Age-related changes in the tiger salamander retina

Tiger salamanders have been used in visual science because of the large size of their cells and the ease of preparation and maintenance of in vitro retinal preparations. We have found that salamanders over 27 cm in length show a variety of visual abnormalities. Compared to smaller animals (15-23 cm), large animals exhibited a decrease in visual responses determined by tests of the optomotor reflex. Small animals responded correctly an average of 84.5% of the time in visual testing at three light levels compared to an average of 68.4% for the large animals with the poorest visual performance at the lowest level of illumination. In addition, large animals contained (i) histological degeneration of the outer retina, in particular, loss and disruption of outer segments and abnormalities of the retinal pigmented epithelium, (ii) loss of cells, including photoreceptors, by apoptosis as evaluated with the TUNEL technique, and (iii) an increase in the number of macrophages and lymphocytes within the retina as determined by morphological examination. These histological changes were present in all large animals and all quadrants of their retinas. In contrast, small animals showed virtually no retinal degeneration, no TUNEL-positive cells, and few immune-like cells in the retina. Since large animals are also older animals. the visual changes are age-related. Loss of visual function and histological degeneration in the outer retina also typify aged human eyes. Thus, we propose that large salamanders serve as an animal model for age-related retinal degeneration. In addition to providing a source of aging retina that is readily accessible to experimental manipulation, the salamander provides a pigmented retina with a mixed (2:1, rod:cone) population of photoreceptors, similar to the degeneration-prone parafoveal region of the human eye.     

Median nerve neuropathy from an old lunate dislocation

We performed a histopathologic and immunohistochemical study of eyes obtained at autopsy of an 84-year-old man from a family with X-linked cone degeneration in which affected members have a 6.5-kilobase deletion in the red cone pigment gene. At his most recent ocular examination, at age 71 years, this patient had had a visual acuity of 20/200 OU, fundus changes suggestive of macular degeneration, borderline-normal full-field rod electroretinograms, and profoundly reduced full-field cone electroretinograms. Histopathologic examination demonstrated marked loss of cone and rod photoreceptors and the retinal pigment epithelium in the central macula. The peripheral cone population was reduced, while the peripheral rod population was relatively preserved. Immunohistochemical examination with an antibody to both red and green cone opsin and an antibody to blue cone opsin disclosed a prominent loss of the red and green cone population and preservation of the blue cone population. These findings show that a defect in the red cone pigment gene can result in extensive degeneration of the red and green cone population across the retina.     

Rod outer segment renewal in the retinae of deep-sea fish

Outer segment renewal involves the synthesis of disc material in the photoreceptor inner segments, the shedding of the tips of the photoreceptor outer segments, and their phagocytosis by the retinal pigment epithelial cells. It has been suggested that in the retinae of deep-sea fish no renewal of outer segments may take place. In order to assess outer segment renewal in deep-sea fish retinae we counted (i) periciliary vesicles in rod inner segments as a parameter for disc-synthesis activity and (ii) phagosomes in retinal pigment epithelial cells as a parameter of shedding and phagocytosis in 12 species of deep-sea fish with multibank or single bank retinae. We also measured the lengths of rod outer segments in order to evaluate the balance between synthesis and phagocytotic activity. In four of these species (Synaphobranchus kaupi, Nematonurus armatus, Coryphaenoides guentheri and Halosauropsis macrochir) we further recorded size-related changes of these parameters and their relation to the position of a given rod within the banks in the retina. The number of periciliary vesicles was highest in inner segments of the most vitread bank and in the periphery of the retina. Phagosomes were most abundant in retinal pigment epithelial cells of the central retina. Long rod outer segments were most frequently recorded in the peripheral retina indicating that in this region new synthesis may outbalance shedding. Vitread rod outer segments were only slightly longer than sclerad ones. Larger animals had shorter rod outer segments than small ones. We present evidence that rod outer segment renewal takes place in the retina of all deep-sea fish. Vitread rods may be more active in this respect than sclerad ones.     

ERG campimetry using a multi-input stimulation technique for mapping of retinal function in the central visual field

The m-sequence stimulation technique that has previously been described allows simultaneous recording of many local electroretinograms (ERGs). For topographical mapping of cone function in the central retina ERG traces from 61 retinal areas were recorded during a 4-min period in 20 normal volunteers and 4 patients with age-related macular degeneration, Stargardt's disease, choroidal atrophy, and polymyalgia associated with visual field loss. The local photopic luminance response was analyzed in each of the 61 areas. In the 2 patients with macular disease central defects were detected, in the remaining patients local defects were found outside the macular region. The method of multifocal ERG recordings provides the possibility of objective testing of visual fields when the outer retinal layers are affected.     

