Vision loss, among the most feared complications of diabetes, is primarily caused by diabetic retinopathy, a disease that manifests in well-recognized, characteristic microvascular lesions. The reasons for retinal susceptibility to damage in diabetes are unclear, especially considering that microvascular networks are found in all tissues. However, the unique metabolic demands of retinal neurons could account for their vulnerability in diabetes. Photoreceptors are the first neurons in the visual circuit and are also the most energy-demanding cells of the retina. Here, we review experimental and clinical evidence linking photoreceptors to the development of diabetic retinopathy. We then describe the influence of retinal illumination on photoreceptor metabolism, effects of light modulation on the severity of diabetic retinopathy, and recent clinical trials testing the treatment of diabetic retinopathy with interventions that impact photoreceptor metabolism. Finally, we introduce several possible mechanisms that could link photoreceptor responses to light and the development of retinal vascular disease in diabetes. Collectively, these concepts form the basis for a growing body of investigative efforts aimed at developing novel pharmacologic and nonpharmacologic tools that target photoreceptor physiology to treat a very common cause of blindness across the world.

While the concept of a dermal light sense has existed for over a century, little progress has been made in our understanding of the mechanisms underlying dispersed photoreception and the evolutionary histories of dispersed photoreceptor cells. These cells historically have been difficult to locate and positively identify, but modern molecular techniques, integrated with existing behavioral, morphological, and physiological data, will make cell identification easier and allow us to address questions of mechanism and evolution. With this in mind, we propose a new classification scheme for all photoreceptor cell types based on two axes, cell distribution (aggregated vs. dispersed) and position within neural networks (first order vs. high order). All photoreceptor cells fall within one of four quadrants created by these axes: aggregated/high order, dispersed/high order, aggregated/first order, or dispersed/first order. This new method of organization will help researchers make objective comparisons between different photoreceptor cell types. Using integrative data from four major phyla (Mollusca, Cnidaria, Echinodermata, and Arthropoda), we also provide evidence for three hypotheses for dispersed photoreceptor cell function and evolution. First, aside from echinoderms, we find that animals often use dispersed photoreceptor cells for tasks that do not require spatial vision. Second, although there are both echinoderm and arthropod exceptions, we find that dispersed photoreceptor cells generally lack morphological specializations that either enhance light gathering or aid in the collection of directional information about light. Third, we find that dispersed photoreceptor cells have evolved a number of times in Metazoa and that most dispersed photoreceptor cells have likely evolved through the co-option of existing phototransduction cascades. Our new classification scheme, combined with modern investigative techniques, will help us address these hypotheses in great detail and generate new hypothesis regarding the function and evolution of dispersed photoreceptor cells.

The cyclic GMP-gated channel responds to changes in free intracellular cGMP, and as a result, it plays a central role in the phototransduction process in rod and cone photoreceptor cells. Recent biochemical, immunochemical, and molecular biology studies indicate that this channel consists of a complex of two distinct subunits and one or more associated proteins. Primary structural analysis indicates that the a and (3 subunits contain a cGMP-binding domain, an even number of membrane-spanning segments, a voltage sensor motif and a pore region. The latter two features are found in voltage-gated channels and suggest that these two classes of channels have evolved from the same ancestral channel protein. A working model for the membrane topography of the channel subunits is proposed based on immunogold labeling studies and sequence analysis. Recent studies also indicate that calmodulin binds to the 240 kDa protein of the channel complex and modulates the sensitivity of the channel for cGMP in a Ca2+-dependent manner. The molecular properties of the channel complex and the possible role of Ca2+-calmodulin modulation of the channel during photoactivation and photorecovery are discussed in relation to the current mechanism of phototransduction in photoreceptor cells

Recoverin is a 23 kDa Ca2+binding protein that has been detected primarily in vertebrate photoreceptors. The role of recoverin in phototransduction has been investigated using a variety of biochemical methods. Initial reports suggesting that recoverin regulates photoreceptor guanylyl cyclase have not been confirmed. Instead, recoverin appears to determine the lifetime of lightstimulated phosphodiesterase activity, perhaps by regulating rhodopsin phosphorylation. Retinal recoverin is heterogeneously fatty acylated at its ammo-terminus. The amino-terminal fatty acid appears to be involved in the interaction of recoverin with photoreceptor membranes.

