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

and/or to monitor changes in RPE cells over time. Jessica Morgan has already shown that it is possible to use AO to track the time course of lipofuscin autofluoresence bleaching and RPE damage following exposure to bright light.

Imaging Retinal Ganglion Cells

The retinal image is conveyed to the brain through an array of 17 or more parallel ganglion cell pathways in the primate. We know remarkably little about the functional significance of many of these morphologically distinct ganglion cell classes. One of the major impediments to learning more about them is that the less numerous cells are difficult to target with a microelectrode that is capable of recording from only one single cell at a time. The development of optical methods to record from many ganglion cells

simultaneously in the intact living eye could greatly accelerate our understanding of ganglion cell function. There are numerous technical hurdles to overcome before this is feasible, but AO retinal imaging has now solved an important one: it is now possible to image ganglion cells along with some of their subcellular structure in vivo in nonhuman primate retina. This capability may also eventually prove useful in the study of diseases such as glaucoma, in which blindness results from the death of ganglion cells. New ways to image ganglion cells at a microscopic spatial scale may lead to earlier diagnosis and delivery of therapy.

Dan Gray, working in the AO group at the University of Rochester, imaged the dendritic morphology of macaque ganglion cells retrogradely labeled with fluorescent dye (rhodamine dextran) in vivo, with a transverse resolution of 1.6 mm (characterized as full width at half maximum;

Figure 9). Although in some cases, they were able to distinguish ganglion cell bodies at different depths in the retina, which is a valuable way to distinguish different classes, the depth resolution is modest, about 115 mm. Viral vector (e.g., AAV2)-mediated-gene delivery is another promising approach for delivering fluorophores (e.g., EGFP) to single cells. It requires a simpler intravitreal rather than an intracranial injection and can produce fluorescence that is essentially permanent in each cell that is transduced by the virus. Melissa Geng and Jason Porter at University of Rochester in collaboration with John Flannery at UC Berkeley combined this method with fluorescence AOSLO in the living rat eye (Figure 10). Another possibility is to use transgenics to create an animal whose cells express a fluorophore. Charles Lin and colleagues used this method to image microglia cells in living mouse eye. Work is underway to extend the viral vector method to nonhuman primate and to achieve expression of activity-dependent fluorophores such as G-CaMP (a green flurescent protein calcium probe) in single ganglion cells. These methods may ultimately allow AO retinal imaging to monitor the functional responses of ganglion cells.

Imaging Retinal Vasculature

Many retinal diseases, such as diabetic retinopathy or wet macular degeneration, have important effects on the retinal

vascular structure and blood flow. AO combined with fluorescein angiography allows imaging of even the fine structure of the retinal capillary bed (Figure 11(a)). Austin Roorda, then at the University of Houston, showed that AO retinal imaging could image leukocyte motion in the smallest retinal capillaries without the need for fluorescein (Figure 11(b)). Steve Burns at University of Indiana has even succeeded in imaging the flow of erythrocytes by tracking movement of single erythrocytes in successive images of a single scan line superimposed on an oriented parallel to a small vessel.

Imaging Retinal Disease

The use of an AO to image retinal disease is one of the most exciting and also one of the most challenging applications. AO is at its best when the pupil is completely dilated and the ocular media are clear and scatter free. The challenge of imaging retinal disease lies in the fact that patients often have small pupils, poor fixation, and/or large amounts of light scatter. Despite these limitations, progress has been made in the deployment of AO in the clinical arena.

For many retinal degenerative diseases, the structural changes of the cone mosaic and the RPE mosaic of patients have been impressively documented in vivo. The diseases affecting cones include not only those relevant

to color blindness mentioned previously, but also more common ones such as retinitis pigmentosa and photoreceptor dystrophy. As per the examples shown in Figure 12, cone loss can occur as a patchy, random dropout of cones that otherwise appear normal. In other cases, presumably when the mosaic has had the opportunity to remodel, the mosaic completely tiles the retina but the cones are larger and their density is reduced. Because the structure of the cone mosaic can be documented in living eyes, the specific structure in individual eyes can be correlated with the genotype of the patient, and also with functional measurements such as Humphrey visual field sensitivity, multifocal ERG, and contrast sensitivity (Figure 13).

Retinal degenerative diseases affect many other retinal structures, besides photoreceptors and RPE. As described in this article, AO imaging has enabled the accumulation of normative data of many retinal structures in both human and nonhuman primates, as well as data from animal disease models. These data will advance the clinical investigation of retinal degenerative disease. For example, the structure of lamina cribrosa was imaged in both normal primate eye and eye with experimentally induced glaucoma. Ultimately, AO imaging may play a role in the diagnosis of glaucoma and other retinal diseases in clinical practice.

