Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

Rod–Cone Pathway

A consequence of the high sensitivity of the rod bipolar pathway is that it saturates at modest light levels where the rods themselves are not saturated. To capture light-evoked signals from rods until they themselves saturate additional pathways are required. Studies of the ultrastructure of the outer plexifom layer reveal that gap junctions exist between rods and rods, and rods and cones. The nature of the rod-to- rod gap junction is unclear, but has been proposed to dissipate small rod signals into the network allowing signal averaging at the cost of a twofold elevation in visual threshold. The rod-to-cone gap junctions have been studied in more detail and are found in a majority of rods. Evidence points to the expression of the gap junction subunit connexin 36 in cones, but not in rods. Thus, the electrical gap junctions between rods and cones must be heterologous. Given the lower input impedance of cones, signal transfer will preferably travel from rods to cones, which would allow these signals to be relayed to ganglion cells through the cone circuitry.

Physiological recordings have indicated robust rod signals in the cone photoreceptors of several species. In particular, measurements from dark-adapted retina indicate that rod-to-cone coupling must exist, going against the idea that, to maximize visual sensitivity, the rods and cones should be uncoupled in darkness. Recordings from mouse retinal whole mounts suggest that the signals through this pathway are 10-fold less sensitive than for the rod bipolar pathway. These physiological data correspond well with psychophysical experiments indicating a secondary rod pathway with a 10-fold lower sensitivity than the primary pathway.

Rod–OFF Pathway

A third rod pathway has been identified in some species of mammals. Under conditions where the rod–bipolar and rod–cone pathways are blocked or eliminated, OFF signals with rod sensitivity have been shown to persist in the

mouse retina. Direct synaptic contacts have been identified anatomically in mice, rats, and rabbits. However, direct contacts between rods and OFF bipolar cells have not yet been reported in higher mammals. It is possible that an OFF rod pathway evolved as a specialization in nocturnal rodents to sense dark objects against a light sky.

The rod–OFF pathway is believed to be active at 5–10-fold higher light intensities than the rod bipolar pathway. Its prominence as a signaling circuit in rodents appears to vary based on the percentage of the OFF cone bipolar cell population that sees direct signals from rods. It is now believed that between 5% and >25% of cone OFF bipolar cells may see direct contacts from rods, the upper end suggesting that this pathway may play an integral role in visual processing.

Despite attempts by various groups to characterize the physiological response properties of the rod pathways, the exact sensitivities and operating ranges of each pathway remain unclear. The lack of physiological evidence elucidating the role of these pathways in rod vision arises from common signaling mechanisms used by each. Thus, genetic or pharmacological manipulation of each rod-signaling pathway will also influence other pathways, making it impossible to unambiguously identify the properties of each. Nevertheless, the evidence now points to the rod bipolar pathway as the primary carrier of singlephoton responses near absolute visual threshold, with an5–10-fold reduction in the sensitivity of the rod–cone and rod–OFF pathways that may carry signals at higher light levels where cone function is merged.

Cone Pathways

Our understanding of the cone pathways is far less complete than our understanding of rod pathways in the mammalian retina. The physiological properties of the cones and cone pathways remain as one of the frontiers in retinal neurobiology, especially as cone function dominates our visual experience. To date, as many as nine types of cone bipolar cells have been identified in mammalian retinas, which may connect to as many as 10–15 types of retinal ganglion cells. Other than the specific pathway for S-cones, there is presently little understanding of specific circuits that carry cone responses to ganglion cells.

Adaptation to Mean Background Light

A common feature of all sensory systems is the ability to adapt to increases in the mean level of a stimulus by reducing the gain of the system. Such sensitivity adjustments allow the sensory system to remain maximally responsive as the stimulus intensity changes. Both rods and cones in the retina, as well as their circuitry,

exhibit adaptive mechanisms that are designed to increase the dynamic range of the receptor. However, in the context of the dynamic range of the visual system, the influences of adaptation on the lower limit of scotopic and upper limit of photopic vision are opposite. To retain maximal sensitivity near absolute visual threshold, the retina must maximize its gain for the single-photon response and any adaptive mechanism would allow the rod circuitry to aid in the transition from scotopic to mesopic vision. Conversely, the upper light limits of our visual experience are ultimately defined by adaptation in the cone photoreceptors, which continue to operate even when a majority of the photopigment is bleached.

