Carbenoxolone blocks the light-evoked rise in intracellular calcium in isolated melanopsin ganglion cell photoreceptors.

BACKGROUND: Retinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs). These ganglion cell photoreceptors send axons to several central targets involved in a variety of functions. Within the retina ipRGCs provide excitatory drive to dopaminergic amacrine cells via glutamatergic signals and ipRGCs are coupled to wide-field GABAergic amacrine cells via gap junctions. However, the extent to which ipRGCs are coupled to other retinal neurons in the ganglion cell layer via gap junctions is unclear. Carbenoxolone, a widely employed gap junction inhibitor, greatly reduces the number of retinal neurons exhibiting non-rod, non-cone mediated light-evoked Ca(2+) signals suggesting extensive intercellular coupling between ipRGCs and non-ipRGCs in the ganglion cell layer. However, carbenoxolone may directly inhibit light-evoked Ca(2+) signals in ipRGCs independent of gap junction blockade. METHODOLOGY/PRINCIPAL FINDINGS: To test the possibility that carbenoxolone directly inhibits light-evoked Ca(2+) responses in ipRGCs, the light-evoked rise in intracellular Ca(2+) ([Ca(2+)](i)) was examined using fura-2 imaging in isolated rat ipRGCs maintained in short-term culture in the absence and presence of carbenoxolone. Carbenoxolone at 50 and 100 µM concentrations completely abolished the light-evoked rise in [Ca(2+)](i) in isolated ipRGCs. Recovery from carbenoxolone inhibition was variable. CONCLUSIONS/SIGNIFICANCE: We demonstrate that the light-evoked rise in [Ca(2+)](i) in isolated mammalian ganglion cell photoreceptors is inhibited by carbenoxolone. Since the light-evoked increase in [Ca(2+)](i) in isolated ipRGCs is almost entirely due to Ca(2+) entry via L-type voltage-gated calcium channels and carbenoxolone does not inhibit light-evoked action potential firing in ipRGCs in situ, carbenoxolone may block the light-evoked increase in [Ca(2+)](i) in ipRGCs by blocking L-type voltage-gated Ca(2+) channels. The ability of carbenoxolone to block evoked Ca(2+) responses must be taken into account when interpreting the effects of this pharmacological agent on retinal or other neuronal circuits, particularly if a change in [Ca(2+)](i) is the output being measured.

A mouse M-opsin monochromat: retinal cone photoreceptors have increased M-opsin expression when S-opsin is knocked out.

Mouse cone photoreceptors, like those of most mammals including humans, express cone opsins derived from two ancient families: S-opsin (gene Opn1sw) and M-opsin (gene Opn1mw). Most C57Bl/6 mouse cones co-express both opsins, but in dorso-ventral counter-gradients, with M-opsin dominant in the dorsal retina and S-opsin in the ventral retina, and S-opsin 4-fold greater overall. We created a mouse lacking S-opsin expression by the insertion of a Neomycin selection cassette between the third and fourth exons of the Opn1sw gene (Opn1sw(Neo/Neo)). In strong contrast to published results characterizing mice lacking rhodopsin (Rho⁻/⁻) in which retinal rods undergo cell death by 2.5 months, cones of the Opn1sw(Neo/Neo) mouse remain viable for at least 1.5 yrs, even though many ventral cones do not form outer segments, as revealed by high resolution immunohistochemistry and electron microscopy. Suction pipette recordings revealed that functional ventral cones of the Opn1sw(Neo/Neo) mouse not only phototransduce light with normal kinetics, but are more sensitive to mid-wavelength light than their WT counterparts. Quantitative Western blot analysis revealed the basis of the heightened sensitivity to be increased M-opsin expression. Because S- and M-opsin transcripts must compete for the same translational machinery in cones where they are co-expressed, elimination of S-opsin mRNA in ventral Opn1sw(Neo/Neo) cones likely increases M-opsin expression by relieving competition for translational machinery, revealing an important consequence of eliminating a dominant transcript. Overall, our results reveal a striking capacity for cone photoreceptors to function with much reduced opsin expression, and to remain viable in the absence of an outer segment.