In the human eye, domination of the electroretinogram (ERG) by the b−wave and other postreceptor components ordinarily obscures all but the first few milliseconds of the rod photoreceptor response to a stimulating flash. However, recovery of the rod response after a bright test flash can be analyzed using a paired-flash paradigm in which the test flash, presented at time zero, is followed at time t by a bright probe flash that rapidly saturates the rods (Birch et al., 1995). In ERG experiments on normal subjects, the hypothesis that a similar method can be used to obtain the full time course of the rod response to test flashes of subsaturating intensity was tested. Rod-only responses to probe flashes presented at varying times t after the test flash were used to derive a family of amplitudes A(t) that represented the putative rod response to the test flash. These rod-only responses to the probe flash were obtained by computational subtraction of the cone-mediated component of each probe flash response. With relatively weak test flashes (11–15 scot-td-s), the time course of the rod response to the test flash derived in this manner was consistent with a four-stage impulse response function of time-to-peak ≃170 ms. A(170), the amplitude of the derived response at 170 ms, increased with test flash intensity (Itest) to a maximum value Amo and exhibited a dependence on Itest given approximately by the relation, A(170)/Amo = 1 - exp(-kItest), where k = 0.092 (scot-td-s)−1. In steady background light, the falling (i.e. recovery) phase of the derived response began earlier, and the sensitivity parameter k was reduced several-fold from its dark-adapted value. As the sensitivity, kinetics, and light-adaptation properties of the derived response correspond closely with those of photocurrent flash responses previously obtained from isolated rods in vitro, it was concluded that the response derived here from the human ERG approximates the course of the massed in vivo rod response to a test flash.

The effect of light adaptation on the period of photocurrent saturation induced by a bright stimulating flash was examined in rod photoreceptors of the larval-stage tiger salamander (Ambystoma tigrinum). Using suction electrodes, photocurrent responses to brief flashes were recorded from single, isolated rods in the presence and absence of steady background illumination. Background light decreased the saturation period (T) measured at fixed flash intensity (fixed If) and in this respect light-adapted the saturating response. Effects of the background on responses to weak (i.e. subsaturating) and bright flashes were compared with changes in a parameter, where ΔT is the decrease in saturation period, and where TR* is the slope of the line that relates T and ln If in a given state of adaptation. Dark- and light-adapted responses to flash intensities and , respectively, exhibited similar absolute peak photocurrent and falling-phase kinetics when and satisfied the relation, , where Ib is the background intensity. It is argued that ψ approximates the relative PDE*/R* gain of transduction, i.e. the relative peak level of activated cGMP phosphodiesterase (PDE*) produced by a given, small amount of photoactivated visual pigment (R*). Interpreted on this view, the results imply that light adaptation derives largely from a decrease in PDE*/R* gain, rather than from the stimulation of guanylate cyclase activity. The data are consistent with the possibility that modulation of the lifetime of PDE* underlies the background dependence of ψ.

Desensitization of cGMP-activated channels was examined in excised, inside-out patches obtained from rod photoreceptors. Cyclic GMP was applied using a rapid-flow system in which concentration jumps are complete within 10–50 ms. In outer-segment patches containing many channels, the cGMP-dependent conductance reached a steady plateau that was maintained for tens of seconds in the presence of cGMP; thus, there was no indication of slow desensitization. However, rapid desensitization on the scale of milliseconds could not be ruled out because of limited speed of access of cGMP to the inner face of the patch membrane. To test for rapid desensitization, inner-segment patches containing only a single cGMP-activated channel were used. In these one-channel patches, there was no change in activity of the channel with time from its earliest onset after application of cGMP, indicating that rapid desensitization also did not occur.

Although inositol trisphosphate (InsP3) is a key substance in phototransduction of invertebrate photoreceptors, its intracellular concentration remains unknown. The purpose of this study was to assay its concentration and the concentration of its precursor, phosphatidylinositol bisphosphate (PtdInsP2), within squid photoreceptors. Rhabdomeric membranes were purified and their PtdInsP2 content measured with a phosphate assay after the extracted phospholipids were deacylated and separated by ion-exchange chromatography. At least 75% of the total PtdInsP2 found in the retinal homogenate was associated with the plasma membranes of the rhabdomeric microvilli, where PtdInsP2 was 3.1 ± 0.7% of the total phospholipids, a level comparable to values published for rat brain. In terms of rhodopsin, microvillar membranes contained 3.7 ± 0.9 mol PtdInsP2/mol rho. The InsP3 content of living retinas was measured with a radioreceptor assay. The basal content of dark-adapted retinas was 0.15 ± 0.05 InsP3/rho, equivalent to 30 ± 9 nmol/g tissue that is about twice that of rat brains. Flash illumination (≈lms in duration) that photoactivated 1% of rhodopsin increased the level about fivefold to 0.68 ± 0.22 lnsP3/rho. Corresponding decrease in PtdInsP2 was undetectable as it was within measurement errors. For PtdInsP2, the measured content corresponds to 5.6 ± 1.4 mM within the volume of rhabdomere. Maximal light-induced concentration of InsP3 is calculated to be 1.2 ± 0.4 mM within the cytoplasm of the distal segment. Each photoactivated rhodopsin leads to the formation of 500 InsP3 molecules when measured 15 s after the flash. Thus, high concentration of InsP3 in these cells is primarily due to restricted intracellular volumes rather than to high amplification by the enzyme cascade. The InsP3 concentration within squid photoreceptors is the highest yet reported for any transducing cell and may indicate the involvement of relatively low affinity receptors or channels during invertebrate phototransduction.