See also: Color Blindness: Acquired; Color Blindness: Inherited; Optical Coherence Tomography.

Further Reading

Arathorn, D. W., Yang, Q., Vogel, C. R., et al. (2007). Retinally stabilized cone-targeted stimulus delivery. Optics Express 15: 13731–13744.

Artal, P., Chen, L., Fernandez, E. J., et al. (2004). Neural compensation for the eye’s optical aberrations. Journal of Vision 4: 281–287.

Biss, D. P., Sumorok, D., Burns, S. A., et al. (2007). In vivo fluorescent imaging of the mouse retina using adaptive optics. Optics Letters 32: 659–661.

Carroll, J., Neitz, M., Hofer, H., Neitz, J., and Williams, D. R. (2004). Functional photoreceptor loss revealed with adaptive optics: An alternate cause of color blindness. Proceedings of the National Academy of Sciences of the United States of America 101: 8461–8466.

Carroll, J., Gray, D. C., Roorda, A., and Williams, D. R. (2005). Recent advances in retinal imaging with adaptive optics. Optics and Photonics News 16: 36–42.

Chen, L., Artal, P., Gutierrez, D., and Williams, D. R. (2007). Neural compensation for the best aberration correction. Journal of Vision 7(9): 1–9.

Geng Y., Greenberg K. P., Wolfe R., Gray D. C., et al. (2009). In vivo imaging of microscopic structures in the rat retina. Investigative Ophthalmology and Visual Science 50: 5872–5879.

Gray, D. C., Wolfe, R., Gee, B. P., et al. (2008). In vivo imaging of the fine structure of rhodamine-labeled macaque retinal ganglion cells. Investigative Ophthalmology and Visual Science

49: 467–473.

Hofer, H., Artal, P., Singer, B., Aragon, J. L., and Williams, D. R. (2001). Dynamics of the eye’s wave aberration. Journal of Optical

Society of America A, Optics, Image Science, and Vision

18: 497–506.

Hofer, H., Carroll, J., Neitz, J., Neitz, M., and Williams, D. R. (2005). Organization of the human trichromatic cone mosaic. Journal of Neuroscience 25: 9669–9679.

Hofer, H., Singer, B., and Williams, D. R. (2005). Different sensations from cones with the same photopigment. Journal of Vision 5: 444–454.

Liang, J., Williams, D. R., and Miller, D. T. (1997). Supernormal vision and high-resolution retinal imaging through adaptive optics. Journal of Optical Society of America A, Optics, Image Science, and Vision

14: 2884–2892.

Martin, J. A. and Roorda, A. (2005). Direct and noninvasive assessment of parafoveal capillary leukocyte velocity. Ophthalmology 112: 2219–2224.

Morgan, J. I., Dubra, A., Wolfe, R., Merigan, W. H., and Williams, D. R. (2009). In vivo autofluorescence imaging of the human and macaque retinal pigment epithelial cell mosaic. Investigative Ophthalmology and Visual Science 50: 1350–1359.

Morgan, J. I., Hunter, J. J., Masella, B., et al. (2008). Light-induced retinal changes observed with high-resolution autofluorescence imaging of the retinal pigment epithelium. Investigative Ophthalmology and Visual Science 49: 3715–3729.

Porter, J., Guirao, A., Cox, I. G., and Williams, D. R. (2001). Monochromatic aberrations of the human eye in a large population.

Journal of Optical Society of America A, Optics, Image Science, and Vision 18: 1793–1803.

Porter, J., Queener, H., Lin, J., Thorn, K., and Awwal, A. A. S. (2006). Adaptive Optics for Vision Science: Principles, Practices, Design and Applications. Hoboken, NJ: Wiley-Interscience.

Roorda, A. and Williams, D. R. (1999). The arrangement of the three cone classes in the living human eye. Nature 397: 520–522.

Thibos, L. N., Hong, X., Bradley, A., and Cheng, X. (2002). Statistical variation of aberration structure and image quality in a normal population of healthy eyes. Journal of Optical Society of America A, Optics, Image Science, and Vision 19: 2329–2348.

Yoon, G. Y. and Williams, D. R. (2002). Visual performance after correcting the monochromatic and chromatic aberrations of the eye.

Journal of Optical Society of America A, Optics, Image Science, and Vision 19: 266–275.

Zhong, Z., Petrig, B. L., Qi, X., and Burns, S. A. (2008). In vivo measurement of erythrocyte velocity and retinal blood flow using adaptive optics scanning laser ophthalmoscopy. Optics Express 16: 12746–12756.

Glossary

Mu¨ller cell – A glial cell in the vertebrate retina that spans from the vitreal surface to the external limiting membrane, with apical processes that extend into the interphotoreceptor matrix. These cells perform multiple functions in the retina including the processing of visual retinoids.

