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.     

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.     

Characterization of gene expression, genomic structure, and chromosomal localization of Hells (Lsh)

We studied the development of multibank rod retinae by monitoring the size-related addition of new layers of rod inner and outer segments in four species of deep-sea fishes and found two different growth paradigms. In the mesopelagic Chauliodus sloani, new banks of rod inner and outer segments are added as long as the fish increases in size, as observed earlier by Locket (1980). By contrast, in three bathybenthic species (Antimora rostrata, Corvphaenoides (Coryphaenoides) guentheri, and Coryphaenoides (Nematonurus) armatus), the final complement of banks is reached when the specimens have grown to between 20 and 47% of their maximal size, suggesting that the visual system is mature only after this stage. Increase in retinal area, density of rod nuclei, and densities of rod inner and outer segments were also studied in these and additional species. Taken together with previous data on rod proliferation patterns and outer segment membrane synthesis, our findings indicate that at least in species with no continual addition of new banks, there is no major functional difference between the innermost and outermost banks of rod inner and outer segments. While Chauliodus spends all its life in the mesopelagic environment, the three bathybenthic species live in this environment during early development and descend towards greater depths only upon maturation. We speculate that this coincides with the stage when the full complement of rod banks is formed in the retina, as a possible prerequisite for a life outside the reach of sunlight.     

Immunomodulatory effects of linomide in animals immunized with immunopathogenic retinal antigens: dissociation between different immune functions

Linomide (LS-2616, quinoline-3-carboxamide) has been reported to exert a diverse range of effects on the immune system. On one hand, this drug was found to stimulate the immune system and to enhance activities such as DTH or allograft rejection. On the other hand, linomide was shown to inhibit the induction of experimental autoimmune encephalomyelitis and myasthenia gravis, as well as the development of diabetes in non-obese diabetic (NOD) mice. Here we report the effects of linomide in animals immunized with uveitogenic retinal antigens. Treatment with linomide completely inhibited the development of experimental autoimmune uveoretinitis (EAU) in mice immunized with interphotoreceptor retinoid-binding protein and markedly suppressed EAU in rats immunized with S-antigen (S-Ag). In addition, linomide-treated rats exhibited reduced antibody production and lymphocyte proliferative response to S-Ag. In contrast to these suppressive activities, linomide treatment did not affect the development of adoptively transferred EAU in rats and moderately enhanced the DTH reactions to S-Ag in immunized rats in which EAU and other immune responses to this antigen were suppressed.     

Glucose metabolism in photoreceptor outer segments. Its role in phototransduction and in NADPH-requiring reactions

Glucose metabolism in the photoreceptor rod outer segment produces both ATP (GTP) and NADPH to support phototransduction and NADPH-requiring processes in this organelle. Glycolysis in isolated bovine rod outer segments produces 44.0 +/- 6.4 nmol of ATP/min/mg of protein or 5.7 mM ATP/min. This rate of ATP production is more than sufficient to maintain the basal rate of cGMP synthesis (0.86 mM cGMP/min) in the dark requiring 1.7 mM ATP/min. Following photoexcitation, the 4.5-fold increase in the turnover of cGMP requires an ATP synthesis rate of up to 7.7 mM ATP/min (Ames, A., Walseth, T. F., Heyman, R. A., Barad, M., Graeff, R. M., and Goldberg, N. D. (1986) J. Biol. Chem. 261, 13034-13042). Under these conditions the rate of ATP production by glycolysis as measured in isolated rod outer segments is not sufficient for the regeneration of cGMP. Additional energy is most likely provided by the phosphocreatine shuttle which transports high energy phosphate groups in the form of creatine phosphate from the rod inner segment to the rod outer segment for conversion to ATP. The hexose monophosphate pathway in bovine rod outer segments can produce up to 39.8 +/- 2.2 nmol of NADPH/min/mg of protein. This rate of NADPH production is sufficient to support both the reduction of retinal to retinol (1.2 +/- 0.2 nmol of NADPH/min/mg of protein) following the photobleaching of rhodopsin and glutathione reduction (1.1 +/- 0.1 nmol of NADPH/min/mg of protein) for the protection of rod outer segments from oxidative damage. These studies provide insight into the contribution of anaerobic glycolysis and the hexose monophosphate pathway in providing energy and nucleotides for phototransduction and other outer segment processes.     

