriminate between bovine Tr alpha and Tc alpha. Immunohistochemical studies on chicken retinas revealed that the antibody against bovine Tr alpha specifically stained the rod outer segments. On the other hand, the antibody against bovine Tc alpha uniformly stained the outer segments of the double cones and all types of single cones, while the single cones were immunohistochemically classified into three types by using a combination of antibodies against bovine rhodopsin and chicken iodopsin. Immuno-blot analyses demonstrated that the antibody against Tc alpha recognized a single band of chicken photoreceptor protein, whose molecular weight (42,000) was in good agreement with that of bovine Tc alpha (41,000). The antibody against Tr alpha recognized a protein having the same molecular weight as that of bovine Tr alpha (39,000). These observations strongly suggested that all types of chicken cone cells have a single common Tc alpha (42 kDa) structurally related to bovine Tc alpha, though each cone cell type has a distinct visual pigment.     

Disease sequence from mutant rhodopsin allele to rod and cone photoreceptor degeneration in man

Mutations in the gene encoding rhodopsin, the visual pigment in rod photoreceptors, lead to retinal degeneration in species from Drosophila to man. The pathogenic sequence from rod cell-specific mutation to degeneration of rods and cones remains unclear. To understand the disease process in man, we studied heterozygotes with 18 different rhodopsin gene mutations by using noninvasive tests of rod and cone function and retinal histopathology. Two classes of disease expression were found, and there was allele-specificity. Class A mutants lead to severely abnormal rod function across the retina early in life; topography of residual cone function parallels cone cell density. Class B mutants are compatible with normal rods in adult life in some retinal regions or throughout the retina, and there is a slow stereotypical disease sequence. Disease manifests as a loss of rod photoreceptor outer segments, not singly but in microscopic patches that coalesce into larger irregular areas of degeneration. Cone outer segment function remains normal until >75% of rod outer segments are lost. The topography of cone loss coincides with that of rod loss. Most class B mutants show an inferior-nasal to superior-temporal retinal gradient of disease vulnerability associated with visual cycle abnormalities. Class A mutant alleles behave as if cytotoxic; class B mutants can be relatively innocuous and epigenetic factors may play a major role in the retinal degeneration.     

Multifocal rod electroretinograms

PURPOSE: To assess the feasibility of obtaining reliable multifocal rod electroretinograms (ERGS) and to compare them to full-field ERGs. METHODS: Multifocal rod ERGs were recorded using a stimulus array of 61 hexagons. The minimum number of dark, blank frames between flashes was varied from 0 (a minimum of 13.3 msec between flashes) to 21 (a minimum of 293 msec between flashes). Full-field ERGs were obtained using trains of flashes designed to simulate the multifocal sequences. Flashes were blue (W47B), except in a few cases in which red (W26) was used to check for cone intrusion. Flash intensities varied from -1 to 1.7 log scot td-s. RESULTS: Dark-adapted, multifocal ERGs to blue flashes had a small, early component followed by a larger, late component. The early component showed little change in amplitude with increasing intensity. Comparisons with the full-field ERGs indicated that the early component was the focal response. The larger, late component was the response to stray light, and it can be suppressed with the addition of a surround. The focal response was from a relatively circumscribed retinal region. This is shown by comparing the multifocal rod responses from a patient with retinitis pigmentosa to her behaviorally measured rod visual field. CONCLUSIONS: By choosing conditions (namely, flashes of moderate intensity with a surround) to minimize the effects of stray light, multifocal rod ERGs can be recorded with sufficient localization to be clinically useful. However, the signal-to-noise ratio of these multifocal rod ERGs was poorer than for multifocal cone responses for comparable recording periods because of the need for blank frames and the slower recovery of the rods to successive presentations.     

The effect of peripherin/rds haploinsufficiency on rod and cone photoreceptors

Haploinsufficiency because of a null mutation in the gene encoding peripherin/rds has been thought to be the primary defect associated with the photoreceptor degeneration seen in the retinal degeneration slow (rds) mouse. We have compared the effects of this haploinsufficiency on rod and cone photoreceptors by measuring the levels of rod- and cone-specific gene expression, by determining the relative rates of rod and cone degeneration, and by electroretinography. These analyses were performed at ages before and after the onset of degeneration of the photoreceptor cells. The data were consistent in demonstrating that measures for cone photoreceptors are relatively spared in comparison to comparable measures for rod photoreceptors. Blue cones were retained in higher number than red/green cones for the first 3 months of the degeneration. Our results indicate that the haploinsufficiency present in rds/+ mice has a greater impact on the rod than on the cone photoreceptor, a finding that likely reflects the tight regulation of peripherin/rds and the need for two functional alleles to assemble the structure of the rod outer segment and/or differences between the ultrastructure of the rod and cone outer segments.     

