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

the ETDRS vision chart, a halving of the visual angle, over a 1-year follow-up. At that time (fall, 2008), therefore, laser treatment, involving focal application of small, relatively light-intensity argon laser burns to areas of vascular leakage in the maculae of eyes with diabetic macular edema, and in particular clinically significant macular edema, was considered the treatment of choice. Application of this treatment, however, requires a degree of caution. Because eyes with clinically significant macular edema may not have suffered a loss of central vision (and most eyes with macular edema in the ETDRS had visual acuity at, or better than, 20/40), and because the laser treatment technique employed in this study involves the placement of moderately intense, small-diameter laser burns directly to microaneurysms identified ophthalmoscopically or by fluorescein angiography, placement of such burns to lesions very close to the center of the macula may result in damage to central vision. The use of focal laser photocoagulation for lesions very close to the macular center therefore does require an element of clinical judgment. Hence, the ETDRS recommended that laser treatment for clinically significant macular edema should be considered, but did not establish such treatment as an absolute standard of care.

Results of Other Clinical Trials

Another multicenter RCT, the Branch Vein Occlusion Study, found a significant beneficial effect of focal argon laser photocoagulation for macular edema, and for retinal neovascularization, in this disorder. However, eyes with central retinal vein occlusion, a much more severe condition, received no benefit from focal laser photocoagulation in the Central Vein Occlusion Study.

Focal laser treatment was also shown to be of clear benefit for the treatment of macular edema resulting from branch retinal vein occlusion in another RCT, the Branch Vein Occlusion Study. However, it was ineffective for central retinal vein occlusion in the Central Vein Occlusion Study, yet another RCT. There is no evidence for a beneficial effect of laser treatment for macular edema from any other cause.

As the pathogenesis of macular edema appears to have an inflammatory component, anti-inflammatory therapies have been widely used as treatment for some forms of this disorder. The ETDRS evaluated aspirin at a dose of 650 mg day 1, compared to placebo for severe nonproliferative diabetic retinopathy and for diabetic macular edema and found no beneficial effect. In aphakia or pseudophakia cystoid macular edema, one RCT has demonstrated the efficacy of treatment with a topical nonsteroidal anti-inflammatory eyedrop, although some clinicians combine this with a topical steroid. More recently, a number of investigators have used intravitreal

injections of triamcinolone, a steroid molecule, for several forms of macular edema. Although several papers have reported good results of this procedure for diabetic macular edema, a very recent paper detailing the results of a large, multi-institutional RCT in the Diabetic Retinopathy Clinical Research Network found that, over a 2-year followup, intravitreal triamcinolone, at 1 and 4 mg doses, was significantly less effective in this disorder than focal laser treatment. These doses of intravitreal triamcinolone have been evaluated for their efficacy in treating macular edema resulting from branch or central retinal vein occlusion in the Standard Care versus COrticosteroid for REtinal vein occlusion (SCORE) study, two multi-institutional randomized, controlled clinical trials sponsored by the U.S. National Eye Institute. For central retinal vein occlusion, these studies found that triamcinolone injected intravitreally in either a 1 or a 4 mg dose produced a significantly higher number of eyes that gained three or more lines of vision over a 1-year follow-up interval than observation alone. One milligram was concluded to be the preferred dose for this indication because of its smaller number of adverse effects. This result is the first time that any treatment has been reported as significantly effective in improving vision in macular edema from a central retinal vein occlusion. However, the concurrent SCORE clinical trial showed that neither of these two steroid doses injected intravitreally produced gains in vision for eyes with macular edema secondary to branch retinal vein occlusion that were superior to those obtained by focal argon laser photocoagulation, the previously accepted standard of care for that entity. Reasons for the different efficacies of steroids and laser for macular edema in diabetic retinopathy and in central and branch retinal vein occlusions remain to be explained.

Another intravitreal steroid, fluocinolone, has been used successfully as a long-term, slow-release implant inserted surgically in the treatment of macular edema resulting from chronic uveitis. Like other steroids, but to a somewhat greater degree, the fluocinolone implant can result in substantial elevations of intraocular pressure, and in cataract formation. A full evaluation by RCT of the fluocinolone implant for macular edema in diabetes is underway, but clinical trials using this steroid implant involving other disorders in which macular edema is present have not yet been carried out.