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.     

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.     

Epitope mapping of twelve monoclonal antibodies against the phenolic glycolipid-I of M. leprae

Epitope mapping of 12 monoclonal antibodies (MAbs) directed to the trisaccharide part of the phenolic glycolipid-I (PGL-I) of Mycobacterium leprae was carried out by using the set of chemically synthesized sugar-BSA conjugates. The results can be summarized as follows: mAb (1-21), mAb (1-24) and mAb (1-25) recognized the outer (nonreducing end) monosaccharide of the trisaccharide chain of PGL-I. However, the affinity of these MAbs to the outer monosaccharide was weak. They required the contributions of some parts of the second sugar for enough affinity. MAbs ml 6A12, ml 8A2, ml 8B2, and PG2 B8F recognized the outer disaccharide. MAb F47-21-3 recognized the outer disaccharide and some parts of the third sugar. MAb SF 1 recognized the trisaccharide of PGL-I. MAb 3D1-A9 recognized the phenol group and the structure around the branching point on the carrier protein in addition to the trisaccharide. MAbs DZ 1 and 2G3-A8 had unique characters which recognized the inner part of the sugar chain. MAb DZ 1 recognized the inner (reducing end) disaccharide. MAb 2G3-A8 recognized the inner monosaccharide, phenol group and the structure around the branching point on the carrier protein. All of the MAbs tested, except for ml 6A12, recognized the anomeric configurations in the sugar parts they recognized; ml 6A12 recognized the anomeric configuration only within the outer disaccharide. This set of MAbs, which were well defined on their binding specificity, promises to be an effective tool for the immunological study of PGL-I and the clinical assessment of leprosy.     

Molecular properties of the cGMP-gated channel of rod photoreceptors

The cGMP-gated channel of the rod photoreceptor cell plays a key role in phototransduction by controlling the flow of Na+ and Ca2+ into the outer segment in response to light-induced changes in cGMP concentrations. The rod channel is composed of two homologous subunits designated as alpha and beta. Each subunit contains a core region of six putative membrane spanning segments, a cGMP binding domain, a voltage sensor-like motif and a pore region. In addition the beta-subunit contains an extended N-terminal region that is identical in sequence to a previously cloned retinal glutamic acid rich protein called GARP. Three spliced variants of GARP (the GARP part of the beta channel subunit; full length free GARP; and a truncated form of GARP) are expressed in rod cells and localized within the outer segments. Immunoaffinity chromatography has been used to purify the channel from detergent solubilized rod outer segments. A significant fraction of the rod Na+/Ca(2+)-K+ exchanger copurifies with the channel as measured by western blotting suggesting that the channel can interact with the exchanger under certain conditions.     

Integrated Child Development Services Programme--need for reappraisal

BACKGROUND: Lipofuscin granules in the retinal pigment epithelium are lipid protein aggregates which are thought to represent the lifelong accumulation of the non-degradable end products from the phagocytosis of photoreceptor outer segments. Given the increasing evidence for a key role for vitamin A in the formation of ocular lipofuscin, the fluorophores generated by reacting vitamin A with lipid were assessed. METHODS: Reaction mixtures consisting of vitamin A (retinol) or its aldehyde (retinal) and (a) isolated rod outer segments, (b) the lipid extract of rod outer segments, (c) protein, or (d) liposomes were incubated at either pH 4.5 or 7.0 for up to 42 days. The fluorescence characteristics and mobility of the chloroform soluble fluorophores generated were compared with those extracted from purified human lipofuscin. Finally, the effect of lysosomal degradation on fluorophores generated in the above mixtures was assessed. RESULTS: Major spectral changes were observed when ROS or liposomes were incubated with retinal. These changes were pH dependent and did not occur if retinal was replaced with retinol. A number of the fluorophores generated exhibited similar fluorescence characteristics and chromatographic mobility to those of lipofuscin. Neither the presence of protein nor exposure to lysosomal enzymes had any effect on the spectral profile or fluorophore mobility of the fluorophores generated. CONCLUSIONS: These results suggest that some of the chloroform soluble fluorophores of lipofuscin are formed as a direct reaction product of retinal and lipid.     