Opsin visual pigment – A member of the G-protein- coupled receptor superfamily that functions as the light receptor in rods and cones. These pigments represent a complex between an opsin protein and a visual chromophore (see below).

Outer segment – An elongated light-sensitive structure attached to the connecting cilium of rod and cone photoreceptors. The rod outer segment in humans comprises a stack of approximately 1000 membranous disks. These disks are loaded with rhodopsin or cone opsin visual pigments.

Rpe65 – An abundant, membrane-associated protein in retinal pigment epithelium cells that functions as a retinoid isomerase. In particular, Rpe65 catalyzes the conversion of an all-trans-retinyl ester to 11-cis-retinol and a free fatty acid.

Visual chromophore – The light-absorbing molecular species in an opsin protein. The most common visual chromophore in vertebrates is 11-cis-retinaldehyde, which isomerizes to all-trans-retinaldehyde upon absorption of a photon.

Vision in vertebrates is provided by two types of photoreceptor cells, rods and cones. Rods mediate vision in dim light while cones mediate high-resolution color vision in bright light. Approximately 95% of photoreceptors in the human retina are rods. Nonetheless, cones are far more important for vision in humans. With the advent of artificial lighting, humans spend much of the time under conditions where the rod photoresponse is saturated and vision is mediated by cones. Both rods and cones contain a lightsensitive structure called the outer segment (OS) comprising a stack of densely packed membranous disks. These disks are packed with rhodopsin or cone-opsin visual pigment. The light-absorbing chromophore in most opsin pigments is 11-cis-retinaldehyde (11-cis-RAL). Absorption of a photon induces its isomerization to all-trans-retinaldehyde (all-trans- RAL), which activates the opsin pigment and stimulates the visual transduction cascade. After a brief period of activation,

the pigment dissociates to yield free all-trans-RAL and apoopsin, which is no longer light sensitive. The pigment is regenerated by recombination of apo-opsin with another 11-cis-RAL. To sustain light sensitivity, all-trans-RAL released by bleached opsin pigments is converted back to 11-cis-RAL by a multi-step enzyme pathway called the visual cycle (Figure 1).

Visual Cycle in Retinal Pigment

Epithelium Cells

All but the first step of the visual cycle takes place within cells of the retinal pigment epithelium (RPE), an epithelial monolayer adjacent to photoreceptor OS. In brief, all- trans-RAL is reduced by all-trans-retinol dehydrogenase (all-trans-RDH) in rod and cone OS to all-trans-retinol (all-trans-ROL) or vitamin A. The all-trans-ROL is released by the OS into the extracellular space or interphotoreceptor matrix (IPM), where it is bound to interphotoreceptor retinoid-binding protein (IRBP). The all-trans-ROL is taken up by apical processes of RPE cells, where it binds to cellular retinol binding protein type-1 (CRBP1). HoloCRBP1 is the substrate for lecithin:retinol acyl transferase (LRAT), which transfers a fatty acid from phosphatidylcholine in internal membranes to the all-trans-ROL. The resulting fatty-acyl ester of all-trans-ROL (all-trans-RE) is the substrate for Rpe65-isomerase. Rpe65 catalyzes hydrolysis of the carboxylate ester and utilizes the energy released for isomerization of all-trans-ROL to 11-cis-retinol (11-cis-ROL). The final catalytic step in the visual cycle is oxidation of 11-cis-ROL, by one of several 11-cis-ROL dehydrogenases (11-cis-RDH) to 11-cis-RAL. Both 11-cis- ROL and 11-cis-RAL are bound to cellular retinaldehyde binding protein (CRALBP) in the RPE. The 11-cis-RAL is released by the apical RPE to the IPM, where it binds IRBP. IRBP carries the 11-cis-RAL to the photoreceptor OS, where it is taken up and recombines with apo-opsin to reform a rhodopsin or cone-opsin pigment.

A Role for Mu¨ller Cells in Visual Pigment Regeneration

Evidence suggests that Mu¨ller glial cells in the retina also participate in the processing of visual retinoids. Mu¨ller cells span nearly the full thickness of the retina. The apical microvilli of Mu¨ller cells extend into the IPM and hence are well situated to exchange visual retinoids

with rod and cone OS, similar to the apical microvilli of RPE cells. Importantly, Mu¨ller cells contain several proteins involved in the processing of visual retinoids. These include (1) CRALBP, (2) RPE-retinal guanine- nucleotide-binding protein (G-protein) coupled receptor (RGR-opsin), a non-photoreceptor opsin that effects light-dependent regulation of the visual cycle in RPE cells, (3) CRBP1, and (4) retinol dehydrogenases types 10 and 11. Mu¨ller cells neither express Rpe65 nor LRAT, which are both present in RPE cells. Therefore, Mu¨ller cells do not simply duplicate the function of RPE cells in the regeneration of visual chromophore.