Localization of guanylate cyclase-activating protein 2 in mammalian retinas

Guanylate cyclase-activating proteins (GCAP1 and GCAP2) are thought to mediate the intracellular stimulation of guanylate cyclase (GC) by Ca2+, a key event in recovery of the dark state of rod photoreceptors after exposure to light. GCAP1 has been localized to rod and cone outer segments, the sites of phototransduction, and to photoreceptor synaptic terminals and some cone somata. We used in situ hybridization and immunocytochemistry to localize GCAP2 in human, monkey, and bovine retinas. In human and monkey retinas, the most intense immunolabeling with anti-GCAP2 antibodies was in the cone inner segments, somata, and synaptic terminals and, to a lesser degree, in rod inner segments and inner retinal neurons. In bovine retina, the most intense immunolabeling was in the rod inner segments, with weaker labeling of cone myoids, somata, and synapses. By using a GCAP2-specific antibody in enzymatic assays, we confirmed that GCAP1 but not GCAP2 is the major component that stimulates GC in bovine rod outer segment homogenates. These results suggest that although GCAP1 is involved in the Ca2+-sensitive regulation of GC in rod and cone outer segments, GCAP2 may have non-phototransduction functions in photoreceptors and inner retinal neurons.     

The eyes of deep-sea fish. II. Functional morphology of the retina

Three different aspects of the morphological organisation of deep-sea fish retinae are reviewed: First, questions of general cell biological relevance are addressed with respect to the development and proliferation patterns of photoreceptors, and problems associated with the growth of multibank retinae, and with outer segment renewal are discussed in situations where there is no direct contact between the retinal pigment epithelium and the tips of rod outer segments. The second part deals with the neural portion of the deep-sea fish retina. Cell densities are greatly reduced, yet neurohistochemistry demonstrates that all major neurotransmitters and neuropeptides found in other vertebrate retinae are also present in deep-sea fish. Quantitatively, convergence rates in unspecialised parts of the retina are similar to those in nocturnal mammals. The differentiation of horizontal cells makes it unlikely that species with more than a single visual pigment are capable of colour vision. In the third part, the diversity of deep-sea fish retinae is highlighted. Based on the topography of ganglion cells, species are identified with areae or foveae located in various parts of the retina, giving them a greatly improved spatial resolving power in specific parts of their visual fields. The highest degree of specialisation is found in tubular eyes. This is demonstrated in a case study of the scopelarchid retina, where as many as seven regions with different degrees of differentiation can be distinguished, ranging from an area giganto cellularis, regions with grouped rods to retinal diverticulum.     

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.     

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.     

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.     

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.     

Pathogenesis of neuroimmunologic diseases. Experimental models

Development of the retina, like that of other tissues, occurs via an orderly sequence of cell division and differentiation, producing the functional retina. In teleost fish, however, cell division and differentiation in the retina continue throughout the life of the animal in two distinct ways. Stem cells in a circumferential germinal zone at the periphery of the retina give rise to all retinal cell types and progenitor cells located throughout the retina in the outer nuclear layer (ONL) produce new rod photoreceptors. These processes in adult retina recapitulate in space the embryonic events responsible for forming the retina. Analysis of these events in an African cichlid fish, Haplochromis burtoni, confirmed that cone photoreceptors differentiate first, followed by rod photoreceptors. Correspondingly, at the margin of the eye, cone photoreceptors differentiate nearer to the margin than do rods. Control of photoreceptor production is not understood. Here we present the time of appearance and distribution pattern of GABA and vimentin which are candidates for the control of retinal cell division and differentiation. Antibody staining reveals that both GABA and vimentin exhibit unique patterns of expression during embryonic retinal development. Vimentin immunoreactivity is evident throughout the retina in a spoke-like pattern between developmental Days 4 and 7, as both cone and rod photoreceptors are being formed. GABA is expressed in horizontal cells between Days 5 and 7, corresponding to the onset of rod differentiation in time and in position within the retina. Moreover, the wave of GABAergic staining in the horizontal cells parallels the wave of rod differentiation across the embryonic retina of H. burtoni. Thus, GABA may play a role in the development of rod photoreceptors.     

