Ágoston Szél

Two different visual pigments in one retinal cone cell

The retina of the mouse, rabbit, and guinea pig is divided into a superior area dominated by green-sensitive (M) cones and an inferior area in which cones possess practically only short wavelength-sensitive (S) photopigments. The present study shows that the transitional zone between these retinal areas is populated by cones labeled by both the M and S cone photopigment-specific antibodies COS-1 and OS-2. It is concluded that the overwhelming majority of the transitional cones express both visual pigments. A small population of the transitional cones was strongly labeled exclusively by OS-2 (genuine S cones). The results indicate that, in contrast to the generally accepted idea of one visual pigment per one cone cell, cones of certain mammalian species can express different opsins simultaneously under natural conditions. We speculate that the coexpression may be due to the overlapping of regulatory factors determining the M and S fields.

Two cone types of rat retina detected by anti-visual pigment antibodies

The presence of two distinct cone types was demonstrated in the retina of the rat using two cone-specific monoclonal anti-visual pigment antibodies. Cones labelled by antibody COS-1 constituted the large majority (about 93%) of cones, and are most probably responsible for the green photopic sensitivity of the rat. About 7% of the cones were recognized by antibody OS-2, and are thought to be blue-sensitive elements. While OS-2 positive cones were evenly distributed throughout the retina, there were slight differences in the distribution of COS-1 positive cones. The cones made up about 0.85% of all photoreceptor cells. Although the OS-2 positive cones occur in a very low number (0.05% of all photoreceptors) and probably do not appreciably contribute to the photopic system of the rat, their presence in the rat strengthens the presumption that most mammalian species exhibit a dual cone system with a shortwave and a middle-to-longwave sensitivity.

Distribution of cone photoreceptors in the mammalian retina

The retina of mammals contains various amounts of cone photoreceptors that are relatively evenly distributed and display a radially or horizontally oriented area of peak density. In most mammalian species two spectrally different classes of cone can be distinguished with various histochemical and physiological methods. These cone classes occur in a relatively constant ratio, middle‐to‐longwave sensitive cones being predominant over short‐wave cones. Recent observations do not support the idea that each cone subpopulation is uniformly distributed across the retina. With appropriate type‐specific markers, unexpected patterns of colour cone topography have been revealed in certain species. In the mouse and the rabbit, the “standard” uniform pattern was found to be confined exclusively to the dorsal retina. In a ventral zone of variable width all cones express short‐wave pigment, a phenomenon whose biological significance is not known yet. Dorso‐ventral asymmetries have been described in lower vertebrates, matching the spectral distribution of light reaching the retina from various sectors of the visual field. It is not clear, however, whether the retinal cone fields in mammals carry out a function similar to that of their counterparts in fish and amphibians. Since in a number of mammalian species short‐wave cones are the first to differentiate, and the expression of the short‐wave pigment seems to be the default pathway of cone differentiation, we suggest that the short‐wave sensitive cone fields are rudimentary areas conserving an ancestral stage of the photopigment evolution.

Rom-1 is required for rod photoreceptor viability and the regulation of disk morphogenesis.

The homologous membrane proteins Rom-1 and peripherin-2 are localized to the disk rims of photoreceptor outer segments (OSs), where they associate as tetramers and larger oligomers. Disk rims are thought to be critical for disk morphogenesis, OS renewal and the maintenance of OS structure, but the molecules which regulate these processes are unknown. Although peripherin-2 is known to be required for OS formation (because Prph2−/− mice do not form OSs; ref. 6), and mutations in RDS (the human homologue of Prph2) cause retinal degeneration, the relationship of Rom-1 to these processes is uncertain. Here we show that Rom1−/− mice form OSs in which peripherin-2 homotetramers are localized to the disk rims, indicating that peripherin-2 alone is sufficient for both disk and OS morphogenesis. The disks produced in Rom1−/− mice were large, rod OSs were highly disorganized (a phenotype which largely normalized with age) and rod photoreceptors died slowly by apoptosis. Furthermore, the maximal photoresponse of Rom1−/− rod photoreceptors was lower than that of controls. We conclude that Rom-1 is required for the regulation of disk morphogenesis and the viability of mammalian rod photoreceptors, and that mutations in human ROM1 may cause recessive photoreceptor degeneration.

Monoclonal antibody-recognizing cone visual pigment

Monoclonal antibodies were raised to a crude photoreceptor-membrane suspension from chicken retinas. Clones producing antibodies against cone outer segments were selected by screening with immunocytochemistry on semithin sections of the retina. One monoclonal antibody, called COS-1, specifically labelled outer segments of double cones and one type of single cones; outer segments of rods and several single cones were not stained. On immunoblots of retinal photoreceptor membranes, this antibody recognized a protein with an apparent molecular mass of 33,000. The visual pigment character of the 33,000 protein was indirectly established by another monoclonal antibody, OS-2, which labelled all outer segments on semithin sections and four bands (33,000-, 36,000-, 38,000-40,000- and a composite band between 66,000-72,000 MW) on immunoblots. Of these, the 36,000- and the 72,000 MW protein bands were identified with an anti-rhodopsin polyclonal antibody as rhodopsin monomer and dimer. Monoclonal antibody OS-2 is assumed therefore to represent an antibody against a common epitope of all visual pigments of the chicken. The monoclonal antibody COS-1 was found to bind to certain cone outer segments of many other vertebrate species as well.