Anti-VEGF Therapies and Macular Edema

Because the VEGFs were first recognized as a family of molecules that enhanced vascular permeability and the breakdown of blood–tissue barriers, before their recognition as angiogenic agents, the development of anti-VEGF agents with efficacy against neovascular retinal and choroidal diseases has been followed by clinical studies of

these agents as potential treatments for macular edema. RCTs involving ranibizumab (Lucentis, Genentech), a humanized anti-VEGF monoclonal antibody injected intravitreally for diabetic macular edema and for macular edema in retinal vein occlusions are currently underway. A very similar monoclonal antibody, bevacizumab (Avastin, Genentech), developed initially as a cancer therapy, has also been employed in some preliminary clinical trials. One possible objection to the putative role of VEGF in diabetic macular edema, relating to the absence of significant VEGF gene polymorphisms in diabetic macular edema cases, compared to the presence of such changes in cases of severe preproliferative and proliferative diabetic retinopathy, has been discussed above. Another is the clinical observation that diabetic macular edema often occurs in the absence of retinal neovascularization and, conversely, neovascularization may occur without macular edema. If excessive VEGF secretion is essential to the cause of both types of diabetic retinal lesion, then both should occur together much more often than not. However, the issue may be much more complex, and its resolution will require, among other things, the completion of the ongoing clinical trials. There have been a number of reports describing encouraging results from much smaller trials. A report of a small, phase 2 trial of bevacizumab for diabetic macular edema from the Diabetic Retinopathy Clinical Research Network described suggestive evidence of a beneficial effect on macular thickness by OCT and visual acuity in patients injected with this agent, compared to focal argon laser photocoagulation. This trial, however, was quite small and complex in organization (a total of 100 patients, randomized into five treatment groups of 20 each, receiving various combinations of different doses of bevacizumab with or without laser therapy). A final determination of the efficacy of this mode of therapy must therefore await the completion of larger, longer controlled clinical trials.

Other agents that are currently being investigated for the treatment of diabetic macular edema include VEGFTNF receptor-associated protein (TRAP; aflibercept, Regeneron), a VEGF receptor that is solubilized by being complexed to an immunoglobulin and that, upon intravitreal injection, acts by sponging VEGF molecules from the vitreous, and sirolimus (rapamycin), an antibiotic with immunomodulatory and anti-inflammatory properties, that is also capable of interfering with neovascularization.

A Puzzling Question

The use of OCT evaluations to measure macular thickness and to determine pathologic changes in macular anatomy has led to an unexpected finding. It has been generally assumed, that macular thickness beyond the

normal range, and visual acuity, were inversely, and fairly closely, correlated, and central macular thickness determined by OCT has been used as an endpoint (usually a secondary endpoint) of several clinical trials of new therapies for diabetic retinopathy and some other diseases. Although a rough correlation does exist, the Diabetic Retinopathy Clinical Research Network and others who have investigated this question have found that, for diabetic macular edema, the correlation is surprisingly poor (Figure 6). We, and others, have found that this poor correlation extends to macular edema in other disorders as well, and our own preliminary results suggest that the slope of the correlation curve of central macular thickness versus visual acuity differs for diabetic macular edema and for pseudophakia macular edema (Irvine–Gass syndrome).

The reasons for this unexpected observation are yet to be established. What is the effect of intracellular fluid accumulation versus large amounts of intercellular edema fluid, as in cystoid macular edema? Does edema of much longer duration have an adverse effect on visual acuity, by contrast with more acute occurrences? What kinds of anatomic alterations have an adverse effect on the visual acuity outcome? As a rule, patients whose fluorescein angiograms show extensive nonperfusion of the perifoveal capillary network, or who have a large lipid plaque in the center of the macula, will have poor visual acuity

OCT center point thickness ( m)

Figure 6 Best-corrected visual acuity, measured on the Early Treatment Diabetic Retinopathy Study (ETDRS) chart, of a large series of patients from the multicenter Diabetic Retinopathy Clinical Research Network, and correlated with macular central point thickness (measured by time domain OCT). The solid line indicates the best-fitting linear correlation curve, while the dashed lines indicate the 95% confidence interval. Although a correlation exists, it is surprisingly poor. Reprinted from DRCR. net Study Group (2007). Relationship between optical coherence tomography-measured central retinal thickness and visual acuity in diabetic macular edema. Ophthalmology 114: 525–536, with permission from Elsevier.

