The continuous function of vertebrate photoreceptors requires regeneration of their visual pigment following its destruction upon activation by light (photobleaching). For rods, the chromophore required for the regeneration of rhodopsin is derived from the adjacent retinal pigmented epithelium (RPE) cells through a series of reactions collectively known as the RPE visual cycle. Mounting biochemical and functional evidence demonstrates that, for cones, pigment regeneration is supported by the parallel supply with chromophore by two pathways-the canonical RPE visual cycle and a second, cone-specific retina visual cycle that involves the Müller glial cells in the neural retina. In this article, we review historical information that led to the discovery of the retina visual cycle and discuss what is currently known about the reactions and molecular components of this pathway and its functional role in supporting cone-mediated vision.

Vertebrate rod and cone photoreceptors require continuous supply of chromophore for regenerating their visual pigments after photoactivation. Cones, which mediate our daytime vision, demand a particularly rapid supply of 11-cis retinal chromophore in order to maintain their function in bright light. An important contribution to this process is thought to be the chromophore precursor 11-cis retinol, which is supplied to cones from Müller cells in the retina and subsequently oxidized to 11-cis retinal as part of the retina visual cycle. However, the molecular identity of the cis retinol oxidase in cones remains unclear. Here, as a first step in characterizing this enzymatic reaction, we sought to determine the subcellular localization of this activity in salamander red cones. We found that the onset of dark adaptation of isolated salamander red cones was substantially faster when exposing directly their outer vs. their inner segment to 9-cis retinol, an analogue of 11-cis retinol. In contrast, this difference was not observed when treating the outer vs. inner segment with 9-cis retinal, a chromophore analogue which can directly support pigment regeneration. These results suggest, surprisingly, that the cis-retinol oxidation occurs in the outer segments of cone photoreceptors. Confirming this notion, pigment regeneration with exogenously added 9-cis retinol was directly observed in the truncated outer segments of cones, but not in rods. We conclude that the enzymatic machinery required for the oxidation of recycled cis retinol as part of the retina visual cycle is present in the outer segments of cones.

The Arg838Ser mutation in retinal membrane guanylyl cyclase 1 (RetGC1) has been linked to autosomal dominant cone-rod dystrophy type 6 (CORD6). It is believed that photoreceptor degeneration is caused by the altered sensitivity of RetGC1 to calcium regulation via guanylyl cyclase activating proteins (GCAPs). To determine the mechanism by which this mutation leads to degeneration, we investigated the structure and function of rod photoreceptors in two transgenic mouse lines, 362 and 379, expressing R838S RetGC1. In both lines, rod outer segments became shorter than in their nontransgenic siblings by 3-4 weeks of age, before the eventual photoreceptor degeneration. Despite the shortening of their outer segments, the dark current of transgenic rods was 1.5-2.2-fold higher than in nontransgenic controls. Similarly, the dim flash response amplitude in R838S+ rods was larger, time to peak was delayed, and flash sensitivity was increased, all suggesting elevated dark-adapted free cGMP in transgenic rods. In rods expressing R838S RetGC1, dark-current noise increased and the exchange current, detected after a saturating flash, became more pronounced. These results suggest disrupted Ca2+ phototransduction feedback and abnormally high free-Ca2+ concentration in the outer segments. Notably, photoreceptor degeneration, which typically occurred after 3 months of age in R838S RetGC1 transgenic mice in GCAP1,2+/+ or GCAP1,2+/- backgrounds, was prevented in GCAP1,2-/- mice lacking Ca2+ feedback to guanylyl cyclase. In summary, the dysregulation of guanylyl cyclase in RetGC1-linked CORD6 is a "phototransduction disease," which means it is associated with increased free-cGMP and Ca2+ levels in photoreceptors.SIGNIFICANCE STATEMENT In a mouse model expressing human membrane guanylyl cyclase 1 (RetGC1, GUCY2D), a mutation associated with early progressing congenital blindness, cone-rod dystrophy type 6 (CORD6), deregulates calcium-sensitive feedback of phototransduction to the cyclase mediated by guanylyl cyclase activating proteins (GCAPs), which are calcium-sensor proteins. The abnormal calcium sensitivity of the cyclase increases cGMP-gated dark current in the rod outer segments, reshapes rod photoresponses, and triggers photoreceptor death. This work is the first to demonstrate a direct physiological effect of GUCY2D CORD6-linked mutation on photoreceptor physiology in vivo It also identifies the abnormal regulation of the cyclase by calcium-sensor proteins as the main trigger for the photoreceptor death.