Red, green, and red-green hybrid pigments in the human retina: correlations between deduced protein sequences and psychophysically measured spectral sensitivities

To analyze the human red, green, and red-green hybrid cone pigments in vivo, we studied 41 male dichromats, each of whose X chromosome carries only a single visual pigment gene (single-gene dichromats). This simplified arrangement avoids the difficulties of complex opsin gene arrays and overlapping cone spectral sensitivities present in trichromats and of multiple genes encoding identical or nearly identical cone pigments in many dichromats. It thus allows for a straightforward correlation between each observer's spectral sensitivity measured at the cornea and the amino acid sequence of his visual pigment. For each of the 41 single-gene dichromats we determined the amino acid sequences of the X-linked cone pigment as deduced from its gene sequence. To correlate these sequences with spectral sensitivities in vivo, we determined the Rayleigh matches to different red/green ratios for 29 single-gene dichromats and measured psychophysically the spectral sensitivity of the remaining green (middle wavelength) or red (long wavelength) cones in 37 single-gene dichromats. Cone spectral sensitivity maxima obtained from subjects with identical visual pigment amino acid sequences show up to a approximately 3 nm variation from subject to subject, presumably because of a combination of inexact (or no) corrections for variation in preretinal absorption, variation in photopigment optical density, optical effects within the photoreceptor, and measurement error. This variation implies that spectral sensitivities must be averaged over multiple subjects with the same genotype to obtain representative values for a given pigment. The principal results of this study are that (1) approximately 54% of the single-gene protanopes (and approximately 19% of all protanopes) possess any one of several 5'red-3'green hybrid genes that encode anomalous pigments and that would be predicted to produce protanomaly if present in anomalous trichromats; (2) the alanine/serine polymorphism at position 180 in the red pigment gene produces a spectral shift of approximately 2.7 nm; (3) for each exon the set of amino acids normally associated with the red pigment produces spectral shifts to longer wavelengths, and the set of amino acids normally associated with the green pigment produces spectral shifts to shorter wavelengths; and (4) changes in exons 2, 3, 4, and 5 from green to red are associated with average spectral shifts to long wavelengths of approximately 1 nm (range, -0.5 to 2.5 nm), approximately 3.3 nm (range, -0.5 to 7 nm), approximately 2.8 nm (range, -0.5 to 6 nm), and approximately 24.9 nm (range, 22.2-27.6 nm).     

Difference in molecular properties between chicken green and rhodopsin as related to the functional difference between cone and rod photoreceptor cells

Using low-temperature spectroscopy, we have investigated the photobleaching process of chicken green, a green-sensitive cone visual pigment present in chicken retina, and compared it to that of rhodopsin, a rod visual pigment. Like rhodopsin, chicken green converts to all-trans-retinal and opsin through batho, lumi, and meta I, II, and III intermediates. However, all of the intermediates of chicken green except lumi, are less stable than the corresponding intermediates of rhodopsin. While early intermediates, batho and lumi are similar in absorption maxima between chicken green and rhodopsin, the meta intermediates of chicken green are about 20 nm blue shifted from those of rhodopsin. Low-temperature time-resolved spectroscopy was applied to estimate the thermodynamic properties of meta intermediates, and it indicated that the less stable properties of meta II and III intermediates of chicken green originate from the smaller activation enthalpies. The decay of the meta II intermediate of chicken green is greatly suppressed when a chicken green sample is irradiated at alkaline conditions while the net charge becomes similar to that of rhodopsin at neutral conditions. These results strongly suggest that the functional properties of chicken green that are different from those of rhodopsin are regulated by the dissociative amino acid residue(s).     