Immunotoxin-induced ablation of melanopsin retinal ganglion cells in a non-murine mammalian model.

In mammals, non-image-forming visual functions, including circadian photoentrainment and the pupillary light reflex, are thought to be mediated by the combination of rods, cones, and the melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs). Although several genetic models have been developed to clarify the individual roles of the rod, cone, and ipRGC systems in mediating non-image visual function, assessing the in vivo role(s) of the ipRGCs has been complicated by the possibility of ontogenetic issues in these genetically modified animal models. In the present study, we describe the development and validation of an immunotoxin that specifically targets the ipRGC population in the mature mammalian retina. This ipRGC immunotoxin, consisting of saporin conjugated to a melanopsin polyclonal antibody, was evaluated with respect to its effectiveness and specificity in depleting the ipRGC population in the fully developed rat retina. The results showed that the ipRGC toxin rapidly and permanently depleted approximately 70% of the ipRGC population, without inducing appreciable changes in the cell number or morphology of any of the non-melanopsin-containing retinal cell populations investigated. These findings suggest that the newly developed ipRGC immunotoxin provides a potent method for achieving relatively rapid, permanent, and selective depletion of the ipRGC population in a non-murine model system. The development of this ipRGC-ablation method is the next step in elucidating the role of ipRGCs in mediating non-visual and circadian light-resetting responses in a wide range of non-murine mammalian models.

Expressions of rod and cone photoreceptor-like proteins in human epidermis.

Previous reports have suggested the existence of photoreceptors for visible radiation at the surface of the human body. Rhodopsin is a well-known photosensitive protein found in the rod cells of the retina and detects light/dark contrast. Cone opsins are also photosensitive receptors in the cone cells of the retina and detect colour. Here, we describe immunochemical studies using anti-rhodopsin and anti-opsin antibodies on human skin. Both mouse retina and human epidermis showed clear immunoreactivity with each antibody. Interestingly, immunoreactivity against longer-wavelength opsin antibody was observed in the basal layer of the epidermis, while immunoreactivity against rhodopsin and shorter-wavelength opsin was observed in the upper layer. PCR analysis confirmed the expression of rhodopsin-like and opsin-like genes in human retina and the skin. These results suggest that a series of proteins, which play a crucial role in visual perception, are expressed in human epidermis.

Synaptic contact between melanopsin-containing retinal ganglion cells and rod bipolar cells.

PURPOSE: Evidence indicates that the melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs) receive input from rods and cones, which are thought to modulate the irradiance detecting system driving entrainment of the circadian system and pupillomotor control. This study was performed to identify retinal cells that have synaptic contact with ipRGCs. METHODS: Immunohistochemistry and high-power confocal microscopy were used to generate stacks of digital images of sections stained with antibodies against melanopsin, protein kinase C (PKCalpha), tyrosine hydroxylase (TH), presynaptic terminal markers (C-terminal binding protein 2 [CtBP2], vesicular monoamine transporter 2 [VMAT2] and postsynaptic marker (glutamate receptor subunit 4 [GluR4]). Results were analyzed in a computer-based three-dimensional reconstruction program for cellular contacts. RESULTS: Markers and melanopsin rod bipolar processes were found to have axosomatic and axodendritic contact with melanopsin-containing RGCs. Typically, three to four contacts were found on the soma of the melanopsin-containing RGCs, together with contacts on proximal dendrites. Contacts visualized by only CtBP2 immunoreactivity could also be demonstrated on melanopsin cell bodies and processes representing contacts with other types of bipolar cells. At the border of the inner plexiform layer (IPL) and inner nuclear layer (INL), where melanopsin processes stratify, contacts between melanopsin and TH or VMAT2 immunoreactivity processes were observed. CONCLUSIONS: Through confocal microscopy and computer-based three-dimensional analyses, this study demonstrates that melanopsin-containing RGCs have synaptic contact with PKC/CtBP2-containing rod bipolar cells and TH/VMAT2-immunoreactive amacrine cells through axodendritic and axosomatic contact, supporting electrophysiological observations that rods and cones signal to the melanopsin-containing intrinsically photosensitive RGCs.

