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Signal integration of converging neural circuits is poorly understood. One example is in the retina where the integration of rod and cone signaling is responsible for the large dynamic range of vision. The relative contribution of rods versus cones is dictated by a complex function involving background light intensity and stimulus temporal frequency. One understudied mechanism involved in coordinating rod and cone signaling onto the shared retinal circuit is the hyperpolarization activated current (Ih) mediated by hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels expressed in rods and cones.Ihopposes membrane hyperpolarization driven by activation of the phototransduction cascade and modulates the strength and kinetics of the photoreceptor voltage response. We examined conditional knock-out (KO) of HCN1 from mouse rods using electroretinography (ERG). In the absence of HCN1, rod responses are prolonged in dim light which altered the response to slow modulation of light intensity both at the level of retinal signaling and behavior. Under brighter intensities, cone-driven signaling was suppressed. To our surprise, conditional KO of HCN1 from mouse cones had no effect on cone-mediated signaling. We propose thatIhis dispensable in cones because of the high level of temporal control of cone phototransduction. Thus, HCN1 is required for cone-driven retinal signaling only indirectly by modulating the voltage response of rods to limit their output.SIGNIFICANCE STATEMENTHyperpolarization gated hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels carry a feedback current that helps to reset light-activated photoreceptors. Using conditional HCN1 knock-out (KO) mice we show that ablating HCN1 from rods allows rods to signal in bright light when they are normally shut down. Instead of enhancing vision this results in suppressing cone signaling. Conversely, ablating HCN1 from cones was of no consequence. This work provides novel insights into the integration of rod and cone signaling in the retina and challenges our assumptions about the role of HCN1 in cones.

The voltage-gated potassium channel responsible for controlling photoreceptor signaling is a heteromeric complex of Kv2.1 subunits with a regulatory Kv8.2 subunit. Kv2.1/Kv8.2 channels are localized to the photoreceptor inner segment and carry IKx, largely responsible for setting the photoreceptor resting membrane potential. Mutations in Kv8.2 result in childhood-onset Cone Dystrophy with Supernormal Rod Response (CDSRR). We generated a Kv8.2 knockout (KO) mouse and examined retinal signaling and photoreceptor degeneration to gain deeper insight into the complex phenotypes of this disease. Using electroretinograms we show that there is a tradeoff between delayed or reduced signaling from rods depending on the intensity of the light stimulus, consistent with reduced capacity for light-evoked changes in membrane potential. The delayed response was not seen ex vivo where extracellular potassium levels are the same, so we conclude the in vivo alteration is influenced by ionic imbalance. We observed mild retinal degeneration. Signaling from cones was reduced but there was no loss of cone density. Loss of Kv8.2 altered responses to flickering light with responses attenuated at high frequencies and altered in shape at low frequencies. The Kv8.2 KO line on an all-cone retina background had reduced cone signaling associated with degeneration. We conclude that Kv8.2 is required by rods and cones for responding to dynamic changes in lighting. The timing and cell type affected by degeneration is different in the mouse and human but there is a window of time in both for therapeutic intervention.

Delivering therapeutics to the posterior segment of the eye is challenging due to various anatomical and physical barriers. While significant improvements have been realized by introducing direct injections to diseased sites, these approaches come with potential side effects that can range from simple inflammation to severe retinal damage. The topical instillation of drugs remains a safer and preferred alternative for patients’ compliance. Here, we report the synthesis of penetratin-complexed, redox-responsive hyaluronic acid-based nanogels for the triggered release and delivery of therapeutics to the posterior part of the eye via topical application. The synthesized nanogels were shown to release their load only when exposed to a reducing environment, similar to the cytoplasm. As a model drug, visual chromophore analog, 9-cis-retinal, was loaded into nanogels and efficiently delivered to the mouse retina’s photoreceptors when applied topically. Electroretinogram measurements showed a partial recovery of photoreceptor function in all treated eyes versus untreated controls. To the best of our knowledge, this report constitutes the first attempt to use a topically applied triggered-release drug delivery system to target the pigmented layer of the retina, in addition to the first attempt to deliver the visual chromophore topically.