Bleaching adaptation in rod photoreceptors is mediated by apo-opsin, which activates phototransduction with effective activity 10⁵- to 10⁶-fold lower than that of photoactivated rhodopsin (meta II). However, the mechanism that produces such low opsin activity is unknown. To address this question, we sought to record single opsin responses in mouse rods. We used mutant mice lacking efficient calcium feedback to boosts rod responses and generated a small fraction of opsin by photobleaching ∼1% of rhodopsin. The bleach produced a dramatic increase in the frequency of discrete photoresponse-like events. This activity persisted for hours, was quenched by 11-cis-retinal, and was blocked by uncoupling opsin from phototransduction, all indicating opsin as its source. Opsin-driven discrete activity was also observed in rods containing non-activatable rhodopsin, ruling out transactivation of rhodopsin by opsin. We conclude that bleaching adaptation is mediated by opsin that exists in equilibrium between a predominant inactive and a rare meta II-like state.SIGNIFICANCE STATEMENT Electrophysiological analysis is used to show that the G-protein-coupled receptor opsin exists in equilibrium between a predominant inactive and a rare highly active state that mediates bleaching adaptation in photoreceptors.

Rhodopsin is the critical receptor molecule which enables vertebrate rod photoreceptor cells to detect a single photon of light and initiate a cascade of molecular events leading to visual perception. Recently, it has been suggested that the F45L mutation in the transmembrane helix of rhodopsin disrupts its dimerization in vitro To determine whether this mutation of rhodopsin affects its signaling properties in vivo, we generated knock-in mice expressing the rhodopsin F45L mutant. We then examined the function of rods in the mutant mice versus wild-type controls, using in vivo electroretinography and transretinal and single cell suction recordings, combined with morphologic analysis and spectrophotometry. Although we did not evaluate the effect of the F45L mutation on the state of dimerization of the rhodopsin in vivo, our results revealed that F45L-mutant mice exhibit normal retinal morphology, normal rod responses as measured both in vivo and ex vivo, and normal rod dark adaptation. We conclude that the F45L mutation does not affect the signaling properties of rhodopsin in its natural setting.

Purpose

Diabetes appears to induce a visual cycle defect because rod dysfunction is correctable with systemic treatment of the visual cycle chromophore 11-cis-retinaldehyde. However, later studies have found no evidence for visual cycle impairment. Here, we further examined whether photoreceptor dysfunction is corrected with 11-cis-retinaldehyde. Because antioxidants correct photoreceptor dysfunction in diabetes, the hypothesis that exogenous visual chromophores have antioxidant activity in the retina of diabetic mice in vivo was tested.

Methods

Rod function in 2-month-old diabetic mice was evaluated using transretinal electrophysiology in excised retinas and apparent diffusion coefficient (ADC) MRI to measure light-evoked expansion of subretinal space (SRS) in vivo. Optokinetic tracking was used to evaluate cone-based visual performance. Retinal production of superoxide free radicals, generated mostly in rod cells, was biochemically measured with lucigenin. Diabetic mice were systemically treated with a single injection of either 11-cis-retinaldehyde, 9-cis-retinaldehyde (a chromophore surrogate), or all-trans-retinaldehyde (the photoisomerization product of 11-cis-retinaldehyde).

Results

Consistent with previous reports, diabetes significantly reduced (1) dark-adapted rod photo responses (transretinal recording) by ∼18%, (2) rod-dominated light-stimulated SRS expansion (ADC MRI) by ∼21%, and (3) cone-dominated contrast sensitivity (using optokinetic tracking [OKT]) by ∼30%. Both 11-cis-retinaldehyde and 9-cis-retinaldehyde largely corrected these metrics of photoreceptor dysfunction. Higher-than-normal retinal superoxide production in diabetes by ∼55% was also significantly corrected following treatment with 11-cis-retinaldehyde, 9-cis-retinaldehyde, or all-trans-retinaldehyde.