Photochemical and biochemical properties of chicken blue-sensitive cone visual pigment

Through low-temperature spectroscopy and G-protein (transducin) activating experiments, we have investigated molecular properties of chicken blue, the cone visual pigment present in chicken blue-sensitive cones, and compared them with those of the other cone visual pigments, chicken green and chicken red (iodopsin), and rod visual pigment rhodopsin. Irradiation of chicken blue at -196 degrees C results in formation of a batho intermediate which then converts to BL, lumi, meta I, meta II, and meta III intermediates with the transition temperatures of -160, -110, -40, -20, and -10 degrees C. Batho intermediate exhibits an unique absorption spectrum having vibrational fine structure, suggesting that the chromophore of batho intermediate is in a C6-C7 conformation more restricted than those of chicken blue and its isopigment. As reflected by the difference in maxima of the original pigments, the absorption maxima of batho, BL, and lumi intermediates of chicken blue are located at wavelengths considerably shorter than those of the respective intermediates of chicken green, red and rhodopsin, but the maxima of meta I, meta II, and meta III are similar to those of the other visual pigments. These facts indicate that during the lumi-to-meta I transition, retinal chromophore changes its original position relative to the amino acid residues which regulate the maxima of original pigments through electrostatic interactions. Using time-resolved low-temperature spectroscopy, the decay rates of meta II and meta III intermediates of chicken blue are estimated to be similar to those of chicken red and green, but considerably faster than those of rhodopsin. Efficiency in activating transducin by the irradiated chicken blue is greatly diminished as the time before its addition to the reaction mixture containing transducin and GTP increases, while that by irradiated rhodopsin is not. The time profile is almost identical with those observed in chicken red and green. Thus, the faster decay of enzymatically active state is common in cone visual pigments, independent of their spectral sensitivity.     

Histamine receptor-independent muscle relaxation elicited by a series of histamine H2-receptor agonists on the isolated guinea pig duodenum: a study into the mechanism of action

To assess the dolphin's capacity for color vision and determine the absorption maxima of the dolphin visual pigments, we have cloned and expressed the dolphin opsin genes. On the basis of sequence homology with other mammalian opsins, a dolphin rod and long-wavelength sensitive (LWS) cone opsin cDNAs were identified. Both dolphin opsin cDNAs were expressed in mammalian COS-7 cells. The resulting proteins were reconstituted with the chromophore 11-cis-retinal resulting in functional pigments with absorption maxima (lambdamax) of 488 and 524 nm for the rod and cone pigments respectively. These lambdamax values are considerably blue shifted compared to those of many terrestrial mammals. Although the dolphin possesses a gene homologous to other mammalian short-wavelength sensitive (SWS) opsins, it is not expressed in vivo and has accumulated a number of deletions, including a frame-shift mutation at nucleotide position 31. The dolphin therefore lacks the common dichromatic form of color vision typical of most terrestrial mammals.     

Spatial and temporal expression of cone opsins during monkey retinal development

The primate retina requires a coordinated series of developmental events to form its specialized photoreceptor topography. In this study, the temporal expression of cone photoreceptor opsin was determined in Macaca monkey retina. Markers for mRNA and protein that recognize short wavelength (S) and long/medium wavelength (L/M) opsin were used to determine (1) the temporal and spatial patterns of opsin expression, (2) the spatial relationship between S and L/M cones at the time of initial opsin expression, and (3) the relative time of cone and rod opsin expression (Dorn et al. [1995] Invest. Ophthalmol. Vis. Sci. 36:2634-2651). Adult cone outer segments were recognized by either L/M or S opsin antiserum. Of all adult cone inner segments, 88-90% contained L/M opsin mRNA, whereas 10-12% contained S opsin mRNA. Fetal cones initially showed cell membrane as well as outer segment labeling for opsin protein, but cell membrane labeling disappeared by birth. No cones at any age contained markers for both S and L/M opsin mRNA or protein. S and L/M opsin protein appeared in the fovea at fetal day 75. Once opsin expression progressed beyond the fovea, both mRNA and protein for S opsin were consistently detected more peripherally than L/M opsin. Cones at the peripheral edge of S opsin expression had basal telodendria that appeared to reach toward neighboring cones. Because interactions between cone populations could organize the cone mosaic, the spatial relationship between S cones and the first cones to express L/M protein was analyzed quantitatively by using double-label immunocytochemistry. No consistent relationship was found between these two cone populations. Cones are generated at least 1 week before rods across monkey retina. However, rod opsin protein appears in and around the fovea at fetal day 66, 1 week before cone opsin protein. This suggests that independent local factors control differentiation in these two photoreceptor populations.     

Switch in rod opsin gene expression in the European eel, Anguilla anguilla (L.)