Adaptation: Rod Pathways

Adaptation within a neural circuit with considerable convergence will begin centrally and move peripherally. Under these circumstances, downstream cells will be the first to detect sufficient signal to adapt for the weakest stimuli, and the rod pathways are no exception. In the rod bipolar pathway, weak background light begins to reduce the gain of ganglion cells and AII amacrine cells before adaptation is detectable in rod bipolar cells or rod photoreceptors. Some mechanisms that provide this gain reduction have been identified, particularly at the rod-to-rod bipolar, and the rod bipolar-to-AII amacrine synapses. For instance, at the rod-to-rod bipolar synapse, the influx of Ca2þ through mGluR6 transduction channels reduces the bipolar cell gain for subsequent stimulation. In addition, at the rod bipolar-to-AII amacrine synapse depression mediated by depletion of the available pool of vesicles can be evoked at individual synapses by single-photon responses. These adaptive mechanisms allow the rod bipolar pathway to extend its range to higher light levels where they merge with the other rod pathways. However, the extension of the dynamic range of vision to lower light levels requires that the retina remains maximally responsive to single photons, and thus these adaptive mechanisms would impair absolute threshold.

Adaptation: Cone Pathways

It has been well documented that adaptation of ganglion cell responses in the cone pathways occurs at lower light levels than where adaptive features of cone pathways have been documented. Cone and horizontal cell recordings have demonstrated that cone adaptation is observed at light levels that exceed those required for the adaptation of cone-driven signals in ganglion cells. This adaptation of the postsynaptic cone circuitry, in turn, prevents these pathways from saturating, thereby allowing the extension of the cone operating range through mesopic to photopic vision. Adaptation, both in the cones and in the postreceptoral circuitry, have been found to be mutually

exclusive. Ultimately, the upper limits of cone vision are directly imposed by receptoral adaptation of the cones themselves and not by the postsynaptic circuitry.

Conclusions

The vertebrate retina has developed many strategies to maximize the dynamic range of vision. At the initial stages of light detection the evolution of two photoreceptor types, the rods and cones, allows the visual system to signal a wide range of light intensities. By dividing the output of these receptors across many retinal pathways, each of which is subject to its own optimization and adaptation, the human eye is capable of providing the brain with information that extends the range of vision to encompass approximately 12 orders of magnitude of light intensity.

See also: Anatomically Separate Rod and Cone Signaling Pathways; Information Processing: Bipolar Cells; Information Processing: Ganglion Cells; Morphology of Interneurons: Bipolar Cells; Phototransduction: Adaptation in Cones; Phototransduction: Adaptation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Rod Photoreceptor Cells: Soma and Synapse.

Further Reading

Barlow, H. B. (1956). Retinal noise and absolute threshold. Journal of the Optical Society of America 46: 634–639.

Barlow, H. B., Levick, W. R., and Yoon, M. (1971). Responses to single quanta of light in the retinal ganglion cells of the cat. Vision Research Supplement 3: 87–101.

Baylor, D. A., Lamb, T. D., and Yau, K. W. (1979). Responses of retinal rods to single photons. Journal of Physiology 288: 613–634.

Dacheux, R. F. and Raviola, E. (1986). The rod pathway in the rabbit retina: A depolarizing bipolar and amacrine cell. Journal of Neuroscience 6: 331–345.

Dunn, F. A. and Rieke, F. (2008). Single photon absorptions evoke synaptic depression in the retina to extend the operational range of rod vision. Neuron 57: 894–904.

Dunn, F. A., Lankheet, M. J., and Rieke, F. (2007). Light adaptation in cone vision involves switching between receptor and post-receptor sites. Nature 449: 603–606.

Field, G. D. and Rieke, F. (2002). Nonlinear signal transfer from mouse rods to bipolar cells and implications for visual sensitivity. Neuron 34: 773–785.

Hecht, S., Schlaer, S., and Pirenne, M. H. (1942). Energy, quanta, and vision. Journal of General Physiology 25: 819–840.

Hornstein, E. P., Verweij, J., Li, P. H., and Schnapf, J. L. (2005). Gap-junctional coupling and absolute sensitivity of photoreceptors in macaque retina. Journal of Neuroscience 25: 11201–11209.

Okawa, H. and Sampath, A. P. (2007). Optimization of single-photon response transmission at the rod-to-rod bipolar synapse. Physiology 22: 279–286.

Sampath, A. P. and Rieke, F. (2004). Selective transmission of single photon responses by saturation at the rod-to-rod bipolar synapse. Neuron 41: 431–443.

Singer, J. H., Lassova, L., Vardi, N., and Diamond, J. S. (2004). Coordinated multivesicular release at a mammalian rod synapse.