The distribution of proliferating cell nuclear antigen-immunoreactive cells in the pineal organ of the rainbow trout Oncorhynchus mykiss.

Cell proliferation in the pineal organ of the immature rainbow trout Oncorhynchus mykiss was investigated by immunocytochemical demonstration of the proliferating cell nuclear antigen (PCNA) together with photoreceptor-specific opsin. Numerous PCNA-immunoreactive cells were found throughout the pineal end-vesicle and stalk. Two types of PCNA-immuno-reactive cells were distinguished: intensely stained, large ovoid and round cells, and mildly stained, slender fusiform cells. The ovoid type of the former cell was found often in the apical region and the round type in the basal region of the epithelium, while the latter fusiform cells were scattered through the apical and middle regions. Occasionally, close approaches were found between the opsin-immunoreactive photoreceptor outer segments and the PCNA-immunoreactive cells, which expressed mildly stained, nuclear and cytoplasmic signals. In addition, overlaps of the opsin-immunoreactive outer segments with the BrdU-labelled cells were occasionally found within the pineal epithelium. These findings suggest that the proliferation and neurogenesis of the pineal photoreceptor cells might persist also in the adult rainbow trout, thus maintaining highly sensitive, photo-signal transduction mechanisms for melatonin synthesis.

Exon-skipping variant of RGR opsin in human retina and pigment epithelium.

An extraneous exon-skipping mRNA encodes an altered form of a light-absorbing opsin in human retina and pigment epithelium (RPE). The predicted protein variant differs from full-length RPE-retinal G protein-coupled receptor (RGR) by having an in-frame deletion of exon 6, which contains the entire sixth transmembrane domain. To verify that the exon 6-deleted RGR protein (RGR-d) exists in human retinas, we have produced RGR-d antibody probes. In Western blot assays, the RGR-d protein was detected in retinas of a large proportion ( approximately 53%) of individual donors, including patients with age-related macular degeneration (AMD). The relative abundance of RGR-d varied significantly between individuals. The altered protein is expressed in RPE cells and has a more basal subcellular localization that is remarkably different from that of normal RGR opsin. The presence of this exon-skipping variant of RGR in humans may contribute to the progressive derangement of the RPE.

Retinal regional differences in photoreceptor cell death and regeneration in light-lesioned albino zebrafish.

Teleost fish regenerate retinal cells from a population of inner nuclear layer (INL) stem cells. To characterize photoreceptor regeneration in zebrafish (Danio rerio), adult albino fish were subjected to constant intense light to cause photoreceptor cell death. Retinal morphometry was performed on histological sections of control and light-lesioned albino retinas to compare the extent of light damage in the ventral, central and dorsal retinal regions. In addition, opsin immunohistochemistry and TUNEL were used to compare photoreceptor cell death in these different retinal areas, while PCNA immunolabeling quantified the cell proliferation that precedes the photoreceptor regeneration. Transgenic albino; Tg(alpha1-tubulin:egfp) zebrafish were also exposed to the intense light in order to examine regeneration-related gene expression changes. The light-lesioned retinas are characterized by extensive rod and cone photoreceptor cell death in the central and dorsal regions. In contrast, many of the rods and cones survive in the ventral retina. The highest levels of INL cell proliferation, which occurs subsequent to photoreceptor death, correspond to the retinal regions that suffer the greatest levels of photoreceptor damage. In the ventral retina, where photoreceptor cell death is minimal, cell proliferation is confined to the ONL. In addition, EGFP expression from the alpha1-tubulin promoter is increased in Müller glial cells in the light-damaged central and dorsal retina, while transgene expression in the ventral retina is restricted to small, round INL cells. Furthermore, expression of the HuC/D neuronal antigen is detected in a subpopulation of the Müller cells in the light-damaged superior retinal region. These data demonstrate that adult albino zebrafish display retinal regional differences in photoreceptor cell death and in the regeneration-related INL cell proliferation response. The high levels of INL cell proliferation and alpha1-tubulin:egfp transgene expression in the Müller cells may be graded in response to the degree of photoreceptor cell death. This suggests that the levels of photoreceptor damage may directly influence cell responses in the underlying retinal layers.