Conclusions

Collectively, data suggest that retinaldehydes improve photoreceptor dysfunction in diabetic mice, independent of the visual cycle, via an antioxidant mechanism.

Purpose

Light detection in retinal rod photoreceptors is initiated by activation of the visual pigment rhodopsin. A critical, yet often-overlooked, step enabling efficient perception of light is rhodopsin dephosphorylation mediated by protein phosphatase 2A (PP2A). PP2A deficiency has been reported to impair rhodopsin regeneration after phosphorylation by G protein receptor kinase 1 (GRK1) and binding of arrestin (Arr1), thereby delaying rod dark adaptation. However, its effects on the viability of photoreceptors in the absence of GRK1 and Arr1 remain unclear. Here, we investigated the effects of PP2A deficiency in the absence of GRK1 or Arr1, both of which have been implicated in Oguchi disease, a form of night blindness.

Methods

Rod-specific mice lacking the predominant catalytic Cα-subunit of PP2A were crossed with the Grk1-/- or Arr1-/- strains to obtain double knockout lines. Rod photoreceptor viability was analyzed in histological cross-sections of the retina stained with hematoxylin and eosin, and rod function was evaluated by ex vivo electroretinography.

Results

PP2A deficiency alone did not impair photoreceptor viability up to 12 months of age. Retinal degeneration was more pronounced in rods lacking GRK1 compared to rods lacking Arr1, and degeneration was accelerated in both Grk1-/- or Arr1-/- strains where PP2A was also deleted. In Arr1-/- mice, rod maximal photoresponse amplitudes were reduced by 80% at 3 months, and this diminution was enhanced further with concomitant PP2A deficiency.

Conclusions

These results suggest that although PP2A is not required for the survival of rods, its deletion accelerates the degeneration induced by the absence of either GRK1 or Arr1.

Two-photon vision arises from the perception of pulsed infrared (IR) laser light as color corresponding to approximately half of the laser wavelength. The physical process responsible for two-photon vision in rods has been delineated and verified experimentally only recently. Here, we sought to determine whether IR light can also be perceived by mammalian cone photoreceptors via a similar activation mechanism. To investigate selectively mammalian cone signaling in mice, we used animals with disabled rod signal transduction. We found that, contrary to the expected progressive sensitivity decrease based on the one-photon cone visual pigment spectral template, the sensitivity of mouse cone photoreceptors decreases only up to 800 nm and then increases at 900 nm and 1000 nm. Similarly, in experiments with the parafoveal region of macaque retinas, we found that the spectral sensitivity of primate cones diverged above the predicted one-photon spectral sensitivity template beyond 800 nm. In both cases, efficient detection of IR light was dependent on minimizing the dispersion of the ultrashort light pulses, indicating a non-linear two-photon activation process. Together, our studies demonstrate that mammalian cones can be activated by near IR light by a nonlinear two-photon excitation. Our results pave the way for the creation of a two-photon IR-based ophthalmoscope for the simultaneous imaging and functional testing of human retinas as a novel tool for the diagnosis and treatment of a wide range of visual disorders.

Calcium regulates the response sensitivity, kinetics and adaptation in photoreceptors. In striped bass cones, this calcium feedback includes direct modulation of the transduction cyclic nucleotide-gated (CNG) channels by the calcium-binding protein CNG-modulin. However, the possible role of EML1, the mammalian homolog of CNG-modulin, in modulating phototransduction in mammalian photoreceptors has not been examined. Here, we used mice expressing mutant Eml1 to investigate its role in the development and function of mouse photoreceptors using immunostaining, in-vivo and ex-vivo retinal recordings, and single-cell suction recordings. We found that the mutation of Eml1 causes significant changes in the mouse retinal structure characterized by mislocalization of rods and cones in the inner retina. Consistent with the fraction of mislocalized photoreceptors, rod and cone-driven retina responses were reduced in the mutants. However, the Eml1 mutation had no effect on the dark-adapted responses of rods in the outer nuclear layer. Notably, we observed no changes in the cone sensitivity in the Eml1 mutant animals, either in darkness or during light adaptation, ruling out a role for EML1 in modulating cone CNG channels. Together, our results suggest that EML1 plays an important role in retina development but does not modulate phototransduction in mammalian rods and cones.