Nature Neuroscience 7: 826–833.

Smith, R. G., Freed, M. A., and Sterling, P. (1986). Microcircuitry of the dark-adapted cat retina: Functional architecture of the rod–cone network. Journal of Neuroscience 6: 3505–3517.

Sterling, P., Freed, M. A., and Smith, R. G. (1988). Architecture of rod and cone circuits to the on-beta ganglion cell. Journal of Neuroscience 8: 623–642.

van Rossum, M. C. and Smith, R. G. (1998). Noise removal at the rod synapse of mammalian retina. Visual Neuroscience 15: 809–821.

Glossary

AP20187 – A small molecule modeled on a dimer of the immunosuppressive drug FK506. One molecule of AP20187 can bind with high affinity to two FK506binding protein (Fv) domains. Thus, Fv domains can be used to produce fusion proteins that will dimerize in the presence of AP20187. This in turn can be used to control the activity of proteins or enzymes whose activities are influenced by dimerization, such as caspases.

Bardet–Biedl syndrome – An autosomal recessive disorder characterized by obesity, retinal degeneration, polydactyly, hypogonadism, developmental delay, and mental retardation. Genes implicated in this syndrome are involved in ciliary transport processes.

Caspase-9 – An initiator caspase in the apoptotic cascade. Caspases are a family of cysteine proteases, which play essential roles in programmed cell death. Once activated, caspase-9 triggers activation of other caspases, precipitating the apoptotic process.

Optical coherence tomography (OCT) –

A technique that permits three-dimensional imaging within tissues that scatter light. The technique is frequently used in ophthalmology to noninvasively image the cell layers of the retina. Peripherin/RDS – A transmembrane glycoprotein found in the outer segment of both rod and cone photoreceptor cells. It is thought to be a structural

protein important for disk morphogenesis. Mutations in the gene encoding peripherin/RDS are associated with a variety of autosomal dominant retinal dystrophies; also referred to in publications as rds, peripherin, or peripherin-2.

Rab proteins and Arf4 – The members of the Ras superfamily of monomeric guanine-nucleotide-binding proteins (G proteins), which are involved in the regulation of membrane trafficking.

Retinal degeneration – A phenotype associated with many different retinal disorders, involving progressive death of retinal cells, usually of a specific cell type.

Retinitis pigmentosa – A hereditary retinal dystrophy characterized by defective dark adaptation, progressive loss of peripheral vision that may eventually extend to loss of central vision, and the appearance of black pigment in the fundus.

RNA helicase Ddx39 – A member of the DEAD box protein family of putative RNA helicases. Family members are characterized by the conserved motif Asp-Glu-Ala-Asp (D-E-A-D), and are involved in RNA metabolism.

Stargardt’s disease – An autosomal recessive juvenile-onset form of macular dystrophy arising from mutations in the ABCA4 gene. The ABCA4 gene product is expressed in photoreceptor cells and is thought to be an ATP-dependent transporter for N-retinylidene-PE.

Introduction

The amphibian retina has many unique properties that have intrigued visual scientists for decades. Many studies have been conducted on the retina of Xenopus laevis, a frog species commonly used as a laboratory animal. These frogs were introduced to the research community in the 1930s and soon became widely available, after the discovery that they can be induced to lay eggs by injection of human chorionic gonadotropin (known as the Hogben test for pregnancy). As a research subject, X. laevis have advantages over other amphibians in that they have low maintenance requirements (they are entirely aquatic and do not require live food), and they can easily be induced to lay eggs. This made them a desirable model organism for developmental biologists studying fertilization and embryonic development; however, they have also been adopted by vision researchers as a model amphibian retina. The large size of the principle rod photoreceptors makes them highly amenable to biochemical, morphological, and electrophysiological studies (Figure 1). Early studies on the properties of the X. laevis visual system laid the groundwork for the use of this animal as a model organism for the study of visual disorders, while more recently developed techniques for genetic manipulation have resulted in X. laevis models of inherited retinal disease that are particularly amenable to certain forms of analysis.