A Second Source of Chromophore for Cones

Several lines of evidence suggest that an alternate source of chromophore precursor is available to cones but not rods. When frog retinas were separated from the RPE, cone opsins, but not rhodopsin, regenerated spontaneously after a photobleach. After photobleaching, isolated salamander cones recovered sensitivity with the addition of either 11-cis-ROL or 11-cis-RAL, while isolated rods only recovered sensitivity with the addition of 11-cis- RAL. Mu¨ller cells in primary culture were shown to take up all-trans-ROL and synthesize 11-cis-ROL, which they secreted into the medium. Salamander cones were shown to dark-adapt and regenerate visual chromophore in isolated retinas separate from the RPE. The intrinsic capacity of cones to recover sensitivity and regenerate

visual chromophore in salamander retinas was lost after exposure to a selective Mu¨ller-cell toxin (a-aminoadipic acid), suggesting that Mu¨ller cells play a role in these processes.

Functional Differences between Rods and Cones

Although morphologically similar, rods and cones differ greatly in sensitivity, dynamic range, and speed of the photoresponse. Rods are single-photon detectors and show saturation of the photoresponse under relatively dim background illumination, producing 500 or more photoisomerizations per second. At saturation, all cyclic guanosine monophosphate (cGMP)-gated cation channels are closed and the rod no longer responds to light. Cones, on the other hand, are 100-fold less sensitive than rods but still exhibit a photoresponse to a light flash under bright background illuminations producing up to 106 photoisomerizations per second. The response kinetics of cones is also several-fold faster than rods. These disparities reflect differences in the properties of rhodopsin and the cone-opsin pigments. Rhodopsin is exceedingly quiet, with a spontaneous activation rate in the dark of one thermal isomerization every 2000 years. Cone opsins are much noisier than rhodopsin for two reasons. First, the spontaneous isomerization rate of cone opsin is 10 000fold higher than of rhodopsin. Second, 11-cis-RAL spontaneously dissociates from cone-opsins. A dark-adapted

red cone contains approximately 10% apo-cone-opsin due to spontaneous dissociation of chromophore. In contrast, 11-cis-RAL combines almost irreversibly with apo-rhodopsin in rods. Apo-opsin, which results from dissociation of 11-cis-RAL, activates the transduction cascade, producing the phenomenon of dark light. These effects contribute to the noisy background of cones and explain their much lower sensitivity than rods.

Although the rod response is saturated in bright light, photon capture by rhodopsin continues unabated. Thus, in a rod-dominant retina under daylight conditions, the vast majority of photoisomerization events contributes nothing to useful vision. Under these circumstances, cones must compete with rods for the limited supply of 11-cis-RAL. The reversible binding of 11-cis-RAL to apo- cone-opsins and its irreversible binding to apo-rhodopsin confers a tendency of rods to steal chromophore from cones. This tendency aggravates the competition between rods and cones for limited chromophore.

An Alternate Retinoid Isomerase Activity

in Cone-Dominant Retinas

Studies on the biochemistry of visual pigment regeneration in cone-dominant retinas suggest a mechanism whereby cones may escape competition from rods for visual chromophore. Experiments were performed on separated retinas and RPE from mice and cattle, as model roddominant species, and from chickens and ground squirrels, as model cone-dominant species. Measurement of endogenous retinoids showed that the rod-dominant species contain high levels of all-trans-retinyl esters (REs) in the RPE but no detectable REs in the retina. Cone-dominant species contain REs in both the RPE and retina, with predominantly 11-cis-REs in the retina. The activities of retinoidprocessing enzymes were measured by incubating total homogenates or microsomes prepared from RPE or retinas with all-trans-ROL, then assaying for synthesis of new retinoids by high-performance liquid chromatography (HPLC). Incubation of RPE homogenates with all-trans- ROL resulted in rapid synthesis of all-trans-REs, due to the activity of LRAT. Only after accumulation of all-trans-REs was 11-cis-ROL synthesis detected, due to the activity of Rpe65. This profile of retinoid synthesis was observed with RPE homogenates from both rodand cone-dominant species.