M opsin phosphorylation in intact mammalian retinas.

The deactivation of visual pigments involved in phototransduction is critical for recovering sensitivity after exposure to light in rods and cones of the vertebrate retina. In rods, phosphorylation of rhodopsin by rhodopsin kinase (GRK1) and the subsequent binding of visual arrestin completely terminates phototransduction. Although signal termination in cones is predicted to occur via a similar mechanism as in rods, there may be differences due to the expression of related but distinct gene products. While rods only express GRK1, cones in some species express only GRK1 or GRK7 and others express both GRKs. In the mouse, cone opsin is phosphorylated by GRK1, but this has not been demonstrated in mammals that express GRK7 in cones. We compared cone opsin phosphorylation in intact retinas from the 13-lined ground squirrel (GS) and pig, cone- and rod-dominant mammals, respectively, which both express GRK7. M opsin phosphorylation increased during continuous exposure to light, then declined between 3 and 6 min. In contrast, rhodopsin phosphorylation continued to increase during this time period. In GS retina homogenates, anti-GS GRK7 antibody blocked M opsin phosphorylation by 73%. In pig retina homogenates, only 20% inhibition was observed, possibly due to phosphorylation by GRK1 released from rods during homogenization. Our results suggest that GRK7 phosphorylates M opsin in both of these mammals. Using an in vitro GTPgammaS binding assay, we also found that the ability of recombinant M opsin to activate G(t) was greatly reduced by phosphorylation. Therefore, phosphorylation may participate directly in the termination of phototransduction in cones by decreasing the activity of M opsin.

Prenatal human ocular degeneration occurs in Leber's congenital amaurosis (LCA2).

BACKGROUND: Leber's congenital amaurosis (LCA) encompasses the most precocious and severe forms of inherited retinal dystrophy, displaying very significant visual handicap at or soon after birth. Among the currently identified mutations, alterations in the gene coding for retinal pigment epithelium 65-kDa protein (RPE65) lead to LCA2. Existing animal models for LCA2 (RPE65(-/-) null mice and naturally occurring RPE65(-/-) Briard dogs) exhibit near normal retinal histology at birth, although no recordable photofunction can be detected. Structural degeneration in both cases occurs with delayed onset, cone death generally preceding that of rods. METHODS: We obtained retinal tissue from a voluntarily aborted embryo of an LCA2 carrier in order to compare histopathology and immunohistochemistry with age-matched normal foetal retina. RESULTS: Compared to normal retinas, affected retina displayed cell loss and thinning of the outer nuclear (photoreceptor) layer, decreased immunoreactivity for key phototransduction proteins, and aberrant synaptic and inner retinal organisation. The gene mutation abolished detectable expression of RPE65 within the retinal pigment epithelium (RPE) of affected eyes, and ultrastructural examination revealed the presence of lipid and vesicular inclusions not seen in normal RPE. In addition, mutant eyes demonstrated thickening, detachment and collagen fibril disorganisation in the underlying Bruch's membrane, and the choroid was distended and abnormally vascularised, in comparison with controls. CONCLUSIONS: Such data contrast with the late-onset ocular changes observed in animal models, indicating caution should be exercised when inferring human retinal pathophysiology from information based on other species.

First identification and localization of a visual pigment in Hydra (Cnidaria, Hydrozoa).