Early Work on X. laevis – Biochemistry, Electrophysiology, and Microscopy

Modern research on the amphibian retina dates back to the extraordinary anatomical studies of Cajal and

Figure 1 Expression of a bovine rhodopsin P23H mutant transgene causes retinal degeneration in Xenopus laevis. Combined confocal/DIC (upper panels) or confocal micrographs (lower panels) of developmental stage 47/48 wild-type retinas (left panels) or transgenic retinas expressing bovine-rhoP23H (right panels). Animals were raised in bright cyclic light for

2 weeks, beginning at fertilization. The 12-mm retinal cyrosections were stained with wheat germ agglutinin (red), B630N anti-rhodopsin (green), and Hoechst 33342 nuclear stain (blue). The central retinas of animals expressing bovine P23H rhodopsin (right panels) have almost no remaining rods, with those that remain having short and irregular outer segments. Note the relatively large diameter (7 mm) of the rod photoreceptors relative to those of the mammalian retina. Due to the relatively large diameter of the photoreceptors, only a single row of nuclei is required in the ONL. RPE, retinal pigment epithelium; OS, outer segment; INL, inner nuclear layer; ONL, outer nuclear layer. Scale = 10 mm.

others in the 1800s that defined many of the principal cell types of the retina. Most early studies dealt with Rana and Bufo species. However, X. laevis have been used as a subject of retinal research dating back more than 50 years. In the 1950s, Wald and co-workers used X. laevis for investigations of visual pigment biochemistry. From the most abundant rod photoreceptors, it was found that the principal pigment exhibited maximal absorbance at 523 nm.

The amphibian retina is also of interest to electrophysiologists. Although the first electroretinogram (ERG) was recorded by Holmgren in the 1860s from a frog eye, X. laevis frogs were not used extensively for electrophysiological studies until the 1970s, with initial studies of X. laevis retinal physiology performed by Ripps and coworkers, correlating visual pigment content and photoreceptor threshold.

In the 1950s and 1960s, detailed analysis of the ultrastructure of photoreceptors was obtained by electron microscopy, leading to the current understanding of photoreceptor disk membrane structure, and the mechanisms of disk membrane synthesis and renewal. Many of these studies utilized amphibian retina, again typically Rana species, although Lanzavecchia examined the ultrastructure of X. laevis rods and cones in 1960. Further studies by Kinney and Fisher further characterized the morphogenesis and ultrastucture of the principal rod photoreceptor in X. laevis.

In the 1940s and 1950s, work by Sperry and co-workers demonstrated regeneration of the optic nerve after transection in various amphibians. Beginning in the late 1960s, Jacobson and co-workers conducted investigations of retinal development in X. laevis, with particular emphasis on the development of retinotectal projections. These studies employed embryonic surgery (e.g., inversion of the eye) to identify the origin of signals for optic nerve axon guidance, and demonstrated that the location of synapses of ganglion cell axons in the optic tectum are specified by the location (dorsal, ventral, nasal, or temporal) of the cell bodies in the retina.

These early investigations established the X. laevis retina as a viable subject for retinal research that is still in use, including ongoing studies of X. laevis disk shedding and renewal, electrophysiology, biochemistry, and retinal development, that are further improving our understanding of retinal function. Additionally, several studies discussed below have directly modeled human retinal disorders in

X.laevis, in order to better understand retinal dysfunction.

X. laevis as a Model for Vitamin A Deprivation

One of the first instances of modeling retinal disease in X. laevis was a study by Witkovsky and co-workers on vitamin A deprivation in tadpoles. This model reproduced features of night blindness (i.e., decreased rod sensitivity) seen in vitamin-A-deprived patients. Among other novel findings, the authors demonstrated that bleaching of photopigment causes a greater reduction in photoreceptor sensitivity than can be accounted for by reduction in pigment quantity alone. This was accounted for in later studies, which demonstrated that opsin has a greater tendency to activate the visual transduction cascade than rhodopsin. More recently, studies on vitamin A deprivation in X. laevis have been continued by Solessio and co-workers, who have also pioneered the use of psychophysical measurements of X. laevis visual sensitivity.

X. laevis as a Model for Glaucoma

Early studies by Jacobson and Keating involving optic nerve transection in X. laevis are similar to paradigms

currently used in research of optic nerve injury or glaucoma, although more typically the research subject is a mammal. However, unlike a mammalian optic nerve, a severed X. laevis optic nerve regenerates (a property that would be highly desirable in glaucoma patients). Research focused on X. laevis models of retinal regeneration of this type is currently being continued by Belecky-Adams and co-workers, who recently identified a possible role of the RNA helicase Ddx39 in the regulation of stem cells in the retina.