Following bright flashes, rod photoreceptors exhibit a period of photocurrent saturation that increases linearly with the logarithm of flash intensity. In a recent report, Pepperberg et al. (1992) presented evidence that the slope of the function relating the saturation period (T) to the natural logarithm of flash intensity (In If) represents the exponential lifetime (τ) of photoactivated visual pigment: τ = ΔT/Δ[ln If]. In salamander rods, 11 -cis 9-desmethylretinal combines with opsin to form 9-desmethyl rhodopsin. Dim flash responses mediated by this analogue visual pigment exhibited slow recovery kinetics relative to those of native pigment (Corson et al., 1991). This observation raises the hypothesis that the physiological lifetime of photoactivated 9-desmethyl rhodopsin is substantially longer than that of native visual pigment. To test this hypothesis, we have examined the relation between the period of photocurrent saturation and flash intensity in salamander rods containing a mixture of the two pigments. Brief stimuli at two widely separated wavelengths (440 and 640 nm) elicited saturating photocurrent responses that were preferentially mediated by 9-desmethyl rhodopsin or residual native pigment, respectively. Plots of T vs. In If revealed a linear increase in the period of response saturation over a large range of saturating intensities at both wavelengths. However, the slope of the relation between T and In If with 440-nm flashes was more than twice as large (4.1 ± 0.5 s, n = 5) as that measured with 640-nm flashes (1.7 ± 0.4 s). For rods subjected only to bleaching of the native pigment, or to bleaching and resensitization with 11-cis retinal, the slope of the relation between T and In If remained independent of wavelength and indistinguishable from that of native pigment in unbleached cells. The data provide support for the hypothesis that the slope parameter τ represents the lifetime of photoactivated pigment, and specifically suggest that the lifetime of photoactivated 9-desmethyl rhodopsin is abnormally long.

Illumination greatly increases the GTPase activity in homogenates of squid (Loligo) whole retinas or rhabdomeric membranes. Adenylylimidodiphosphate inhibits the light-insensitive (but not the light-sensitive) GTPase activity in these homogenates. Illumination also greatly increases the binding of GTP[γS] to the rhabdomeric membranes. This binding at saturating illuminations indicates that there are approximately 10–100 times more rhodopsin molecules than G-protein molecules in squid photoreceptors. Each light-activated rhodopsin molecule activates about 10 G-protein molecules which might provide amplification for the first stage of the phototransduction cascade.

A method for constructing nonlinear models for light adaptation in the retina is introduced. The components of the models are linear filters and static (instantaneous) nonlinear elements configured in a feedback arrangement. The signals in the models are combined through algebraic addition or multiplication. We apply the method to model light adaptation measured in turtle horizontal cells. Given a particular wiring diagram for the components, the functional forms of the static nonlinearities and frequency responses of the linear filters are determined by constraining the model to give temporal frequency responses (linear regime behavior) consistent with a family of linear feedback models which has been shown to provide a good description of adaptation in these cells. Two particular models, quite different in structure, are presented. Each model responds to perturbations around a mean light level as a feedback circuit in which the gain (strength) of feedback is adjusted to be proportional to the mean light level, but neither model has a separate pathway for measuring the mean light level. Thus, each of these nonlinear models embeds an entire family of linear models parametric in mean light level. Harmonic distortion in the responses of these models to sinusoidal input is found to be qualitatively consistent with physiological data. An alternative class of nonlinear models in which feedback gain is set by a separate slow pathway which tracks the mean light level is ruled out on the basis of its incorrect steady-state input-output behavior. The methods presented can be used to develop specific physical models for light adaptation based on the chemical kinetics of phototransduction or on nonlinear neural feedback. The relevance of the nonlinear models and construction techniques to modeling phototransduction is discussed.