A strikingly different profile was observed when retina homogenates from cone-dominant chicken or ground squirrels were incubated with all-trans-ROL under similar conditions. Addition of all-trans-ROL resulted in rapid synthesis of 11-cis-REs and 11-cis-ROL, with negligible initial synthesis of all-trans-REs. Addition of palmitoyl coenzyme A (palm CoA) to the assay mixture increased synthesis of 11-cis-REs, again with minimal initial synthesis

of all-trans-REs. Synthesis of 11-cis-ROL and 11-cis-REs from all-trans-ROL without prior formation of all-trans- REs suggests that the mechanism of retinoid isomerization is different in retina versus RPE. Instead of converting an all-trans-RE to 11-cis-ROL and a free fatty acid, as catalyzed by Rpe65 in RPE cells, cone-dominant retinas appear to catalyze the direct conversion of all-trans-ROL to 11-cis- ROL. This energetically unfavorable reaction appears to be driven by mass action through secondary esterification of the 11-cis-ROL product. Here, the energy of isomerization is indirectly supplied by hydrolysis of the thio-ester in palm CoA versus hydrolysis of the carboxyl ester in phosphatidylcholine, as catalyzed by Rpe65. The isomerase machinery in retinas may therefore involve two catalytic activities, as depicted in Figure 2. One is an isomerase that catalyzes the passive interconversion of all-trans-ROL and 11-cis- ROL. This enzyme has not yet been identified. The second is a palm-CoA-dependent RE synthase. Several proteins with acyl CoA:retinol acyltransferase (ARAT) activity have been described. At least one of these, diacylglycerol acyltransferase type-1 (DGAT1), is expressed in the retina.

11-cis-ROL Dehydrogenase Activity in Cones but Not in Rods

As discussed above, cones can regenerate visual pigments and restore light sensitivity with the addition of either 11-cis-RAL or 11-cis-ROL, while rods can only regenerate rhodopsin pigment and recover sensitivity with addition of 11-cis-RAL. Since 11-cis-RAL is the visual chromophore for both cone and rod pigments, these observations suggest that cones, but not rods, express an 11-cis-RDH activity that oxidizes 11-cis-ROL to 11-cis-RAL. Robust nicotinamide adenine dinucleotide phosphate (NADPH)- dependent 11-cis-RDH activity has been detected in cone

All-trans-ROL 11-cis-ROL 11-cis-RP

Isomerase-2 Ester

synthase

Isomerosynthase HS-CoA

complex

Figure 2 Proposed isomerase-2 complex in Mu¨ller cells. The isomerase in Mu¨ller cells appears to catalyze direct conversion of all-trans-ROL to 11-cis-ROL. This energetically unfavorable reaction appears to be driven by mass action through secondary esterification of 11-cis-ROL to an 11-cis-RE. This synthase uses palm CoA as an acyl donor, and hence is a type of ARAT. The isomerase-2/ARAT complex has been named isomerosynthase to denote its activities. Neither isomerase-2 nor its ARAT partner has been identified to date.

but not rod in photoreceptors. Reduction of all-trans-RAL to all-trans-ROL in bleached photoreceptors requires NADPH, and hence is an energy-consuming process. Oxidation of an 11-cis-ROL to 11-cis-RAL generates an NADPH reducing-equivalent. Since reduction of all-trans-RAL and oxidation of 11-cis-ROL occurs with 1:1 stoichiometry in a cone that is utilizing 11-cis-ROL as a chromophore precursor, the presence of reciprocal NADP+/NADPH-specific dehydrogenases in cones affords a self-renewing supply of dinucleotide substrate at no energy cost to the cell. Eliminating the need for energydependent synthesis of NADPH may remove the metabolic bottleneck of all-trans-RAL-reduction in cones at high rates of photoisomerization.

An Alternate Visual Cycle that Mediates Pigment Regeneration in Cones

The observations outlined above can be explained by the hypothesized alternate visual cycle shown in Figure 3. According to this model, all-trans-ROL-isomerase (isomerase-2) and the 11-cis-ROL-specific ARAT are in Mu¨ller cells, which also express the 11-cis-ROL and 11-cis-RAL binding-protein, CRALBP. The major source of substrate for isomerase-2 is all-trans-ROL released by rods and cones during light exposure. Isomerization of all- trans-ROL to 11-cis-ROL and subsequent esterification of 11-cis-ROL to 11-cis-REs are probably limited by substrate

availability. It has been shown that apo-CRALBP stimulates 11-cis-RE-hydrolase (11-cis-REH) in RPE cells. These observations suggest a regulatory mechanism for the proposed visual cycle: in the dark-adapted state, cones contain fully regenerated opsin pigment and hence do not use 11-cis-ROL. CRALBP is saturated with 11-cis-ROL and the level of 11-cis-REs are high in Mu¨ller cells. Both isomerase-2 and 11-cis-RE-synthase are inactive due to the absence of available substrate. With the onset of light and the bleaching of photopigments, cones begin to take up 11-cis-ROL. This results in desaturation of CRALBP, activation of 11-cis-REH, and mobilization of 11-cis-RE-stores in Mu¨ller cells. Rods and cones begin to release all-trans- ROL, which permits replenishment of 11-cis-REs through activation of isomerase-2 and 11-cis-RE-synthase. This pathway becomes progressively more active with increasing light intensity, availability of all-trans-ROL substrate, and consumption of the 11-cis-ROL product by cones.