X. laevis and Studies of the Transport of Rhodopsin

The first investigations of the mechanism of outer segment renewal utilized amphibians injected with radioactive amino acids in a pulse–chase paradigm that allowed newly synthesized membranes to be visualized by autoradiography. The original experiments by Young and coworkers demonstrated that new disks were formed at the base of the rod outer segment, and subsequently became discontinuous with the outer segment plasma membrane. These studies were further extended by Besharse and Hollyfield, who examined the influence of light on disk synthesis in X. laevis photoreceptors, demonstrating diurnal regulation of disk membrane synthesis. Collectively, these studies demonstrated the extremely high rate of rod photoreceptor disk membrane synthesis in X. laevis retina, estimated at roughly 50-fold higher than in mammalian photoreceptors.

These findings suggested that, due to the enormous rate of outer segment membrane synthesis, the amphibian retina would be an excellent choice for studying the biosynthesis of rhodopsin. This work was pioneered by Papermaster and Dreyer, and subsequently continued by Papermaster and co-workers. These studies largely used Rana and Xenopus species, and resulted in identification of molecular and ultrastructural details of the rhodopsin transport pathway. Components of the biosynthetic machinery and transport mechanisms first identified in amphibia included associated features of the connecting cilium (the pericilliary ridge complex), and vesicles transporting rhodopsin (RTCs or rhodopsin transport carriers). Deretic was able to reconstitute rhodopsin transport in amphibian retinal extracts, and using this assay demonstrated that the rhodopsin transport signal was located in the cytoplasmic C-terminal domain. This in vitro assay and associated methodologies subsequently led to identification of a number of molecules associated with rhodopsin transport, including the small G proteins, rab6, rab8, rab11, and arf4.

However, due to the lack of an effective system for culturing photoreceptors, it was not possible to incorporate molecular biology approaches into the study of

amphibian rhodopsin transport pathways until the 1990s, when techniques for the production of transgenic X. laevis developed by Kroll and Amaya, and cloning of promoters suitable for driving expression in X. laevis rods, allowed the study of rhodopsin transport in a genetically manipulated amphibian retina. Tam and colleagues found that rhodopsin-green fluorescent protein (GFP) fusion proteins expressed in X. laevis retina were transported correctly to rod outer segments. This allowed further identification and in-vivo demonstration of the function of the outer segment localization signal QVAPA, located at the extreme C-terminus of rhodopsin. Several mutations affecting this region cause retinitis pigmentosa (RP). These studies also demonstrated the extraordinary utility of transgenic X. laevis for conducting comparisons between transgenic animals, as numerous primary transgenic animals carrying different transgenes can be generated in a relatively short amount of time. For example, the rhodopsin outer segment localization signal was identified and refined using 15 distinct transgene constructs.

Using the same transgenic X. laevis system, the function of rab8 in rhodopsin transport was explored using dominant-negative and constitutively active mutants. Expression of these mutant rab8-GFP fusion proteins in X. laevis rods generated the first X. laevis models with an inherited retinal degeneration (RD) phenotype, as these fusion proteins proved to be quite toxic to retinal rods, and the phenotype was passed to F1 offspring. The expression of dominant-negative GFP-rab8T22N caused a particularly rapid death of photoreceptors associated with accumulation of rhodopsin-containing intracellular vesicles in the vicinity of the base of the connecting cilium. Although it was not clear at the time, subsequent studies indicate that the resulting phenotype may be closely related to the RD associated with Bardet–Biedel syndrome (BBS). Similar investigations of the small G-protein Arf4, which binds the rhodopsin outer segment localization signal, are ongoing.

The RD observed in this study demonstrated a unique central-to-peripheral distribution subsequently seen in all other X. laevis models of RD. This is associated with the rapid growth of the eye in young X. laevis, which results in continuous addition of new photoreceptors to the peripheral retina. Thus, a single cryosection can demonstrate all stages of photoreceptor degeneration moving from central retina to periphery.

Modeling RP in Transgenic X. laevis

Subsequently, genetically modified transgenic X. laevis was used in a number of studies that directly examined mutations associated with the human disorder RP, an inherited form of RD. The pioneering study involved

transgenic expression of mutant forms of peripherin/rds, a protein found at the periphery of rod outer segment disks. Peripherin/rds and its mutants were expressed as GFP fusion proteins using the rod opsin promoter to drive expression in retinal rods, at levels sufficiently high to cause RD in some cases. Confocal microscopy of the intrinsic GFP fluorescence showed that several fusion proteins had unique localization patterns distinct from wild type that respectively suggested either specific disruptions in normal function, or misfolding and endoplasmic reticulum (ER) retention. Furthermore, electron microscopy revealed unique abnormalities in disk organization, possibly associated with disruption of the normal functions of the peripherin/rds C-terminus.