regardless of macular thickness. Are there more subtle anatomic changes that can be detected by newer, highresolution OCT methods (Figures 1(c), 2(c), and 5(b) and (c); compare with Figure 4(b))? Are there aberrations in photoreceptor structure or orientation that can be detected by high-resolution OCT, or by adaptive

optics techniques? What prognostic information can be obtained by electrophysiologic methods such as the multifocal electroretinogram (Figure 7), that can detect functional alterations in very small regions of the macula? These and other questions remain subjects for investigation in the study of macular edema.

See also: Adaptive Optics; Blood–Retinal Barrier; Breakdown of the Blood–Retinal Barrier; Optical Coherence Tomography.

Further Reading

Aiello, L. P., Avery, R. L., Arrigg, P. G., et al. (1994). Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. New England Journal of Medicine 331:

1480–1487.

Al-Kateb, H., Mirea, L., Xie, X., et al. (2007). Multiple variants in vascular endothelial growth factor (VEGFA) are risk factors for time to severe retinopathy in type 1 diabetes: The DCCT/EDIC genetics study.

Diabetes 56: 2161–2168.

Bearse, M. A., Jr., Adams, A. J., Han, Y., et al. (2006). A multifocal electroretinogram model predicting the development of diabetic retinopathy. Progress in Retinal and Eye Research 25: 425–448.

Diabetic Retinopathy Clinical Research Network (2008). A randomized trial comparing intravitreal triamcinolone acetonide and focal/grid photocoagulation for diabetic macular edema. Ophthalmology 115: 1447–1449.

Diabetic Retinopathy Clinical Research Network, Scott, I. U., Edwards, A. R., et al. (2007). A phase II randomized clinical trial of intravitreal bevacizumab for diabetic macular edema. Ophthalmology 114: 1860–1867.

Drexler, W. and Fujimoto, J. G. (2008). State-of-the-art retinal optical coherence tomography. Progress in Retinal and Eye Research 27(1): 45–88.

Early Treatment Diabetic Retinopathy Study Research Group (1985). Photocoagulation for diabetic macular edema, Early Treatment Diabetic Retinopathy Study Report No. 1. Archives of Ophthalmology 103: 1796–1806.

Frank, R. N. (2004). Medical progress: Diabetic retinopathy.

New England Journal of Medicine 350: 48–58.

Frank, R. N. (2006). Etiologic mechanisms in diabetic retinopathy.

In: Ryan, S. J., Schachat, A. P., Wilkinson, C. P., and Hinton, D. (eds.) Retina, 4th edn., ch. 66, pp. 1240–1270. London: Elsevier.

Gao, B. B., Clermont, A., Rook, S., et al. (2007). Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation. Nature Medicine 13: 181–188.

Gass, J. D. and Norton, E. W. (1966). Cystoid macular edema and papilledema following cataract extraction. A fluorescein fundoscopic and angiographic study. Archives of Ophthalmology 76: 646–661.

Huang, D., Swanson, E. A., Lin, C. P., et al. (1991). Optical coherence tomography. Science 254: 1178–1181.

Jampol, L. M., Sanders, D. R., and Kraff, M. C. (1984). Prophylaxis and therapy of aphakic cystoid macular edema. Survey of Ophthalmology 28(supplement): 535–539.

Schuman, J. S., Puliafito, C. A., and Fujimoto, J. G. (2004). Optical Coherence Tomography of Ocular Diseases, 2nd edn. Thorofare, NJ: Slack.

Tezel, T. H., Del Priore, L. V., Flowers, B. E., et al. (1996). Correlation between scanning laser ophthalmoscope microperimetry and anatomic abnormalities in patients with subfoveal neovascularization. Ophthalmology 103: 1829–1836.

The SCORE Study Research Group (2009). A randomized trial comparing the efficacy and safety of intravitreal triamcinolone with observation to treat vision loss associated with macular edema secondary to central retinal vein occlusion. The Standard Care vs Corticosteroid for Retinal Vein Occlusion (SCORE) Study Report 5.

Archives of Ophthalmology 127: 1101–1114.