The rod photoreceptors of the European eel, Anguilla anguilla (L.), alter their wavelength of maximum sensitivity (lambda max) from c.a. 523 nm to c.a. 482 nm at maturation, a switch involving the synthesis of a new visual pigment protein (opsin) that is inserted into the outer segments of existing rods. We artificially induced the switch in rod opsin production by the administration of hormones, and monitored the switch at the level of mRNA accumulation using radiolabelled oligonuleotides that hybridized differently to the two forms of eel rod opsin. The production of the deep-sea form of rod opsin was detected 6 h after the first hormone injection, and the switch in rod opsin expression was complete within four weeks, at which time only the mRNA for the deep-sea opsin was detectable in the retinal cells. It is suggested that this system could be used as a tractable model for studying the regulatory control of opsin gene expression.     

Origins and antiquity of X-linked triallelic color vision systems in New World monkeys

It is known that the squirrel monkey, marmoset, and other related New World (NW) monkeys possess three high-frequency alleles at the single X-linked photopigment locus, and that the spectral sensitivity peaks of these alleles are within those delimited by the human red and green pigment genes. The three alleles in the squirrel monkey and marmoset have been sequenced previously. In this study, the three alleles were found and sequenced in the saki monkey, capuchin, and tamarin. Although the capuchin and tamarin belong to the same family as the squirrel monkey and marmoset, the saki monkey belongs to a different family and is one of the species that is most divergent from the squirrel monkey and marmoset, suggesting the presence of the triallelic system in many NW monkeys. The nucleotide sequences of these alleles from the five species studied indicate that gene conversion occurs frequently and has partially or completely homogenized intronic and exonic regions of the alleles in each species, making it appear that a triallelic system arose independently in each of the five species studied. Nevertheless, a detailed analysis suggests that the triallelic system arose only once in the NW monkey lineage, from a middle wavelength (green) opsin gene, and that the amino acid differences at functionally critical sites among alleles have been maintained by natural selection in NW monkeys for >20 million years. Moreover, the two X-linked opsin genes of howler monkeys (a NW monkey genus) were evidently derived from the incorporation of a middle (green) and a long wavelength (red) allele into one chromosome; these two genes together with the (autosomal) blue opsin gene would immediately enable even a male monkey to have trichromatic vision.     

Rod influence on hue-scaling functions

Rod influence on hue appearance of spectral lights was characterized by comparing the scaling of red, green, yellow, and blue hue sensations for an 8 degrees-diameter, 7 degrees-eccentric test spot under conditions that minimized (cone plateau) and maximized (dark adapted) rod influence at two mesopic light levels (1.5 and 3.0 log scoptic trolands). At the lower light level, the hue-scaling functions showed that rod signals influenced the spectral range and magnitude of all four primary hues. The rod influence could not be characterized as a ubiquitous augmentation or diminution of any hue over the entire spectrum. This constrains models of rod influence on color vision.     

Mechanisms of spectral tuning in blue cone visual pigments. Visible and raman spectroscopy of blue-shifted rhodopsin mutants

Spectral tuning by visual pigments involves the modulation of the physical properties of the chromophore (11-cis-retinal) by amino acid side chains that compose the chromophore-binding pocket. We identified 12 amino acid residues in the human blue cone pigment that might induce the required green-to-blue opsin shift. The simultaneous substitution of nine of these sites in rhodopsin (M86L, G90S, A117G, E122L, A124T, W265Y, A292S, A295S, and A299C) shifted the absorption maximum from 500 to 438 nm, accounting for 2,830 cm-1, or 80%, of the opsin shift between rhodopsin and the blue cone pigment. Raman spectroscopy of mutant pigments shows that the dielectric character and architecture of the chromophore-binding pocket are specifically altered. An increase in the number of dipolar side chains near the protonated Schiff base of retinal increases the ground-excited state energy gap via long range dipole-dipole Coulomb interaction. In addition, the W265Y substitution causes a decrease in solvent polarizability near the chromophore ring structure. Finally, two substitutions on transmembrane helix 3 (A117G and E122L) act in combination with the other substitutions to alter the binding-pocket structure, resulting in stronger interaction of the protonated Schiff base group with the surrounding dipolar groups and the counterion. Taken together, these results identify the amino acid side chains and the underlying physical mechanisms responsible for a majority of the opsin shift in blue visual pigments.     