The cnidarian Hydra does not possess identified photoreceptive structures or specialized cells for light detection; nevertheless, it shows a marked photosensitivity. So far no evidence has been previously reported about the localization of the proteins involved in the photoresponse. We used polyclonal antibodies and immunofluorescence microscopy on whole-mount Hydra to identify a putative rhodopsin-like protein. Our results show an immunoreactivity in the ectodermal layer of Hydra, which corresponds in position to the nervous epidermal sensory cells. These data provide the first identification of a rhodopsin-like protein in a phylogenetically old invertebrate and give a new insight into the Hydra photoreceptive response.

Immunohistochemical detection of retinal cones in monkey retina: light and electron microscopic study.

PURPOSE: Retinal cones have wave-length-specific visual pigments. To identify subclasses of cones, opsin-specific antibodies were previously established for the immunohistochemical studies of frozen sections. In this study, we produced retinal cone antibodies and examined (1) the specificity of these antibodies with Western blot analysis, (2) the application of these antibodies to paraffin-embedded monkey retinal sections, and (3) the use of these antibodies in light and electron microscopic immunohistochemical analyses of the localization of retinal cones. METHODS: The N-terminal peptide of blue opsin, and the C-terminal peptide of green/red opsin were used as immunogens in New Zealand White rabbits. Immunohistochemical staining was performed using the ABC method and immunogold method. As antigen retrieval treatment, paraffin-embedded cynomolgus monkey retinas were subjected to enzyme and microwave treatment. RESULTS: Both anti-red/green and anti-blue cone opsin antibodies detected 40 kDa native cone opsins in crude retina extract. The red/green and blue cone opsin immunostaining after trypsin treatment revealed that a positive signal was observed in the cone outer segment. Immunogold labeling also showed that gold particles were concentrated on the cone outer segment. CONCLUSION: In the antigen retrieval method, trypsin treatment is the appropriate method to obtain optimal staining of paraffin sections of retina. Using this method, retinal cone in conserved old paraffin sections can be identified immunohistochemically.

Rhodopsin transport in the membrane of the connecting cilium of mammalian photoreceptor cells.

The transport of the photopigment rhodopsin from the inner segment to the photosensitive outer segment of vertebrate photoreceptor cells has been one of the main remaining mysteries in photoreceptor cell biology. Because of the lack of any direct evidence for the pathway through the photoreceptor cilium, alternative extracellular pathways have been proposed. Our primary aim in the present study was to resolve rhodopsin trafficking from the inner to the outer segment. We demonstrate, predominantly by high-sensitive immunoelectron microscopy, that rhodopsin is also densely packed in the membrane of the photoreceptor connecting cilium. Present prominent labeling of rhodopsin in the ciliary membrane provides the first striking evidence that rhodopsin is translocated from the inner segment to the outer segment of wild type photoreceptors via the ciliary membrane. At the ciliary membrane rhodopsin co-localizes with the unconventional myosin VIIa, the product of human Usher syndrome 1B gene. Furthermore, axonemal actin was identified in the photoreceptor cilium, which is spatially co-localized with myosin VIIa and opsin. This actin cytoskeleton of the cilium may provide the structural bases for myosin VIIa-linked ciliary trafficking of membrane components, including rhodopsin.

Cone properties of retinal margin cells in the monkey (Macaca mulatta).