Previous attempts to use rhodopsin-GFP fusion proteins to develop similar models of RP were unsuccessful, most likely due to low expression levels. In order to adapt the system for the study of RD induced by rhodopsin mutants, a system was devised for detection of nonfluorescent transgene products based on nonconserved rhodopsin antibody epitopes, such that epitope tags involving minimal (or no) sequence changes could be introduced. Initially, this system was applied to the study of an RP-causing mutation (Q348ter) that disrupts the previously identified rhodopsin outer segment localization signal. In these studies, the power of the X. laevis system for drawing comparisons between transgenes was further expanded. Rhodopsin mutants defective in signal transduction properties were combined with rhodopsin mutants responsible for RP to dissect the role of rhodopsin signal transduction in rod cell death pathways. The results demonstrated rhodopsin mislocalization was associated with axonal sprouting and cell death, regardless of whether rhodopsin signal transduction properties were inhibited.

The same system was subsequently applied to the study of the rhodopsin mutation P23H, the most common cause of autosomal dominant RP in North America. Despite numerous studies of this rhodopsin mutant in cultured cells and transgenic rodents, there was no clear consensus as to the effects of this mutation on rhodopsin function; in cultured cells, it was classified as a mutant defective in folding and ER exit, while transgenic animal studies suggested it was transported correctly to rod outer segments, where it caused RD that was exacerbated by light.

Studies in transgenic X. laevis compared several different forms of P23H rhodopsin, including P23H rhodopsins based on different species, and P23H rhodopsins defective in signal transduction and chromophore binding. Interestingly, all forms of P23H rhodopsin caused RD, but varied in terms of ER retention, expression level, and light sensitivity. P23H rhodopsins that exhibited dramatic ER retention (X. laevis P23H rhodopsin) caused RD under all circumstances, while P23H rhodopsins that were

transported in small quantities to the OS (bovine P23H rhodopsin) caused RD only on light exposure (Figure 1). Furthermore, for bovine P23H rhodopsin, disruption of the chromophore-binding site was associated with reduced expression levels and RD, regardless of light exposure. This result reconciles the differences seen between previous studies, suggesting that in some forms of P23Hinduced RD, chromophore binding promotes ER exit of newly synthesized rhodopsin. The dramatic sensitivity of these phenotypes to the underlying rhodopsin sequence was confirmed by Zhang and colleagues, who demonstrated light-sensitive RD in an X. laevis rhodopsin that differed from that used previously only in the sequences of the epitope tags. In addition to providing insight into the mechanisms underlying RD, these studies dramatically emphasize the difficulties in extrapolating results reported from a single disease model to human disease states.

A unique finding in this system was the presence of considerable quantities of truncated P23H rhodopsin, in which a significant portion of the N-terminal domain (including the mutated H23 residue) was removed; in fact, this was the dominant species observed in retinas expressing bovine P23H rhodopsin. This truncated species was also previously observed in cultured cells, although in smaller quantities. Identification of this species in other transgenic models would be difficult due to lack of a suitable reagent for detection, but was readily achieved in X. laevis due to the availability of both N- and C-terminal specific antibodies that did not cross-react with endogenous rhodopsin.

Subsequent studies of the same X. laevis models of P23H-rhodopsin-induced RD probed the association of chromophore binding and ER exit. In order to address the question of whether the causative factor in light-induced RD was a reduction in the supply of free 11-cis retinal, or isomerization of 11-cis retinal bound to P23H rhodopsin as chromophore, the sensitivity of RD to different wavelengths of light was examined. It was determined that the profile of light sensitivity was consistent with photoisomerization of rhodopsin (which maximally absorbs green light) rather than free chromophore (with maximal absorbance in the UV). This also brings to mind similar studies of the constitutively active rhodopsin mutant K296E, classified as misfolding by some studies in cultured cells, suggesting that the active conformation of rhodopsin and/or loss of chromophore can be associated with altered kinetics of ER exit.

Studies of additional RP-causing rhodopsin mutations in transgenic X. laevis are ongoing, and have been reported at international meetings, including K296E rhodopsin and the glycosylation-defective mutants T4K and T17M. Glycosylation-defective rhodopsin mutants are also reported to be associated with light-exacerbated RD in X. laevis.