Photoreceptor loss in age-related macular degeneration

PURPOSE: The authors showed previously that parafoveal rods, but not cones, decrease during the course of adulthood in donor eyes that were screened to exclude the grossly visible macular drusen and pigmentary disturbances typical of age-related macular degeneration (AMD). Because AMD begins in the parafovea, this selective loss of rods actually may be subclinical AMD not yet visible in the fundus. If so, AMD must have a predilection for rods over cones. The authors tested this hypothesis by determining the relative numbers of cones and rods in donor eyes with mid-to late-stage AMD and in age-matched controls. METHODS: Thirteen eyes (from seven donors) with grossly visible macular drusen and pigmentary disturbances were either wholemounted for photoreceptor counts or sectioned through the fovea for histopathology and carbonic anhydrase histochemistry to label red-green cones. Eyes were assigned to AMD or control groups on the basis of histopathology and clinical history. RESULTS: Five nonexudative AMD (NE-AMD) eyes from three donors showed sparing of foveal cones and loss of rods and cones in the parafovea. In two donors, rod loss exceeded cone loss at most parafoveal locations, and in one donor, rod density was normal and cone density was reduced. In eight exudative AMD (EX-AMD) eyes from five donors, photoreceptors surviving along the margins of and overlying disciform scars were largely cones. CONCLUSIONS: Photoreceptors are lost in NE-AMD as well as in the more severe exudative form, consistent with functional and clinical studies. The authors propose that rods die in older eyes without evidence of overt retinal pigment epithelial disease. In persons susceptible to AMD, the retinal pigment epithelium becomes dysfunctional. Secondarily, rod loss continues and cones begin to degenerate. Eventually, only degenerate cones remain; ultimately, all photoreceptors may disappear.     

Role of hydroxyl-bearing amino acids in differentially tuning the absorption spectra of the human red and green cone pigments

The human red and green cone pigments differ at either 15 or 16 amino acids, depending upon which polymorphic variants are compared. Seven of these amino acid differences involve the introduction or removal of a hydroxyl group. One of these differences, a substitution of alanine for serine at position 180, was found previously to produce a 5 nm blue shift. To determine the role of the remaining six hydroxyl group differences in tuning the absorption spectra of the human red and green pigments, we have studied six site-directed mutants in which single amino acids from the green pigment have been substituted for the corresponding residues in the red pigment. Blue shifts of 7 and 14 nm were observed upon substitution of phenylalanine for tyrosine at position 277 and alanine for threonine at position 285, respectively. Single substitutions at positions 65, 230, 233, and 309 produced spectral shifts of 1 nm or less. These data are in good agreement with a model based upon sequence comparisons among primate pigments and with the properties of site-directed mutants of bovine rhodopsin. Nonadditive effects observed in comparing the absorption spectra of red-green hybrid pigments remain to be explained.     

Spectral tuning and molecular evolution of rod visual pigments in the species flock of cottoid fish in Lake Baikal

Lake Baikal in Eastern Siberia is the deepest and one of the largest and most ancient lakes in the world. However, even in the deepest regions, oxygenation levels do not fall below 75-80% of the surface levels. This has enabled a remarkable flock of largely endemic teleost fish of the sub-order Cottoidei to colonize all depth habitats. We have previously shown that species that occupy progressively deeper habitats show a blue shift in the peak wavelength of absorbance (lambda max) of both their rod and cone visual pigments; for the rod pigments, a number of stepwise shifts occur from about 516 nm in littoral species to about 484 nm in abyssal species. By sequencing the rod opsin gene from 11 species of Baikal cottoids that include representatives from all depth habitats, we have been able to identify four amino acid substitutions that would account for these shifts. The effect of each substitution on lambda max is approximately additive and each corresponds to a particular lineage of evolution.     

Myasthenia gravis as a prototype autoimmune receptor disease

We recorded full-field electroretinograms before and after vitrectomy in 18 eyes of 18 patients with idiopathic macular hole. The results were compared between affected and fellow eyes in the preoperative and early (within 10 days) and late (3 to 6 months) postoperative periods. No significant changes between affected and control eyes were found in the amplitude of the rod electroretinogram, mixed cone-rod electroretinogram, cone electroretinogram a- and b-waves or 30-Hz flicker electroretinogram in all stages of the study. The peak implicit times of the cone electroretinogram (a- and b-wave) and dark- and light- adapted oscillatory potential (O1-O4), however, were delayed. Also, the amplitude of the oscillatory potentials (O1+O2+O3+O4) was significantly reduced in the early postoperative period. By the late period, all of these changes had resolved. We concluded that electrophysiologic changes were derived from a transitory dysfunction of the inner retina. The possible causes of the electroretinographic changes would include mechanical trauma of the surgery, intravitreous air tamponade or the changes in vitreous electrolytes after surgery. Oscillatory potentials were more sensitive than a- and b-waves in eliciting dysfunction of the inner retina in operate on eyes.