The phototransduction cascade in invertebrates involves the coupling of rhodopsin activation to the action of the enzyme phospholipase C. This step is performed by G-proteins. An antibody against the α-subunit of a mouse Gq type G-protein recognized protein bands in Western blots of lateral eye and ventral nerve photoreceptors of Limulus. The protein bands had an apparent molecular mass of about 42 kDa. The antibody also recognized protein bands of a similar molecular mass in immunoblots of brain and intestine tissue. Immunoreactivity was found in lateral eye frozen sections where it was confined to the rhabdom region. When the antibody was applied to ultrathin sections of ventral nerve photoreceptors, the highest density of labeling was found on the rhabdomeral microvilli, but gold particles were also scattered in the cytoplasm. We conclude that a G-protein of the type Gq participates in the phototransduction of Limulus.

The dark current of single isolated toad rods was monitored by drawing either the inner segment or the outer segment into a suction electrode. The potassium-channel blockers tetraethylammonium (TEA) and 3,4-diaminopyridine (DAP) reduced the amplitude of the dark current when applied to the inner segment. Both drugs were less effective when applied to the outer segment, suggesting that they act at the inner segment to block part of the outward path for the dark current. In addition, DAP affected the kinetics of the light response, possibly by affecting internal pH.

Physiological studies of intracellular messengers frequently employ intracellular injections of large molecules that either monitor or modulate the metabolism of the messenger cascade. Injected molecules have unknown mobility in the cytosol and unknown accessibility to various cytosolic compartments, including those postulated to be traversed by intracellular messenger molecules. In order to determine whether injected molecules have access to the confined spaces through which messenger molecules must diffuse, we injected 5-nm colloidal gold or horseradish peroxidase, or both, into Limulus ventral photoreceptors. Injections were made by applying pressure pulses to the back of an intracellular micropipette that also monitored membrane voltage. The tissue was fixed at varying times after injection and processed for electron microscopy by conventional techniques. Cells fixed 1–3 min after injection contained HRP reaction product only in the cell body. HRP reaction product was found at varying distances down axons in direct relation to the interval between injection and fixation. Colloidal gold particles were found throughout the cell body but not in axons of tissue fixed 1–3 min after injection. Both HRP reaction product and 5-nm colloidal gold particles were observed within the microvillar projections of internal and external rhabdomere, as well as within the extracisternal spaces of endoplasmic reticulum. We conclude that large molecules injected from an intracellular micropipette into an arbitrary locus of ventral photoreceptor cells have access to all of the presumed sites of the phototransduction cascade.

Inside-out patches containing cGMP-gated channels were excised from catfish rod or cone outer segments and held under voltage clamp. The net cGMP-dependent currents elicited by saturating and subsaturating concentrations of cGMP at ±30 mV were measured and the dependence of current upon cGMP concentration was determined. The apparent affinity of the channel for its ligand was estimated by fitting these data with the Hill equation. The concentration of cGMP required to give half the maximum current (K1/2) in rod and cone channels at +30 mV was ~28 μM and ~37 μM, respectively, and was weakly voltage dependent. Thus, cone channels have an intrinsically higher K1/2 than rod channels. For both types of channel, the Hill coefficient was ~2.3. In the presence of calcium-calmodulin, the apparent affinity of the rod channel for cGMP decreased by about twofold, but the apparent affinity of the cone channels was unaffected. These results indicate that the open probability of the cone channel for its ligand cannot be modulated by calmodulin. This represents the first significant departure between rod and cone photoreceptors in mechanisms used by phototransduction and suggests that the β subunit of the cone channel must be different from that of the rod channel.

A protocol was developed to isolate enzymatically photoreceptors from the retina of the squid, Loligo pealei. The procedure routinely results in a high yield of intact cells. Examination of solitary photoreceptors under Nomarski optics revealed that the fine morphological features described in anatomical studies of retinal sections are retained. The distal segment is up to 250 μm long, 4–7 μm wide, covered in part by short microvilli; the inner segment and the cell body, with the initial portion of the axon, are also clearly discernible in solitary cells. Suction electrode measurements performed from the cell body confirmed that responsiveness to light survived cell isolation. Macroscopic membrane currents were measured using the whole-cell tight-seal technique, and the perforated-patch method. Step depolarizations of membrane voltage administered in the dark elicited a slowly activating, sustained outward current. Light stimulation evoked an inward current graded with stimulus intensity; the peak current could amply exceed 1000 pA. Intense photostimulation gave rise to a prolonged inward aftercurrent that lasted for tens of seconds. On-cell patch recording along the intermediate segment and most of the smooth areas of the distal segment showed a large incidence of silent patches, with the occasional presence of voltage-dependent channels. On the other hand, channel activity could be recorded more frequently from electrode placements near the apical tip of the cell, where the presence of microvilli could be confirmed visually. Some patches were unresponsive to voltage Stimulation applied in the dark but produced distinct bursts of channel openings after illumination. The feasibility of single-cell electrophysiology in isolated photoreceptors, together with the growing body of biochemical information on cephalopod preparations, makes squid an attractive model system to investigate the visual process in invertebrates using multiple experimental approaches.