PURPOSE: To characterize a cell population in the monkey retinal margin that was labeled with a cone-specific antibody and to determine the presence of additional markers. METHODS: Retinal whole-mount preparations from infant and adult rhesus monkeys (Macaca mulatta) were immunolabeled by incubation overnight with the primary antibodies 7G6, a cone-specific antibody; SV2, a synaptic-vesicle antibody; and opsin antibodies that recognize either the short or long/middle wavelength-sensitive opsins. RESULTS: The retinal margin cells labeled by 7G6 lay within 1 mm of the ora serrata and differed from 7G6-labeled cones in the central retina. The margin cells possessed a soma, a fiber process, and a terminal enlargement that lay in the plane of the retina; no outer segment was discernible. A total of 5400 and 7252 margin cones cells were found in each of two monkeys. The terminal enlargement and soma of the labeled margin cells also showed SV2 immunoreactivity. Surprisingly, opsin immunoreactivity extended throughout the margin cell, which is consistent with the absence of a discernible outer segment. CONCLUSIONS: Cells with immunoreactive cone properties were found in the margin of the monkey retina. The absence of an outer segment and the presence of somatic opsin and SV2 are reminiscent of features observed in the central cones of fetal monkey retinas. These results suggest that a subpopulation of cones in the retinal margin might fail to mature completely and thus retain juvenile characteristics into adulthood.

Klebsiella pneumoniae 35 and 36 kDa porins are common antigens in different serotypes and induce opsonizing antibodies.

BACKGROUND: Klebsiella pneumoniae is a major cause of neonatal sepsis and nosocomial infections in Mexico. Antibiotic therapy is the first choice for treatment but the increase in multiple resistance strains has forced scientists to look for alternative treatments, such as immunotherapy. In this work, we propose that porins could be a common antigen among four different capsular serotypes of Klebsiella pneumoniae for the production of immune sera with opsonizing capacity. METHODS: The 35 and 36 kDa porins from four different serotypes of the bacteria were isolated by the Nikaido method followed by purification in Sephacryl column chromatography. The 36 kDa of serotype K8 was further purified by electroelution. The 35 and 36 kDa porins were used to obtain rabbit polyclonal antibodies (PolyAb) to the four serotypes and the 36 kDa from K8 for the production of monoclonal antibodies (MoAb). Antigenic reactivity of PolyAb and MoAb were analyzed by ELISA and WB and their opsonizing capacity for human PMN was measured by chemiluminescence (CL) using capsulated and non-capsulated bacteria. RESULTS: Porins from the four strains showe electrophoretic homology and cross reaction by ELISA and WB. CL assays indicated that PolyAb opsonized heterologous strains and that MoAb perform this in the absence of capsule. CONCLUSIONS: K.pneumoniae 35 and 36 kDa porins are common antigens for the four serotypes studied and induce opsonizing antibodies.

Absence of short-wavelength sensitive cones in the retinae of seals (Carnivora) and African giant rats (Rodentia).

Most non-primate mammals have two types of cone: short-wavelength sensitive (S) and middle-to-long-wavelength sensitive (M/L) cones. In two species of African giant rats, Cricetomys gambianus and C. emini, and in two species of earless seals, Phoca hispida and P. vitulina, the retinal cone types and cone distributions were assessed with antibodies specific for the M/L-cone opsin and the S-cone opsin, respectively. All four species were found to completely lack S-cones, while M/L-cones were present in low densities. M/L-cone densities, rod densities and cone/rod ratios were determined across the retina. Cone proportions are about 0.3-0. 5% in C. gambianus, 0.5-0.8% in C. emini, and 1.5-1.8% in P. hispida. An absence of S-cones has previously been reported in a few nocturnal mammals. As earless seals are visually active during night and day, we conclude that an absence of S-cones is not exclusively associated with nocturnality. The functional and comparative aspects are discussed.

Immunoreactive pinopsin in pineal and retinal photoreceptors of various vertebrates.