A New Role for IRBP

IRBP is the major extracellular retinoid-binding protein in retinas. It has been suggested that the primary function of IRBP is to bind all-trans-ROL and 11-cis-RAL during translocation of these retinoids between OS and the RPE. However, loss of IRBP in irbp / mice has only a mild effect on rhodopsin regeneration. Might IRBP play another role? The endogenous ligands of IRBP have been studied.

In light-adapted frog and bovine retinas, IRBP contains higher levels of 11-cis-ROL than 11-cis-RAL, in addition to all-trans-ROL. Moreover, IRBP immunoreactivity was found in association with cone but not rod OS. These observations suggest that the critical function of IRBP may not be exchange of all-trans-ROL and 11-cis-RAL between rods and RPE cells, as previously assumed, but rather exchange of all-trans-ROL and 11-cis-ROL between cones and Mu¨ller cells.

Alternate Visual Cycle in Rod-Dominant

Species

Although clearly present in cone-dominant retinas, the evidence for an alternate visual cycle in Mu¨ller cells of roddominant species is less compelling. Endogenous 11-cis-REs and isomerase-2 catalytic activity were undetectable in homogenates from rod-dominant bovine and mouse retinas. The absence of 11-cis-retinoids in rpe65 / mice has also been put forth as further evidence against the Mu¨ller-cell pathway in this species. However, this observation is difficult to interpret since rpe65 / photoreceptors contain no visual chromophore, hence all-trans-ROL, the substrate for the alternative visual cycle, is not released following light exposure. The effective Michaelis constant (Km) of all-trans-ROL for 11-cis-RE-synthesis by chicken retina homogenates is 13.5 mM. Thus, even in chickens, the alternate visual cycle is only active under conditions that yield high concentrations of all-trans-ROL. Recently, isolated mouse retinas were photobleached and analyzed physiologically for recovery of rod and cone light sensitivity. Cone sensitivity and the cone photoresponse recovered in these retinas despite the absence of RPE cells, while rod sensitivity and the rod response were dramatically attenuated. These physiological observations argue for existence of the alternate visual cycle in mouse retinas. The role of this pathway in rod-dominant retinas should be resolved upon identification of the proteins responsible for isomerase-2 activity, and measurement of their expression levels in rodand cone-dominant retinas.

See also: Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin; Phototransduction: The Visual Cycle.

Further Reading

Chen, Y. and Noy, N. (1994). Retinoid specificity of interphotoreceptor retinoid-binding protein. Biochemistry 33: 10658–10665.

Das, S. R., Bhardwaj, N., Kjeldbye, H., and Gouras, P. (1992). Muller cells of chicken retina synthesize 11-cis-retinol. Biochemical Journal 285: 907–913.

Fain, G. L., Matthews, H. R., Cornwall, M. C., and Koutalos, Y. (2001). Adaptation in vertebrate photoreceptors. Physiological Reviews 81: 117–151.

Jin, M., Li, S., Nusinowitz, S., et al. (2009). The role of interphotoreceptor retinoid-binding protein on the translocation of visual retinoids and function of cone photoreceptors. Journal of Neuroscience 29: 1486–1495.

Jones, G. J., Crouch, R. K., Wiggert, B., Cornwall, M. C., and Chader, G. J. (1989). Retinoid requirements for recovery of sensitivity after visual-pigment bleaching in isolated photoreceptors. Proceedings of the National Academy of Sciences of the United States of America

86: 9606–9610.

Kefalov, V. J., Estevez, M. E., Kono, M., et al. (2005). Breaking the covalent bond – a pigment property that contributes to desensitization in cones. Neuron 46: 879–890.

Mata, N. L., Radu, R. A., Clemmons, R., and Travis, G. H. (2002). Isomerization and oxidation of vitamin A in cone-dominant retinas. A novel pathway for visual-pigment regeneration in daylight. Neuron 36: 69–80.

Mata, N. L., Ruiz, A., Radu, R. A., Bui, T. V., and Travis, G. H. (2005). Chicken retinas contain a retinoid isomerase activity that catalyzes the direct conversion of all-trans-retinol to 11-cis-retinol.

Biochemistry 44: 11715–11721.

Miyazono, S., Shimauchi-Matsukawa, Y., Tachibanaki, S., and Kawamura, S. (2008). Highly efficient retinal metabolism in cones.

Proceedings of the National Academy of Sciences of the United States of America 105: 16051–16056.