The SCORE Study Research Group (2009). A randomized trial comparing the efficacy and safety of intravitreal triamcinolone with standard care to treat vision loss associated with macular edema secondary to branch retinal vein occlusion. The Standard Care vs Corticosteroid for Retinal Vein Occlusion (SCORE) Study Report 6.

Archives of Ophthalmology 127: 1115–1128. Wojtkowski, M., Srinivasan, V., Fujimoto, J. G., et al. (2005).

Three-dimensional retinal imaging with high-speed ultrahigh-resolution optical coherence tomography. Ophthalmology 112: 1734–1746.

Glossary

Ciliary photoreceptors – Visual receptor cells in which the photosensitive region is derived from a cilium, a structure protruding from the cell body and characterized by the internal presence of nine longitudinal microtubules arranged in a circular fashion (and sometimes two additional microtubules in the center). Folding of the membrane of the cilium enhances its surface area to accommodate a great number of photopigment molecules.

Circumpallial nerve – Nerve that loops around the mantle of mollusks. It collects the axons from the eyes and projects to central neural ganglia of the animal.

Cyclic nucleotide-gated channels (CNG channels) – Family of ion channels that are ancestrally related to certain voltage-activated channels, but have evolved a different gating mechanism that responds to the binding of cGMP and cAMP to a site located in the intracellular side. They are responsible for generating the receptor potential in a variety of sensory cells, like olfactory neurons and many photoreceptors.

Diacylglycerol (DAG) – Product of the breakdown of phosphatidylinositol bisphosphate (PIP2) by phospholipase C (PLC). This is the lipid moiety of the molecule and remains membrane bound. It is a prime activator of protein kinase C (PKC).

Guanylate cyclase (GC) – Enzyme responsible for generating the intracellular messenger cyclic guanosine monophosphate (cGMP), using guanosine trisphosphate (GTP) as substrate.

Inositol trisphosphate (IP3) – Soluble, bioactive product of the breakdown of PIP2, resulting from the cleavage of the polar head of this phospholipid. It can diffuse from the membrane to the endoplasmic reticulum (ER), where it binds to IP3 receptors, causing them to expose a calcium-permeable pore and to release calcium contained within the endoplasmic reticulum (ER).

Microvillar photoreceptors – These are also called rhabdomeric photoreceptors. Photoreceptor cells in which the light-sensitive region presents evaginations of the cell membrane in the form of thin cylindrical protrusions, called microvilli. These are internally packed with longitudinal bundles of actin filaments that confer structural stability. The resulting increase in the surface area of the membrane makes

it possible to accumulate a large population of photopigment molecules.

Phosphatidylinositol bisphosphate (PIP2) – Minor phospholipid present in the inner leaflet of the cell membrane. Its enzymatic breakdown by phospholipase C (PLC) yields two bioactive products, namely inositol trisphosphate (IP3) and diacylglycerol (DAG).

Phosphodiesterase (PDE) – Enzyme that cleaves a phosphodiester bond. In vertebrate photoreceptors, PDE breaks down cGMP, forming the inert compound 50-GMP, thus leading to the closure of ion channels gated by cGMP.

Phospholipase C (PLC) – Enzyme that hydrolyzes inositol phospholipids in the membrane. A subclass, known as PLC-b, is activated by a guanine-binding protein (G-protein) of the Gq type.

Protein kinase C (PKC) – A class of enzymes capable of transferring a phosphate from an ATP molecule to a serine or threonine residue in a protein. Members of a subclass of these enzymes, known as conventional PKCs, are activated by calcium and/or diacylglycerol, two messenger molecules whose intracellular concentration increases in microvillar photoreceptors after stimulation with light.

pS, Pico-siemens (10–12 S) – A measurement unit of conductance. The conductance of a body is 1 S, such that upon applying 1 V across it, an electrical current of 1 A would flow.

Transient receptor potential (TRP), transient receptor potential like (TRPL) – Proteins first identified in the eyes of Drosophila as the ion channels subserving the late and the early phase, respectively, of the receptor potential. Genetic mutations leading to the failure to express TRP cause the receptor potential to consist of only the initial, transient phase, hence the name transient receptor potential (TRP).