Inducible RD

In an alternate approach to modeling RP in the transgenic X. laevis retina, Hamm and co-workers designed a druginducible model of RD driven by a modified form of caspase-9. Dimerization and subsequent activation of this caspase-9 transgene is driven by AP20187, a small molecule based on a dimer of FK506. Administration of AP20187 to these transgenic X. laevis induces rapid RD that is not dependent on any particular environmental condition (such as special lighting). The system is designed to examine the effects of rod degeneration on other cell types, including cone photoreceptors and cells of the inner nuclear layer, and to examine the capacity for regeneration of rods in the X. laevis retina. This study was also the first to provide functional (i.e., electrophysiological) data for an X. laevis model of RD.

Interestingly, this model demonstrated a dramatic reduction in electrophysiological responses to stimuli designed to isolate cone function (e.g., a rapid flicker stimulus), despite the fact that no associated cone death was detected. This reduction in cone sensitivity may be associated with the cones themselves, or with other cells associated with the cone pathway and the ERG B-wave (e.g., cone bipolar cells and/or Mu¨ller glia). The restoration of responses to flicker stimuli was associated with a thickening of the inner nuclear layer, and a similar thickening can be observed by optical coherence tomography (OCT) in human RP patients.

Other Transgenic X. laevis Models of Retinal Disease

In addition to rhodopsin and peripherin/rds mutations responsible for RP, gene products related to Stargardt’s disease have been expressed in X. laevis retina as GFP fusion proteins in order to examine their localization properties, although this has not yet resulted in a replication of a RD phenotype.

In studies by Kefalov and colleagues, cone opsins were expressed in X. laevis rods. As cone photoreceptors are considerably noisier than rod photoreceptors, this allowed a determination of the proportion of cone dark noise (activation of transduction in the absence of photons) that is purely due to the cone pigment sequence, and not other aspects of the transduction cascade or photoreceptor environment. Although generating a model of disease was not a goal of this study, the resulting animals could be considered a model for congenital stationary night blindness, which is due to abnormally high activity of the visual transduction cascade in the absence of photons.

X. laevis as a Model for Eye Development/ Developmental Disorders

The rapid development of the X. laevis embryos makes these animals of particular interest to developmental biologists, including those concerned with eye development. The first studies of the development of the X. laevis eye were conducted by Hollyfield through radioactive monitoring of the growth of the developing retina. Chung and colleagues histologically monitored the structural changes in the developing larval X. laevis retina and correlated these changes with electrophysiological changes, notably that while the receptive field of a ganglion cell remains constant in the developing larvae through metamorphosis, the inhibitory peripheral region expands to the entire retina of the adult

X. laevis.

The development of the X. laevis eye has been manipulated by both the overexpression and by the knockdown of transcription factors. El-Hodiri and colleagues have extensively studied the transcriptional regulation of photoreceptor development. More recently, they identified a retinal homeobox gene family member, Rx-L, which regulates photoreceptor-specific gene expression. Expressed in developing embryos, the knockdown of Rx-L expression adversely affected photoreceptor development, causing subtle phenotypes of altered photoreceptor morphology.

Using a similar embryonic transfection paradigm, Knox and colleagues found that overexpression of the transcription factors, Nrl and Nr2e3, in X. laevis retina resulted in an increase in numbers of rods, with concomitant reduction in cone photoreceptors, indicative of the roles of these factors in determining the developing photoreceptor cell fate. This system may prove extremely useful in modeling developmental disorders of the retina with similar underlying mechanisms.

X. laevis Models of Retinal Regeneration

Some recent studies have investigated the fascinating capacity of the X. laevis retina to repair itself after severe traumatic injury. In these studies, the entire retina is excised from an X. laevis tadpole eye. The retina subsequently demonstrates a dramatic capacity to completely regenerate by transdifferentiation of the remaining cells of the retinal pigment epithelium (RPE). Certain aspects of this transdifferentiation can be reproduced in culture, and it appears to be dependent on diffusible factors (possibly fibroblast growth factor 2 (FGF2)) released from the choroid. These results could have implications for traumatic eye injuries such as retinal detachment, retinal degenerative disorders, and glaucoma.

Summary

As an unconventional system for modeling retinal disease, X. laevis presents a number of advantages. As it is quite easy to generate transgenic X. laevis, they are an excellent system for comparing the effects of multiple transgenes. Other advantages include the relative ease of microscopic and electrophysiological studies due to the large size of the photoreceptor cells, regenerative capacity of the retina, and non-cross-reactivity of mammalian antibodies. However, there are also significant disadvantages, such as the current lack of knock-out or gene-replacement capabilities, long generation time (1 year), pseudotetraploid genome, and relatively small eyes, such that it is clearly not an ideal system appropriate for all experiments. Rather, X. laevis models of retinal disease are a very useful addition to the library of systems and models available to vision researchers.