Photoreceptor recovery in retinoid-deprived rats after vitamin A replenishment

Dietary deficiency in the retinoid precursors of the visual pigment chromophore 11-cis retinal results in the synthesis of photoreceptor outer segments containing opsin in excess of the vitamin A available for rhodopsin regeneration. This suggests that vitamin A-free opsin may be incorporated into newly synthesized outer segment disc membranes. If this opsin is functionally intact, it should be possible convert it to rhodopsin in vivo by providing the appropriate retinoids, and the resulting rhodopsin should should be able to mediate visual transduction. Experiments were conducted to evaluate this possibility and to identify the rate-limiting steps in photoreceptor recovery from retinoid depletion. Rates were maintained on diets either containing or lacking retinoid precursors of 11-cis retinal for 23 weeks, at which time outer segment opsin content greatly exceeded the availability of visual cycle retinoids in the retina. The retinoid-deprived animals were then each given a single intramuscular injection of all-trans retinol. At various time intervals after retinol administration, electroretinograms (ERGs) were recorded on some rats, and retinal rhodopsin contents were determined in others. At similar time intervals, blood and retinal pigment epithelial (RPE) retinoid levels and photoreceptor outer segment size were also determined. No significant increase in retinal rhodopsin content was observed up to 8 hr after injection, despite the fact that by 3 hr, blood retinol levels had recovered to more than 30% of normal. By 1 day after injection, however, rhodopsin levels had recovered to 30% of normal and ERG responses showed increases in visual sensitivity commensurate with the recovery of rhodopsin. The lag in rhodopsin recovery was apparently due to delayed uptake of retinol from the blood by the RPE. Photoreceptor outer segment size was reduced by over 50% in the retinoid- deprived rats and did not begin to recover by 1 day. By 1 week, however, outer segment size had returned to an average of 65% of normal. Commensurate with this regrowth of the outer segments, both rhodopsin levels and visual sensitivity increased between 1 and 7 days after vitamin A administration. Because the rates of recovery in rhodopsin levels and visual sensitivity greatly exceeded the normal rate of new opsin synthesis at short time intervals after vitamin A repletion, it appears that the opsin incorporated into the disc membranes of retinoid-deprived rats is able to form functional rhodopsin in vivo when the chromophore is supplied. Regrowth of the outer segments back to their normal size is required for full recovery of visual sensitivity.     

Neutron diffraction studies of retinal rod outer segment membranes

Neutron diffraction measurements on isolated retinal rod outer segments show that most of the visual pigment protein, rhodopsin, is embedded in the hydrophobic core of the disk membrane. A very slight outward shift of protein at the cytoplasmic side of the membrane is associated with pigment bleaching.     

The localization of guanylyl cyclase-activating proteins in the mammalian retina

PURPOSE: To explore the distribution of guanylyl cylase-activating proteins 1 and 2 (GCAP1 and GCAP2) in the mammalian retina. METHODS: Cryostat and vibratome vertical sections and wholemount retinas from mouse, rat, cat, bovine, monkey, and human eyes were prepared for immunocytochemistry and viewing by light and confocal microscopy. RESULTS: In all mammalian retinas investigated, intense GCAP1 immunoreactivity (GCAP1-IR) was seen in cone photoreceptor inner and outer segments, cell bodies, and synaptic regions. Intensity of the GCAP1-IR was strong in inner segments of rods in all species but weaker in outer segments-particularly so in primates and cats. GCAP2 immunoreactivity (GCAP2-IR) was weak in bovine, mouse, and rat cones but was intense in human and monkey cones. In all species except primates, GCAP2 staining was intense in rod inner and outer segments. In primates GCAP2-IR was intense in the rod inner segment but faint in the rod outer segment. A striking difference from the GCAP1 pattern of immunoreactivity was seen with GCAP2 antibodies as far as the inner retina was concerned. GCAP2-IR was evident in certain populations of bipolar, amacrine, and ganglion cells in all species. CONCLUSIONS: GCAP1 and GCAP2, which are involved in Ca2+-dependent stimulation and inhibition of photoreceptor guanylyl cyclase, can be detected in mammalian photoreceptor inner and outer segments, consistent with their physiological function. The occurrence of both GCAPs in the synaptic region of the photoreceptors indicates participation of these proteins in pathways other than regulation of phototransduction. The occurrence of GCAP2 in inner retinal neurons is indicative of second-messenger chemical transduction, possibly in metabotropic glutamate, gamma-aminobutyric acid (GABA) receptor, and nitric oxide-activated neural circuits.     

Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by alpha-toxin

Light triggers the phototransduction cascade by activating the visual pigment rhodopsin (Rho --> Rho*). Phosphorylation of Rho* by rhodopsin kinase (RK) is necessary for the fast recovery of sensitivity after intense illumination. Ca2+ ions, acting through Ca2+-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells. In this report, we describe an in vitro assay system using isolated retinas that is well suited for a variety of biochemical assays, including assessing Ca2+ effects on Rho* phosphorylation. Pieces of bovine retina with intact rod outer segments were treated with pore-forming staphylococcal alpha-toxin, including an alpha-toxin mutant that forms pores whose permeability is modulated by Zn2+. The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules <2 kDa but prevent the loss of proteins, including recoverin (25 kDa). The selective permeability of these pores was confirmed by using the small intracellular tracer N-(2-aminoethyl) biotinamide hydrochloride. Application of [gamma-32P]ATP to alpha-toxin-treated, isolated retina allowed us to monitor and quantify phosphorylation of Rho*. Under various experimental conditions, including low and high [Ca2+]free, the same level of Rho* phosphorylation was measured. No differences were observed between low and high [Ca2+]free conditions, even when rods were loaded with ATP and the pores were closed by Zn2+. These results suggest that under physiological conditions, Rho* phosphorylation is insensitive to regulation by Ca2+ and Ca2+-binding proteins, including recoverin.     