Visual cells in the animal kingdom are usually partitioned into two distinct categories on the basis of the structure of the light-sensing organelle: in some photoreceptors, all the machinery necessary to absorb photons and generate a receptor potential is contained in a modified cilium; this is the case of the rods and cones in the vertebrate retina. In other cells, that function is subserved by microvilli, as it

occurs, for example, in the photoreceptors of the compound eye of insects. In both designs, the folding of the membrane greatly increases its surface area to accommodate a large number of photopigment molecules – which are integral membrane proteins – thus conferring a high optical density to the cell, making it an efficient and compact light collector.

It is generally accepted that this dichotomy also marks a sharp boundary between the design of vertebrate versus invertebrate retinae. However, the eyes of several marine mollusks, such as those of Pecten irradians (Figure 1) and Lima scabra challenge such dogma, because they possess a double retina, comprised of microvillar cells in the proximal layer, and ciliary cells in the distal layer. Because each retinal layer gives rise to a separate branch of the optic nerve, which in turn produces either ON or OFF neuronal discharges in response to light, both cell types were thought

(a)

Cornea

Lens

Retina

(b)

Figure 1 (a) The eye of the scallop, Pecten irradian, is of the simple type, possessing a single cornea and lens. The shiny appearance of the pupil is due to the presence of a reflector at the back of the eye, which helps focus light onto the retina.

(b) Cryosection of a fixed eye. The retina is comprised of two separate layers, proximal (pr) and distal (dr), each giving rise to a separate branch of the optic nerve.

to be visual receptors. This conjecture was corroborated by intracellular recordings of light-evoked responses in the intact retina. Surprisingly, these light responses have opposite polarities: proximal cells depolarize – as all other invertebrate photoreceptors were known to do – while distal cells hyperpolarize. Because those measurements were conducted under conditions designed to hamper synaptic transmission, it was unlikely that some of the light responses may have been evoked indirectly. Lingering doubts about the coexistence of functionally and structurally different primary visual receptors were dispelled by recordings of light-dependent changes in membrane voltage and membrane current in enzymatically isolated microvillar and ciliary cells.

Microvillar (Rhabdomeric) Photoreceptors

Excitation

The basic properties of the light response in microvillar visual receptors of mollusks are generally similar to those found in other invertebrate eyes. Light produces a depolarizing receptor potential, which is due to an increase in membrane conductance. Because there are no local second-order neurons, light information must be directly encoded by action potentials (Figure 2) propagating along the axons

25 mV

2 s

Light

Figure 2 Current-clamp recording of the light response in an isolated microvillar photoreceptor of Lima. The membrane voltage was measured through a patch electrode in the wholecell configuration. Flashes of 100-ms duration were presented, as indicated at the bottom, increasing their intensity at 0.6 log units (from bottom trace to top). A graded depolarization is evoked by light, eventually triggering one or more action potentials. The resting potential was –54 mV. Traces were offset vertically for clarity.

that directly emanate from the photoreceptor cells and form the circumpallial nerve. The circumpallial nerve loops around the mantle of mollusks, collects the axons from the eyes, and projects to central neural ganglia of the animal. Voltage-dependent calcium currents contribute to the initiation of regenerative spikes, and, together with voltageand calcium-dependent potassium currents, help shape the light response. The photocurrent is segregated to the photosensitive microvillar lobe, as clearly demonstrated by suctionelectrode measurements (Figure 3). The light-sensitive conductance is cationic, permeable to sodium ions, and, to a lesser extent, to K (permeability ratio, pNa:pK, 1.8:1). Additionally, there is a small contribution of calcium ions, which, however, is quantitatively minute (<3% of the lightevoked inward current). In this respect, these cells differ from Drosophila, where the Ca:Na selectivity ratio of lightdependent channels may be as high as 40:1. Such discrepancy may point to the evolution of different strategies for light-triggered calcium mobilization (a phenomenon universally observed in all microvillar receptors tested to date): in Pecten and Lima, like in some other species (e.g., Limulus), photo-induced Ca transients are impervious to the removal of extracellular calcium and reflect release from IP3-sensitive intracellular stores; such a scheme would have little use for a Ca influx pathway. By contrast, other photoreceptors display minimal – if any – internal release and must therefore depend essentially on Ca-permeable channels at the plasma membrane.