See also: The Photoreceptor Outer Segment as a Sensory Cilium; Primary Photoreceptor Degenerations: Retinitis Pigmentosa; Primary Photoreceptor Degenerations: Terminology; Retinal Degeneration through the Eye of the Fly; Secondary Photoreceptor Degenerations: Age-Related Macular Degeneration; Secondary Photoreceptor Degenerations; Zebra Fish as a Model for Understanding Retinal Diseases; Zebra Fish–Retinal Development and Regeneration.

Further Reading

Araki, M. (2007). Regeneration of the amphibian retina: Role of tissue interaction and related signaling molecules on RPE transdifferentiation. Development, Growth and Differentiation 49: 109–120.

Besharse, J. C., Hollyfield, J. G., and Rayborn, M. E. (1977). Turnover of rod photoreceptor outer segments. II. Membrane addition and loss in relationship to light. Journal of Cell Biology 75: 507–527.

Deretic, D., Williams, A. H., Ransom, N., et al. (2005). Rhodopsin

C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4).

Proceedings of the National Academy of Sciences of the United States of America 102: 3301–3306.

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Glossary

Achromatopsia – A disease characterized by defects in the cone photoreceptors resulting in extreme light sensitivity and color blindness or rod monochromacy.

Apoptosis or programmed cell death – A form of cell death characterized by a series of biochemical and morphological changes resulting in the formation of apoptotic bodies and removal by the immune system.

Bystander effect – This describes the transmission of death from mutant or injured cells to healthy neighboring cells.

Cyclic guanosine monophosphate (cGMP) –

A cyclic nucleotide derived from guanosine triphosphate (GTP). cGMP acts as a regulator of the cyclic-nucleotide-gated ion channels in photoreceptors.

Cyclic nucleotide phosphodiesterases (Pde) –

A family of enzymes that hydrolyze the phosphodiester bond in the second-messenger molecules cAMP and cGMP. They regulate the localization, duration, and amplitude of cyclic nucleotide signaling.

Electroretinography (ERG) – A method for evaluating visual response. Electrodes are placed against the cornea and used to measure the electrical responses of various cell types in the retina to a light flash of varying intensity.

GAF domains – A large group of protein domains that bind small molecules; in the Pde proteins these domains bind cyclic nucleotides. The GAF acronym comes from the names of the first three different classes of proteins identified to contain them: cGMP-specific and-regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA.

Gap junctions – The intercellular connections that occur between some types of cells. These junctions allow the movement of various molecules and ions between cells.

Optokinetic response (OKR) – A method for evaluating zebrafish vision. Fish are placed in a small dish in the center of a rotating drum decorated with vertical stripes. If the fish can see, its eyes will follow the rotating stripes with regular reflexive saccades.

Retinitus pigmentosa (RP) – A group of diseases characterized by defects in the rod photoreceptors resulting in night blindness. Progressive RP often results in cone loss and tunnel vision in some cases progressing to total blindness.

Scotopic vision – The low light vision that is produced exclusively by rod function.

Zebrafish or Danio rerio – A tropical freshwater fish of the minnow family that has gained prominence as a scientific animal model. For further information see the Zebrafish Information Network (ZFIN), an online database of zebrafish genetic, genomic, and developmental information.

Introduction

Inherited photoreceptor degenerations are a major cause of incurable blindness. Degenerations can affect rods, causing night blindness, cones, causing color and daylight blindness, or both cell types, leading to complete blindness. Although there are many models of retinal degeneration caused by variety of mutations in different genes, it is still not possible to completely describe the molecular cascade causing cell death in any of these disorders and therefore it is equally difficult to prevent the degenerative process. Fundamental new information about the biochemistry of photoreceptor cell death is required to enhance our understanding of retinal degeneration and to develop new successful therapies. Zebrafish have gained prominence as a model organism for studies of retinal development, disease, and vision because they offer some distinct advantages over other genetically tractable systems. Zebrafish develop rapidly ex utero and can be maintained transparent allowing cells in the retina to be visualized in live larvae in real time using confocal and multiphoton imaging techniques. This provides the opportunity to visualize morphological and biochemical changes occurring in diseased photoreceptors with cellular and subcellular resolution. Here, we describe two mutations in the zebrafish cone phosphodiesterase (pde6c) gene that result in retinal degeneration. These mutants provide a unique opportunity to learn more about the biochemical triggers and inhibitors of cell death within the retina.