Photoreceptor distribution in the retinas of subprimate mammals.

Relevant data on the distribution of color cones are summarized, with special emphasis on the marked dorsoventral asymmetries observed in a number of mammalian species. In addition, an overview is given of studies that demonstrate the coexistence of two visual pigments within the same cone cell. The biological significance of these phenomena is discussed in conjunction with comparative immunocytochemical analyses of subprimate retinas. Based on various cone distribution patterns and temporal and spatial visual pigment coexpression, two models of cone photoreceptor differentiation are suggested.

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.

Short and mid-wavelength cone distribution in a nocturnal Strepsirrhine primate (Microcebus murinus).

Strepsirrhines are of considerable interest for understanding the evolution of cone photoreceptors because they represent the most ancestral living primates. The retina of nocturnal Strepsirrhines is reported to contain a single population of medium/long wavelength (MW/LW) cones whereas short wavelength (SW) cones are totally absent. The area centralis of nocturnal Strepsirrhines also lacks the degree of central specialization seen in the fovea of diurnal primates. In this study of a nocturnal Strepsirrhine, the gray mouse lemur (Microcebus murinus), we used specific antibodies that recognize SW and MW/LW opsins to determine the presence of different cone subtypes and their distribution in relation to that of rods and ganglion cells. The results are compared to two diurnal Haplorhine species, a New World (Callithrix jacchus) and an Old World (Macaca fascicularis) monkey. In the mouse lemur, both antibodies to MW/LW cone opsin (COS‐1 and CERN956) label the same population of cones. A small proportion of SW cones is only stained by the JH455 antiserum whereas the monoclonal OS‐2 antibody shows negative staining. These two antibodies label the same SW cone population in other primates. The extracellular matrix of all cones is also labeled by the peanut agglutinin (PNA) lectin. In mouse lemur retinal wholemounts, peak cone density is localized at the area centralis and ranged from 7,500 to 8,000 cones/mm2. SW cones represent less than 0.2 % of the total cone population and are mainly located in the nasal part of the retina. SW cones show an irregular distribution and densities never exceed 49 cones/mm2. The distribution of neurons in the ganglion cell layer shows a distinct centroperipheral gradient with a peak of 28,000 cells/mm2 at the area centralis. Rod distribution shows a centroperipheral gradient with the peak (850,000 rods/mm2) including and extending slightly dorsal to the area centralis. The theoretical spatial resolution of the mouse lemur (4.9 cycles/degree) is slightly lower to that of other nocturnal primates. The densities of rods, cones, and ganglion cell layer neurons represent a compromise between spatial resolution and sensitivity for both photopic and scotopic vision. J. Comp. Neurol. 438:494–504, 2001.

Binding sites of photoreceptor-specific antibodies COS-1, OS-2 and AO

The chicken red-sensitive cone visual pigment (iodopsin) and several synthetic peptides of cone and rod visual pigments were used to find the binding sites of our photoreceptor-specific antibodies with immunocytochemistry. The ability of iodopsin to block immunolabeling with monoclonal antibodies COS-1 and OS-2 furnished direct evidence that both antibodies are specific to visual pigments. Immunocytochemistry on whole-mount retinas with and without detergent, as well as electron microscopic labeling of cone photoreceptor membranes revealed the binding sites of COS-1 and OS-2 to be on the cytoplasmic side of the membrane. By testing several synthetic peptides, mainly from the C-terminal region of the cone visual pigments, we found that the domain consisting of the last 6 amino acids of the human red/green-, and the chicken red-sensitive cone pigments completely blocked immunolabeling with COS-1, while the sequence consisting of the last 12 amino acids of the human blue cone pigment was effective to block the binding of OS-2. Both monoclonals can be regarded therefore C-terminal specific antibodies. OS-2 was found to bind to the dark-adapted photopigment more strongly than to the light-adapted one. The binding of the polyclonal rhodopsin antibody AO was almost entirely inhibited by the N-terminal synthetic peptide of bovine rhodopsin indicating that this antibody binds primarily to the N-terminal domain of rhodopsin in a tissue environment.

Cones in the retina of the Mongolian gerbil,Meriones unguiculatus: an immunocytochemical and electrophysiological study

Immunocytochemistry revealed in the retina of the Mongolian gerbil three immunologically distinct photoreceptor cell types. Rods comprising about 87% of the total receptor population were selectively recognized by an antirhodopsin serum (AO). The most abundant cone type (11-13% of photoreceptors) was labeled by the monoclonal antibody COS-1, specific in mammals to the middle-to-long-wave sensitive cone visual pigments. A minor cone population (2.5-5% of the cones) reacted with the monoclonal antibody OS-2, shown earlier to bind to the blue cones in mammalian species. Color substitution experiments revealed on the ERG level a color discrimination capability which must be attributed to the cooperative activity of green-sensitive (COS-1 positive) and blue-sensitive (OS-2 positive) cones. We conclude that the Mongolian gerbil has a well developed cone system, and that it may possess dichromatic green-blue color vision.