Characterization of glucose transporter isoforms in the adult and developing human eye

The expression of glucose transporter isoforms (Glut 1, Glut 3, Glut 4, and Glut 5) in the human eye was investigated at various ages ranging between 8 weeks gestation (first trimester) and adult using Western blot and immunohistochemical analyses. Glut 1 and Glut 3 expression and cellular localization patterns were similar to those of human brain. Glut 1 (50-kilodalton protein) was expressed by epithelial cells (retinal pigmented epithelium, choroidal, iridial, and pars planus), which form the blood-eye barrier, retinal Mueller cells, the lens fiber cells, iridial microvascular endothelial cells, and to a lesser extent by the outer segments of the photoreceptor cells in the adult eye. This pattern was conserved throughout development and was evident as early as 8 weeks gestation. In addition, the endothelial cells of vitreous hyaloid vessels expressed Glut 1 at 8 weeks gestation. Glut 3 (50 to 55-kilodalton protein) immunoreactivity was observed only in the adult inner synaptic layer of the retina. Neither Glut 4 nor Glut 5 was expressed in any occular tissue at any age examined. These results suggest that Glut 1 is the main glucose transporter of the human eye and that it is ontogenically conserved. In contrast, Glut 3 is associated with selective neuronal processes, and its expression is developmentally altered.     

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.     

Developmental expression of the rat rod photoreceptor cGMP-gated cation channel

The appearance of cGMP-gated cation channel protein in the postnatal rat retina has been studied by fluorescence immunocytochemistry of radial retinal sections and immunoblots of retinal membrane proteins. Channel immunoreactivity was first detectable with RCNGC1-7H2 monoclonal antibody at postnatal day 7 (PN7) by both methods. Immunocytochemical label in retinal sections was localized to the outer segments, and immunoreactivity increased with increasing age. We also compared the developmental appearance of the cGMP-gated cation channel to that of other phototransduction proteins and developmental markers. RET-P2, a monoclonal antibody recognizing the 39-kDa rds/peripherin disc protein, first labeled outer segments at PN7, coincident with cGMP-gated cation channel expression. Double labeling of the same section of PN7 rat retina with RET-P2 and R309 (a polyclonal antiserum against the rod cGMP-gated cation channel) revealed identical patterns of labelling. Similarly, double labeling with RCNGC1-7H2 and an antibody against the rod cGMP-phosphodiesterase gave coincident labeling, suggesting coordinate expression mechanisms of phototransduction proteins with each other and with outer segment structural proteins.     

Rod outer segment maintenance is enhanced in the presence of bFGF, CNTF and GDNF

We employed a morphological assay of outer segment collapse to determine if growth factors or other supplements directly affect dissociated rod photoreceptors in vitro. The morphological changes in outer segments were correlated with the light responsiveness of rods. Time-lapse video microscopy was used to observe the collapse of rod outer segments from isolated single cells and small clumps of cells. A consistent pattern of outer segment collapse into the inner segment was observed, yielding a convenient assay of the effects of neurotrophic factors on photoreceptor functional maintenance. The functional state of rods, defined as light-responsiveness, was measured with suction electrode recordings and matched with the various stages of outer segment collapse. Ciliary neurotrophic factor (CNTF) and glial cell-line-derived neurotrophic factor (GDNF) at a high concentration, yielded statistically significant improvements in rat outer segment survival times. Basic fibroblast growth factor (bFGF), which rescues photoreceptors in several rodent models of retinal degeneration, produced a significant increase in survival time in the presence of the cofactor heparin. In 4 out of 10 cases using human tisue, bFGF also yielded a significant increase in survival times. When brain-derived neurotrophic factor (BDNF) was applied to rat rods, outer segment survival times did not change. Outer segments collapsed more quickly when either pigment epithelial cell derived factor (PEDF) or sugar N-acetyl D-galactosamine (NAD-gal) were present. Our results show that rod photoreceptors can respond to bFGF, GDNF and CNTF in vitro and provide evidence for a direct effect of these neurotrophic factors on rods. The rapid collapse of isolated photoreceptors in this model provides a convenient means for testing various neurotrophic agents and the induced cellular responses.     