The photo-induced calcium transients of Pecten and Lima are very large, best monitored with low-affinity fluorescent indicators like Fluo 5F and Calcium Green 5N (KD2.3 and 14 mM, respectively; Figure 4); they are initiated

1 nA

106 cps

Figure 4 Simultaneous recording of membrane current under voltage clamp (top trace) and fluorescence of the indicator Calcium Green 5N, which was dialyzed through the patch pipette at a concentration of 75 mM (bottom trace). Emitted light was measured with a photomultiplier tube in photon-counting mode; the calibration bar at the bottom refers to counts per second (cps). A large increase in fluorescence above the initial level (indicated by the dashed line) occurred shortly after activating the epifluorescence beam, coincident with the beginning of the photocurrent.

in the light-transducing microvillar lobe, and precede the activation of the electrical response by 1 ms. Blunting the light-triggered Ca changes with Ca buffers (internal 1,2-bis(o-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid (BAPTA) or high concentration of ethylene glycol- bis-(b-amino-ethyl ether) N,N,N0,N0-tetraacetic acid (EGTA)) interferes with the light response, dramatically reducing its amplitude and slowing down its kinetics. However, although cytosolic Ca levels are controlled by light and

in turn exert a pivotal regulatory function, Ca does not, in all likelihood, directly activate light-dependent channels. Other events that follow phospholipase C (PLC) activation play a fundamental role; light-activated single-channel currents remain temporally viable in excised, perfused insideout patches, pointing to membrane-bound signaling elements, rather than soluble messengers like inositol 1,4,5 triphosphate (IP3) or Ca2+. Diacylglycerol (DAG), the other, membrane-confined product of the breakdown of phosphatidylinositol-bisphosphate (PIP2), is a plausible candidate. In Lima, a variety of DAG analogs, such as 2- dioctanoyl-sn-glycerol (DOG), phorbol 12-myristate 13acetate (PMA), and (–)-indolactam, increase membrane conductance and evoke an inward current (Figure 5) with a similar ionic selectivity as the photoconductance; moreover, DAG analogs and light interact occlusively, suggesting that their effects converge onto a common target. Calcium elevation greatly potentiates and accelerates the effects of DAG analogs, an observation in line with the aforementioned effects of Ca manipulations. The generality of the role of DAG – or some metabolite thereof – in phototransduction remains to be systematically assessed in different species; moreover, even in Lima, the robust effects of DAG analogs fall quantitatively short of the speed and magnitude of the electrical response elicited by light. While the discrepancy could be ascribed to technical limitations in the application of chemicals to the cell, the participation of additional messenger molecules should not be ruled out. In other systems, phosphatidylinositol 4,5 bisphosphate

DOG

200 pA

Figure 5 Diacylglycerol (DAG) analogs stimulate the lightsensitive conductance in Lima microvillar photoreceptors. Puffer-pipette application of the 2-dioctanoyl-sn-glycerol (DOG, 100 mM) activates an inward current, several hundreds of pA in amplitude, in a cell held under voltage clamp. Control application of dimethyl sulfoxide (DMSO) at the same concentration as that used to dissolve DOG is inert (bottom trace). The current activated by DOG and other diacylglycerol DAG analogs has the same ionic selectivity as the photocurrent; moreover, the analogs interact occlusively with light.

(PIP2), long viewed simply as the substrate of phosholipase C-b (PLC-b) and the precursor of IP3 and DAG, has emerged as a signaling molecule in its own right. Because PIP2 levels in the rhabdomeric membrane are bound to drop with light-triggered activation of PLC, a direct participation of this phospholipid in light signaling would call for a negative messenger role, one that helps maintain some element(s) of the transduction cascade in the inactive state. In Lima, intracellular administration of PIP2 specifically antagonizes the light-evoked current while sparing volt- age-dependent currents. Moreover, in excised patches of Pecten rhabdomeric membrane screened for the exclusive presence of light-activated channels, functional depletion of PIP2 by antibodies (to avoid confounding by the concomitant generation of its bioactive hydrolysis products) induces the appearance of single-channel currents, which can be silenced by exogenous replenishment of PIP2. Thus, the visual excitation process may be complex, and involve the interplay of several signaling and modulator molecules, rather than constituting a linear cascade.