Retinal photoreceptor density decreases with age

PURPOSE: To evaluate the effect of age on the density of retinal photoreceptors in humans. METHODS: Fifty-five normal eyes from human donors, with a mean age of 58.7 +/- 19.1 years and an axial length of less than 27 mm, were examined. After opening of the globes by a 16-mm corneoscleral trephination, 25 retinal tissue samples were obtained in four meridians. The photoreceptors were counted on photographs taken from the photoreceptor inner segments after sonographic removal of the outer segments. The cones in the foveal center could not be evaluated. RESULTS: Outside the foveal center, the photoreceptor density decreased significantly with increasing age. In absolute and relative terms, the decline was more marked for the rods (mean rod loss, 236 +/- 63 cells or 0.37% +/- 0.10%/mm2 and year of a mean density of 62.987 rods/mm2) than for the cones (mean cone loss, 5.90 +/- 0.68 cells or 0.18% +/- 0.02%/mm2 and year of a mean density of 3320 +/- 578 cones/mm2). For both cones and rods, the cell loss was more marked at an eccentricity of approximately 5-8 mm than in the retinal periphery at an eccentricity of 14 to 20 mm. There were no significant differences between the superior, inferior, temporal, or nasal meridian nor between women and men. CONCLUSIONS: The photoreceptors decrease in density with increasing age. The annual cell loss of approximately 0.2% to 0.4% is similar to the age-related loss of retinal ganglion cells and pigment epithelium cells. The decline in photoreceptor count affects more rods than cones. It does not prefer special age groups. It is more pronounced at an eccentricity of 5 to 8 mm than in the retinal periphery at an eccentricity of more than 14 mm. It may be important for an age-related decrease in visual functions and for diseases affecting the photoreceptors in elderly patients.     

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.     

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.     

The photovoltage of macaque cone photoreceptors: adaptation, noise, and kinetics

Whole-cell voltage and current recordings were obtained from red and green cone photoreceptors in isolated retina from macaque monkey. It was demonstrated previously that the cone photovoltage is generated from two sources, phototransduction current in the cone outer segment and photocurrent from neighboring rods. Rod signals are likely transmitted to cones across the gap junctions between rods and cones. In this study, the "pure" cone and rod components of the response were extracted with rod-adapting backgrounds or by subtracting the responses to flashes of different wavelength equated in their excitation of either rods or cones. For dim flashes, the pure cone component was similar in waveform to the cone outer segment current, and the rod component was similar to the photovoltage measured directly in rods. With bright flashes, the high frequencies of the rod signal were filtered out by the rod/cone network. The two components of the cone photovoltage adapted separately to background illumination. The amplitude of the rod component was halved by backgrounds eliciting approximately 100 photoisomerizations sec-1 per rod; the cone component was halved by backgrounds of 8700 photoisomerizations sec-1 per cone. Coupling between rods and cones was not modulated by either dim backgrounds or dopamine. Voltage noise in dark-adapted cones was dominated by elementary events other than photopigment isomerizations. The dark noise was equivalent in magnitude to a steady light eliciting approximately 3800 photoisomerizations sec-1 per cone, a value significantly higher than the psychophysical estimates of cone "dark light."     

Myosin VIIa, the product of the Usher 1B syndrome gene, is concentrated in the connecting cilia of photoreceptor cells

Usher syndrome is the most common form of combined deafness and blindness. The gene that is defective in Usher syndrome 1B (USH1B) encodes for an unconventional myosin, myosin VIIa. To understand the cellular function of myosin VIIa and why defects in it lead to USH1B, it is essential to determine the precise cellular and subcellular localization of the protein. We investigated the distribution of myosin VIIa in human and rodent photoreceptor cells and retinal pigment epithelium (RPE), primarily by immunoelectron microscopy, using antibodies generated against two different domains of the protein. In both human and rodent retinae, myosin VIIa was detected in the apical processes of the RPE and in the cilium of rod and cone photoreceptor cells. Immunogold label was most concentrated in the connecting cilium. Here, myosin VIIa appeared to be distributed outside the ring of doublet microtubules near the ciliary plasma membrane. These observations indicate that a major role of myosin VIIa in the retina is in the photoreceptor cilium, perhaps in such a function as trafficking newly synthesized phototransductive membrane or maintaining the diffusion barrier between the inner and outer segments. Our results support the notion that defective ciliary function is the underlying cellular abnormality that leads to cellular degeneration in Usher syndrome.