Pugh, E. N. and Lamb, T. D. (2000). Phototransduction in vertebrate rods and cones: Molecular mechanisms of amplification, recovery and light adaptation. In: Stavenga, D. G., DeGrip, W. J., and Pugh, E. N., Jr (eds.) Handbook of Biological Physics, pp. 184–255. Amsterdam: Elsevier Science B.V.

Radu, R. A., Hu, J., Peng, J., et al. (2008). Retinal pigment epitheliumretinal G protein receptor-opsin mediates light-dependent translocation of all-trans-retinyl esters for synthesis of visual chromophore in retinal pigment epithelial cells. Journal of Biological Chemistry 283: 19730–19738.

Saari, J. C. and Bredberg, D. L. (1987). Photochemistry and stereoselectivity of cellular retinaldehyde-binding protein from bovine retina. Journal of Biological Chemistry 262: 7618–7622.

Stecher, H., Gelb, M. H., Saari, J. C., and Palczewski, K. (1999). Preferential release of 11-cis-retinol from retinal pigment epithelial cells in the presence of cellular retinaldehyde-binding protein.

Journal of Biological Chemistry 274: 8577–8585.

Wang, J. S., Estevez, M. E., Cornwall, M. C., and Kefalov, V. J. (2009). Intra-retinal visual cycle required for rapid and complete cone dark adaptation. Nature Neuroscience 12: 295–302.

Yen, C. L., Monetti, M., Burri, B. J., and Farese, R. V., Jr. (2005). The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerols, waxes, and retinyl esters. Journal of Lipid Research 46: 1502–1511.

Glossary

Neurotransmitter – A chemical substance that is released at synaptic connections that are used to relay, amplify, and modulate signals between cells and neurons.

Photopic vision – The scientific term for color vision mediated by multiple cone photoreceptor types in bright light. In the human retina, photopic vision is tri-chromatic.

Phototransduction – A process by which light is converted into electrical signals in rod and cone photoreceptor cells in the retina of the eye.

Retinal circuitry – It refers to the neuronal pathways in the retina that carry information from photoreceptor cells to ganglion cells.

Scotopic vision – Vision mediated by rod photoreceptors in dim light. Scotopic vision is color blind.

Spatio-temporal vision – A term commonly used to describe the spatial and temporal properties of human visual processing, and the neural mechanisms which underpin them.

Visual adaptation – A process by which vision adjusts to or gets used to a change in overall brightness, color, and other spatio-temporal properties, in order to maximize visual sensitivity.

Rod and Cone Photoreceptors

The process of vision is initiated by the absorption of light by a photoreceptor pigment molecule. The mammalian retina contains two classes of photoreceptors, referred to as rods and cones, each distinct in their structural morphology and in their behavior in response to light. Rod photoreceptors are highly sensitive to light and are capable of responding to a single photon. In contrast, cone photoreceptors are less sensitive to light, but are exquisitely tuned to mediate color and spatio-temporal vision. There are three types of cone photoreceptors in the human retina, each with different, but overlapping, spectral sensitivities, and the interaction of the output from these photoreceptor types mediates the ability to discriminate small color differences.

Based on studies of the evolution of visual pigments, it is hypothesized that rods evolved from cones. A single

amino acid substitution has been shown to exchange the molecular properties of rod and cone visual pigments. This suggests that a single spontaneous amino acid substitution could have been one of the key steps in the divergence of rodand cone-mediated vision. The first step in the formation of rods themselves may have been the spontaneous formation of disk membranes that were separated from the plasma membrane of the outer segment. This step is thought to have increased the light sensitivity of the photoresponse, a physiological property that is typical of rod photoreceptors. The post-receptoral retinal circuitry for rods is superimposed on pre-existing retinal circuitry mediating cone function. This feature of retinal circuitry predates the evolution of the rod photoreceptor itself and likely represents the pathway of a redundant cone photoreceptor that evolved into a rod. As described below, there are multiple sites in the retinal circuitry where rods have the opportunity to penetrate the cone system circuitry.

The absorption of a photon of light by a photoreceptor pigment molecule initiates a sequence of events referred to as the phototransduction cascade. Much of what we know about the specifics of the phototransduction cascade comes from the study of the rod photoresponse. In mammalian rods, the absorption of a photon of light by rhodopsin, the visual chromophore in rods, initiates a sequence of biochemical events that ultimately leads to a decrease in the intracellular level of cyclic guanosine 3,50-monophosphate (cGMP) and the closure of the cGMP-gated ion channels in the outer segment. The resultant closure of the cGMPgated channels in the outer-segment membrane decreases the circulating current by blocking inward current flow. This results in an hyperpolarization of the photoreceptor cell membrane and a decrease in the release of neurotransmitter at the photoreceptor synapse with second-order neurons. The biochemical steps in cone outer-segment phototransduction are thought to be similar to that which occurs in rods. In fact, there are corresponding components in rod and cone phototransduction at each step in the cascade, including rod and cone versions of visual pigment, tranducin, phosphodiesterase, arrestin, kinase, cGMP-gated channels, and Naþ/Kþ, Ca2þ exchangers.