Pinopsin is a pineal specific opsin newly identified in the pineal of birds which has an absorption maximum at 470 nm. As the opsin content of photoreceptors in the pineal complex of several species is not yet known, in the present work, we studied their pinopsin immunoreactivity in various vertebrates from cyclostomes to mammals. We also compared the immunoreactivity of pineal photoreceptors to that of retinal cones and rods of each animal. For the immunocytochemistry, we raised antibodies in rabbits against a 14 amino acids containing part of the chicken pinopsin molecule. The immunoreaction was performed at the electron microscopic level. The pineal organs show a great diversity in vertebrates: there is a pineal organ present from cyclostomes to mammals, in addition, there is a parapineal organ in cyclostomes and fishes, a frontal organ in frogs and a parietal eye in several reptiles. We detected a strong pinopsin immunoreaction on most of the pinealocytes of birds and on the large photoreceptor-type of the pineal of reptiles. Rod-type photoreceptors of the avian retina and a cone of the reptile retina was immunoreactive as well. According to the known absorption maximum of pinopsin, the immunoreactivity may indicate a green-blue light-sensitivity for these photoreceptors. The immunoreactivity was less pronounced or absent in mammals as well as in less differentiated species. The pineal organ of snakes and the parietal eye of reptiles equally failed to exhibit pinopsin immunoreactive photoreceptors, presumably, due to the absence of green-blue light-sensitive photoreceptors of pinopsin-type in these species.

Cytophilic immunoglobulins revisited via natural killer cells.

Cytophilia is one of the biologic properties of the Fc region of immunoglobulins operationally defined as the ability of an antibody molecule to attach to certain cell types before combination with antigen. By attachment of cytophilic antibody to membrane Fc receptors, cells become "armed" with antibodies and able to interact specifically with soluble or particulate antigens. In contrast to this cytophilia, the so-called opsonic antibody binds to FcR of various cell types only after it has complexed with antigen. In spite of knowledge of cytophilic properties of immunoglobulins for more than 30 years and an impressive accumulation of facts regarding the structure and function of cell-surface FcR binding IgG, IgE, or IgA molecules, less progress has been made in understanding the molecular basis of cytophilia. Our extensive studies of the structural and functional aspects of the interaction of IgG in monomeric form with human natural killer cells, via the type IIIA IgG Fc receptor (Fc gamma RIIIA), provide a focal point for revisiting this major pathway of direct and continuous communication between the humoral and cellular compartments of the immune system.

The spatio-temporal pattern of photoreceptor degeneration in the aged rd/rd mouse retina.

Photoreceptor degeneration in the retina of the rd/rd (retinal degeneration) mice has been studied using immunocytochemistry with antisera against cone- and rod-opsin. The rd/rd mice exhibited different regional specific rates of degeneration for rods and cones. As early as postnatal day 25, cells labelled with the rod-opsin and cone-opsin antisera disappeared preferently from the central retina. Whereas in the inferior half of the retina, degeneration subsequently proceeded towards the periphery, this did not occur in the dorsal hemisphere. By the age of 100 days, many cells immunoreactive for the cone-opsin antiserum and a few cells immunoreactive for the rod-opsin antiserum were located in an area of the dorsal retina. The ventral retina lacked labelled elements at this age. Finally, rd/rd mice at one year or 600 days of age contained a similar number of cone-opsin immunopositive cells (approximately 2000-2800 cells), occupying almost the same area in the retina as that found at 100 days of age. A photoreceptor candidate for the entrainment of non-visual photoreception probably remains in the cone population in aged rd/rd mice.

Chlamyrhodopsin represents a new type of sensory photoreceptor.

In order to find optimal light conditions for photosynthetic growth, the green alga Chlamydomonas uses a visual system. An optical device, a rhodopsin photoreceptor and an electrical signal transduction chain that mediates between photoreceptor and flagella comprise this system. Here we present an improved strategy for the preparation of eyespot membranes. These membranes contain a retinal binding protein, which has been proposed to be the apoprotein of the phototaxis receptor. The retinal binding protein, which we named chlamyopsin, was purified and opsin-specific antibodies were raised. Using these antibodies, the opsin was localized in the eyespot region of whole cells during growth and cell division. The opsin cDNA was purified and sequenced. The sequence reveals that chlamyopsin is not a typical seven helix receptor. It shows some homology to invertebrate opsins but not to opsins from halobacteria. It contains many polar and charged residues and might function as a light-gated ion channel complex. It is likely that this lower plant rhodopsin diverged from animal opsins early in opsin evolution.