An additional complexity in the visual excitation scheme of molluskan microvillar receptors is the presence of separate populations of ion channels underlying the photocurrent, as subsequently corroborated in Drosophila where transient-receptor potential (TRP) and transient-receptor potential-like (TRPL) channels were molecularly identified. Two components of the macroscopic photocurrent of Lima can be distinguished by their time course and ionconduction properties. In Pecten, cell-attached patch-clamp recordings in the exposed light-sensitive membrane reveal single-channel currents specifically activated by photostimulation (Figure 6) with a unitary conductance of 48 pS and an additional population of smaller-amplitude currents18 pS. The identity of these single-channel currents as the constitutive elements of the macroscopic photoresponse was confirmed by the similarity of kinetics and light sensitivity, extending down to stimulation intensities that only evoke single-photon responses.

Light Adaptation

Modulation of sensitivity is a fundamental process in photoreceptor function, and enables the cell to maintain responsiveness over widely varying levels of ambient illumination. Once again, calcium had been singled out as a critical player as early as in the 1970s in Limulus, but its downstream effector(s) remained unclear. Protein kinase C (PKC) is a likely mediator, because some PKC subtypes are activated by calcium (and DAG), and also because in Drosophila it is associated with the macromolecular ‘light-transduction complex’. Ca-dependent PKCa is selectively expressed in Lima eyes, as established by Western blot analysis using isoform-specific antibodies, and localizes in the light-sensing lobe of microvillar photoreceptors. Moreover, upon illumination it translocates from the cytosol to the membrane

(a)

Figure 7 Light-induced translocation of PKCa.

(a) Photoreceptor dark adapted for 1 h and directly fixed in paraformaldehyde (left), or illuminated for 3 s just before fixation (right). Anti-PKCa antibodies revealed a different spatial pattern of immunofluorescence in the two conditions, whereas in control cells PKCa is largely distributed diffusely in the cytosol of the microvillar lobe, after light the fluorescence forms a much more pronounced ring around the edges and is nearly absent from the central portion. (b) Intensity profiles of the immunofluorescence, measured along a line cutting across the rhabdomeric lobe.

(a functional assay of its activation), on a similar timescale as the onset of light adaptation (Figure 7). Chemical stimulation of PKC specifically depresses the light response, consistent with its role in desensitization, while pharmacological antagonists of PKC reduced light adaptation. These observations strongly support the involvement of PKC in the calcium-dependent regulation of response sensitivity.

Ciliary Photoreceptors

Photoreceptors of the distal retina function in a profoundly different way. Their resting potential is relatively depolarized ( –35 mV) owing to a high resting gNa/gK

Figure 8 Hyperpolarizing receptor potentials elicited by a 1-s light of increasing intensity (top to bottom trace) in a dissociated ciliary photoreceptor of Pecten, measured with a patch electrode in whole-cell current-clamp mode.

ratio, and illumination produces a hyperpolarizing receptor potential graded with light intensity (Figure 8). The light sensitivity is significantly lower than in proximal photoreceptors, but the purpose here is not range fractionation (unlike rods and cones of the vertebrate retinas). Instead, ciliary photoreceptors play a fundamentally different role from that of their microvillar counterparts: the information output ultimately entails action potentials, which, of course, could not be caused by light-induced hyperpolarization. It is a reduction of illumination that causes firing in these axons: the effect of light is to remove, in a timeand intensity-dependent way, the steady-state inactivation of voltage-dependent calcium channels, such that when illumination is decreased (e.g., when an approaching predator casts a shadow on the animal’s visual field), the return to a depolarized membrane potential triggers a Ca spike. As such, these cells function as dark detectors and activation of the phototransduction cascade serves the function of priming them to respond to light dimming.

Excitation

The hyperpolarizing receptor potential of ciliary visual receptors also arises from an increase in membrane

400 pA

Light

Time (ms)

Figure 9 Light-induced increase in membrane conductance in a ciliary photoreceptor, assessed by superimposing a repetitive voltage step on the steady holding potential (10 mV, 100 Hz, and 0.5 duty cycle). When a light response was elicited, the size of membrane current perturbations grew several fold, indicating the opening of ion channels.

conductance (Figure 9). The reversal potential is –80 mV, close to the calculated value for EK, the equilibrium potential for potassium, and exhibits a near-perfect Nernstian dependency on the concentration of extracellular potassium. Photostimulation, therefore, opens K-selective channels. Cell-attached patch recording on the ciliary appendages of the cell, presumably the light-sensitive organelles, reveals outwardly directed single-channel currents activated by light but not by voltage, with a unitary conductance 26 pS.