Distribution of transforming growth factor-beta isoforms in the mammalian retina

The distribution of transforming growth factor-beta (TGF-beta) was examined in the posterior segment of the monkey, human, and feline eye using antisera to TGF-beta 1, TGF-beta 2, or TGF-beta 3. A number of different antibodies, tissue processing methods, immunolocalization techniques, and microscopic imaging systems were used in an attempt to gain a more comprehensive picture of TGF-beta isoform distribution in the retina and retinal pigmented epithelium (RPE). The results are generally consistent in identifying one or more of the three TGF-beta isoforms in the cytoplasm of a small, overlapping subset of cells. RPE cells, photoreceptors, Mueller cells, ganglion cells, hyalocytes, and cells associated with choroidal and retinal vessels are all represented in this immunoreactive population. No evidence of extracellular labeling was noted. The intracellular distribution of the three isoforms is distinctly different in photoreceptors. Anti-TGF-beta 1 precursor and anti-TGF-beta 2 immunoreactivity is confined primarily to rod outer segments, whereas anti-TGF-beta 3 immunoreactivities are restricted to mitochondria within inner segments. In the RPE, clusters of anti-TGF-beta 2 positive cytoplasmic granules are located near the cells' lateral borders, whereas anti-TGF-beta 3 labeling is concentrated apically. These results provide baseline information from which new hypotheses regarding the function(s) of TGF-beta isoforms in the retina can be formulated.     

Superoxide dismutases in retinal degeneration of WBN/Kob rat

PURPOSE: To examine the relationship between retinal degeneration and superoxide dismutase (SOD) in the degenerative retina of the WBN/Kob rat. METHODS: The retinas of 4-week-old and 10-month-old WBN/ Kob rats were examined with immunohistochemistry and immunoblotting. Wistar Kyoto rats were used as controls. RESULTS: Retinal degeneration began 4 weeks after birth. Four-week-old WBN/Kob rats showed no manganese superoxide dismutase (Mn-SOD) immunoreactivity in the photoreceptor inner segments or copper-zinc superoxide dismutase (CuZn-SOD) immunoreactivity in the outer nuclear layer or photoreceptor inner segments. Control 4-week-old Wistar Kyoto rats showed positive immunoreactivities at these sites. CONCLUSIONS: The retinal degeneration of WBN/Kob rats begins in the outer retina. The lack of SODs in the outer retina may contribute to retinal degeneration in the WBN/Kob rats.     

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.     

Progressive retinal atrophy in miniature longhaired dachshund dogs

A form of generalized progressive retinal atrophy unlike other previously recorded canine retinal dystrophies has been investigated in Miniature Longhaired Dachshund dogs. Segregation patterns in litters from matings involving affected individuals were consistent with simple autosomal recessive inheritance. The earliest ophthalmoscopic signs, appearing at approximately 6 months of age and coinciding in some cases with the onset of nyctalopia, included changes in the granular appearance of the tapetal fundus followed by generalized tapetal hyper-reflectivity and retinal vascular attenuation; later there was irregular loss of pigment in the non-tapetal fundus and optic atrophy. However, there was marked variation in the age of onset and progression of the disease, even within a single litter. The electroretinogram was normal in waveform and latency in four affected littermates at 10 weeks of age but by 9 months was markedly reduced in amplitude in two pups and virtually extinguished in the others. Significant histological changes at 10.5 weeks of age included thinning of the outer nuclear layer, irregularity and attenuation of the rod photoreceptor outer segments and early disorganization of the rod outer segment disc lamellae. By 25 weeks the photoreceptors were grossly degenerate with short rounded inner segments and only residual amounts of outer segment material remaining. This condition in the Miniature Longhaired Dachshund is later in onset than rod-cone dysplasia in Irish Setters but significantly earlier than progressive retinal atrophy in Tibetan Terriers and progressive rod-cone degeneration in, for example, Miniature Poodles. The condition could therefore serve as a potentially useful model for retinitis pigmentosa in man.     

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.