Cone Postreceptoral Circuitry

Consider first the retinal circuitry mediating cone function. Cone photoreceptor cells synapse with bipolar and horizontal cells in the outer plexiform layer of the retina

(see Figure 1). Cones release glutamate at a steady rate in darkness but this rate is slowed in a graded response that is correlated to the change in the outer-segment membrane potential after stimulation by light. The modulation of synaptic transmitter release drives signaling to secondorder bipolar cells.

There are at least nine different types of cone bipolar cells in the mammalian retina that are distinguished on the basis of a number of features, including the number of

IPL GJ

Sublamina b

GCL

Figure 1 Cone signaling pathway in the mammalian retina. Cone photoreceptors respond to light with a graded hperpolarization and synapse with bipolar and horizontal

(not shown) cells in the outer plexiform layer (OPL) of the retina. The release of glutamate at the synaptic terminal is modulated by the change in the outer-segment membrane potential.

Cone signals are delivered to ON and OFF bipolar cells (BC) which carry signals to ON and OFF ganglion cells (GC) in the inner plexiform layer (green and red channels, respectively), thereby maintaining segregated cone pathways through the retina.

ON and OFF bipolar cells provide excitatory and inhibitory inputs to amacrine cells and ganglion cells and respond with a graded depolarization (sign-inverting) or hyperpolarizing (sign-conserving) response, respectively. Cones communicate

laterally with other cones (and rods) via electrical couplings called gap junctions (GJ). Lateral communication is also afforded by horizontal cells in the OPL and by amacrine cells in the IPL. Abbreviations: OS – outer segment, ONL – outer nuclear layer, OPL – outer plexiform layer, INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer, GJ – gap junction.

cone photoreceptors contacting the bipolar cell, the spread of the dendritic terminals, and the stratification of their axon terminals in the inner plexiform layer. The precise function of each of these types of bipolar cells is not well understood but they are presumed to be tuned to process different aspects of the visual stimulus. Morphologically, bipolar cells can be subdivided into classes commonly referred to as diffuse and midget bipolar cells. Diffuse bipolar cells (of which there are at least six types) have broad dendritic spreads and make contact with 5–20 long (L-) and middle (M-) wavelength cones; the number of connections depends on where they are located in the retina. These cells are likely involved in tasks requiring broad spatial averaging and color processing but have poor spatial resolution. In contrast, midget bipolar cells have a narrow dendritic tree and can make contact with a single cone and with a single ganglion cell. This cone-mediated circuitry provides a mechanism for high spatial resolution of a scene and is thought to mediate an acuity channel. The midget bipolar cell also transmits color information by virtue of its one-on-one connections with L- and M-sensitive cones. In addition, bipolar cells that make specialized contacts with short (S-) wavelength sensitive cones have been described in the primate, rat, and mouse retina.

Functionally, there are two broad classes of bipolar cells in the mammalian retina mediating the cone signaling pathway. They are commonly referred to as ON and OFF bipolar cells, because of their expression of either excitatory glutamate receptors (mGluRs) or inhibitory glutamate receptors (iGluRs). ON cone bipolar cells make contact with photoreceptors through invaginations in the cone pedicle and are flanked by a pair of horizontal cell dendrites, an arrangement commonly referred to as a triad. The triads are apposed to a presynaptic ribbon, which is the release site for neurotransmitter mediating signal transfer to second-order neurons. There are typically about 25 invaginations in a cone pedicle, but as many as 50 invaginations have been reported. A recent study has identified the protein pikachurin, a previously unknown dystroglycan-binding protein, as critical for the apposition of photoreceptor and bipolar cells dendrites at the ribbon synapse. OFF bipolar cells make contact directly at the cone pedicle base where up to 500 contacts are made with postsynaptic cells.

ON bipolar cells that express mGluRs are depolarized (sign-inverting) by the light response in cone photoreceptor cells. Their dendritic processes terminate in sublamina B of the inner plexiform layer (see Figure 1). In contrast, OFF bipolar cells express iGluRs. In these cells glutamate expression is linked to Naþ influx. OFF cone bipolar cells are hyperpolarized (sign-conserving) by the light response in cones and have processes that end in sublamina A of the inner plexiform layer. The two types of bipolar cells provide excitatory and inhibitory inputs to downstream cells