Light-signaling in distal photoreceptors diverges sharply from that of their microvillar counterparts: the photoresponse is insensitive to both IP3 and antagonists of the IP3 receptor, and is also impervious to Ca elevation and to Ca buffering. Moreover, the guanine nucleotide-binding alpha Q protein (Gaq) is not expressed in the distal photoreceptors of another member of the Pectinidae. These data strongly argue against the involvement of PLC – the canonical transduction cascade of invertebrate vision.

By contrast, substantial data have accumulated in support of a role for cGMP: intracellular application of cGMP or slowly hydrolyzing analogs (but not cAMP) elicits an outward current (Figure 10(a)) with similar ion-conduction properties as the light-evoked current: the reversal potential and its dependency on [K ]o are the same (Figure 10(a)), and so is the characteristic outward rectification. Both the photocurrent and the current elicited by cGMP are inhibited by the same antagonists, such as l-cis-diltiazem. Furthermore, illumination and exogenous cGMP analogs interact occlusively. Activation of cGMP-dependent currents can be obtained in excised membrane patches, suggesting that cGMP operates directly on the channels. It can be concluded that cGMP is the internal final messenger for visual excitation, as it occurs in vertebrate photoreceptors.

Despite the structural and functional similarities of Pecten distal photoreceptors with rods and cones, a key difference places these receptors in a novel, distinct

subcategory because the photoresponse is due to the opening, rather than the closing, of cGMP-gated channels. This implies that light must elevate cGMP levels, and this calls for an enzymatic machinery different from that of rods and cones. Several clues on the nature of this light-signaling pathway have emerged.

Photopigment, G Protein, and Arrestin

The molecular identity of the photopigment of ciliary receptors was elucidated in the Japanese scallop Patinopecten yessoensis, where a novel form of rhodopsin, dubbed SCOP2, was cloned and localized to the distal retinal layer by in situ hybridization. As in other invertebrates, the photopigment of scallop ciliary receptors is thermally stable upon illumination. The action spectrum, measured by both the late or the early receptor potential, peaks at 500 nm, and rhodopsin photoisomerization red-shifts its absorption curve by 75 nm; as a consequence, the fractional state of the pigment (rhodopsin (R)/metarhodopsin (M)) is a photoequilibrium that can be manipulated by varying the wavelength of illumination. A massive R to M conversion by blue-light illumination gives rise to prolonged hyperpolarizing after-potentials (or outward after-currents under voltage clamp) that can be reset by illumination with red light. Little is known about the detailed mechanisms that terminate the light response, but antibodies against bovine arrestin label a single band in Western blots of Pecten retinal homogenates and decorate ciliary photoreceptors both in cryosections of the eye and in dissociated retinas. Moreover, intracellular dialysis with the same antibodies slow down the falling phase of the photocurrent and allow prolonged after-currents to be elicited by spectrally neutral flashes, indicating that an arrestin-like molecule is implicated in visual excitation turn-off.

Seven transmembrane domain receptors (like rhodopsin), signal through a heterotrimeric G protein, and ciliary photoreceptors are no exception: GTP-g-S, which interferes with G-protein deactivation, because it is resistant to the GTPase activity of Ga and associated GTPase activating proteins (GAPs), causes the flash response to become sustained. Conversely, GDP-b-S inhibits phototransduction. However, the identity of the G-protein is unusual for visual cells: the only detectable Ga form expressed in the distal retina was molecularly identified as a Gao, by Shichida and colleagues in P. yessoensis. A similar Gao, differing in a stretch of 22 amino acids but otherwise identical at the nucleotide level, has been cloned in P. irradians; this was also demonstrated to be confined to the layer of ciliary photoreceptors by in situ hybridization. Physiological and pharmacological data corroborate the participation of Gao in light transduction:

(1) mastoparan peptide activators of Gao induce an outward current, which is suppressible by blockers of the light-sensitive conductance; (2) the light response is