We examined a white-eyed strain of the norpA mutant (norpA;cn bw) and white (w)norpA+ controls using microspectrophotometry (MSP), electron microscopy (EM), and electroretinography (ERG). These studies revealed that light mediates receptor demise in norpA even though norpA lacks phototransduction. Rhodopsin and the rhabdomere which houses it decrease with increasing age in norpA but not in w with rearing on a 12 h light/12-h dark cycle or in constant light. At higher temperature in norpA;cn bw and w reared in constant light, visual pigment decreases, rhabdomeres diminish, and cells die. Importantly, dark rearing blocked visual pigment loss in norpA;cn bw; the M-potential, an ERG reflection of visual pigment level, corroborated this finding. MSP showed that norpA's visual pigment loss was not due to acute loss of metarhodopsin, rhodopsin's photoproduct. NorpA blocks certain processes expected to be light elicited. The alteration of visual pigment as a function of time of day, present in w controls, is absent in white-eyed norpA, suggesting that light-induced depolarization may be necessary to entrain the rhythm. Microspectrofluorometry using the fluorescent dye, Lucifer yellow, suggested that norpA lacks a light-induced uptake mechanism; using control flies, we determined the stimulus parameters required for uptake in vivo. An attempt to “cure” norpA;cn bw by replacement “therapy” using phospholipase C, missing in norpA's phototransduction cascade, was largely unsuccessful.

Efferent fibers from a central circadian clock innervate photoreceptors along the ventral nerve of Limulus and release octopamine when active. We have recorded ERG-like responses from the ventral eye in vivo over several day periods. We have also used intracellular microelectrodes to study changes in ventral photoreceptor function during exogenous applications of octopamine (the putative efferent neurotransmitter), IBMX (a phosphodiesterase inhibitor), and forskolin (an adenylate cyclase activator): (1) Responses to light measured at night from ventral photoreceptors in vivo are greater in amplitude than those recorded during the day; (2) Octopamine and agents that increase intracellular levels of cAMP in ventral photoreceptors decrease the rate of spontaneous (dark) bumps, increase photoreceptor response to light without changing threshold, and often increase the bump duration; and (3) These changes in function of ventral photoreceptors are similar to those that have been observed in the photoreceptor of the lateral eye during circadian clock activity at night, and in vitro in the presence of those same pharmacological agents.

We have developed a new model of phototransduction that accounts for the dynamics of primate and human cone flash responses in both their linear and saturating range. The model incorporates many of the known elements of the phototransduction cascade in vertebrate photoreceptors. The input stage is a new analytic expression for the activation and inactivation of cGMP-phosphodiesterase (PDE). Although the Lamb and Pugh (1992) model (of a delayed ramp for the rising phase of the PDE* response in amphibian rods) provided a good fit for the first 2 log units of stimulus intensity without parameter adjustments, the remaining 4 log units of the data required nonlinear modifications of both delay and gain (slope). We show that this nonlinear behavior is a consequence of the delay approximation and develop a completely linear model to account for the rising phase of amphibian rod photocurrent responses over the full intensity range (~6 log units). We use the same dynamic model to account for primate cone responses by decreasing the time constants of PDE activation and introducing an enhanced inactivation process. This PDE* response activates a nonlinear calcium feedback stage that modulates guanylate cyclase synthesis of cyclic GMP. By adjustment of the throughput and feedback parameters, the full model successfully captures most of the features of the primate and human cone flash responses throughout their dynamic range. Our analysis suggests that initial processes in the transduction cascade may be qualitatively different from comparable processes in rods.

We have investigated by immunofluorescence the appearance of immunoreactive guanosine 3′-5′ cyclic monophosphate phosphodiesterase (cGMP-PDE) during the postnatal development of the retina of the pigmented rat. We show that a sudden increase in immunoreactivity takes place during postnatal day five (P5), when rod outer segments begin to form; immunoreactivity develops rapidly in the following days. Labeling is restricted to the developing photoreceptor outer segments, sparing other retinal cells, as confirmed by electron microscopy immunocytochemistry. In addition, cGMP-PDE immunoreactivity follows a center-to-periphery gradient paralleling photoreceptor differentiation. It appears that cGMP-PDE is expressed when the photoreceptor subcellular compartments are already formed, and represents a specific marker of late photoreceptor differentiation. The appearance of cGMP-PDE during development is temporally correlated with the appearance of other proteins of the phototransduction machinery.