Mitochondrial dysfunction is an important cause of heritable vision loss. Mutations affecting mitochondrial bioenergetics may lead to isolated vision loss or life-threatening systemic disease, depending on a mutation's severity. Primary optic nerve atrophy resulting from death of retinal ganglion cells is the most prominent ocular manifestation of mitochondrial disease. However, dysfunction of other retinal cell types has also been described, sometimes leading to a loss of photoreceptors and retinal pigment epithelium that manifests clinically as pigmentary retinopathy. A popular mouse model of mitochondrial disease that lacks NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4), a subunit of mitochondrial complex I, phenocopies many traits of the human disease Leigh syndrome, including the development of optic atrophy. It has also been reported that ndufs4(−/−) mice display diminished light responses at the level of photoreceptors or bipolar cells. By conducting electroretinography (ERG) recordings in live ndufs4(−/−) mice, we now demonstrate that this defect occurs at the level of retinal photoreceptors. We found that this deficit does not arise from retinal developmental anomalies, photoreceptor degeneration, or impaired regeneration of visual pigment. Strikingly, the impairment of ndufs4(−/−) photoreceptor function was not observed in ex vivo ERG recordings from isolated retinas, indicating that photoreceptors with complex I deficiency are intrinsically capable of normal signaling. The difference in electrophysiological phenotypes in vivo and ex vivo suggests that the energy deprivation associated with severe mitochondrial impairment in the outer retina renders ndufs4(−/−) photoreceptors unable to maintain the homeostatic conditions required to operate at their normal capacity.

Cone photoreceptors are essential for vision under moderate to high illuminance and allow color discrimination. Their fast dark adaptation rate and resistance to saturation are believed to depend in part on an intraretinal visual cycle that supplies 11- cis-retinaldehyde to cone opsins. Candidate enzymes of this pathway have been reported, but their physiologic contribution to cone photoresponses remains unknown. Here, we evaluate the role of a candidate retinol isomerase of this pathway, sphingolipid δ4 desaturase 1 (Des1). Single-cell RNA sequencing analysis revealed Des1 expression not only in Muller glia but also throughout the retina and in the retinal pigment epithelium. We assessed cone functional dependence on Muller cell-expressed Des1 through a conditional knockout approach. Floxed Des1 mice, on a guanine nucleotide-binding protein subunit α transducin 1 knockout ( Gnat1-/-) background to allow isolated recording of cone-driven photoresponses, were bred with platelet-derived growth factor receptor α (Pdgfrα)-Cre mice to delete Des1 in Muller cells. Conditional knockout of Des1 expression, as shown by tissue-selective Des1 gene recombination and reduced Des1 catalytic activity, caused no gross changes in the retinal structure and had no effect on cone sensitivity or dark adaptation but did slightly accelerate the rate of cone phototransduction termination. These results indicate that Des1 expression in Muller cells is not required for cone visual pigment regeneration in the mouse.-Kiser, P. D., Kolesnikov, A.V., Kiser, J. Z., Dong, Z., Chaurasia, B., Wang, L., Summers, S. A., Hoang, T., Blackshaw, S., Peachey, N. S., Kefalov, V. J., Palczewski, K. Conditional deletion of Des1 in the mouse retina does not impair the visual cycle in cones.

Cone photoreceptors mediate daytime vision in vertebrates. The rapid and efficient regeneration of their visual pigments following photoactivation is critical for the cones to remain photoresponsive in bright and rapidly changing light conditions. Cone pigment regeneration depends on the recycling of visual chromophore, which takes place via the canonical visual cycle in the retinal pigment epithelium (RPE) and the Müller cell–driven intraretinal visual cycle. The molecular mechanisms that enable the neural retina to regenerate visual chromophore for cones have not been fully elucidated. However, one known component of the two visual cycles is the cellular retinaldehyde-binding protein (CRALBP), which is expressed both in the RPE and in Müller cells. To understand the significance of CRALBP in cone pigment regeneration, we examined the function of cones in mice heterozygous for Rlbp1, the gene encoding CRALBP. We found that CRALBP expression was reduced by ∼50% in both the RPE and retina of Rlbp1+/− mice. Electroretinography (ERG) showed that the dark adaptation of rods and cones is unaltered in Rlbp1+/− mice, indicating a normal RPE visual cycle. However, pharmacologic blockade of the RPE visual cycle revealed suppressed cone dark adaptation in Rlbp1+/− mice in comparison with controls. We conclude that the expression level of CRALPB specifically in the Müller cells modulates the efficiency of the retina visual cycle. Finally, blocking the RPE visual cycle also suppressed further cone dark adaptation in Rlbp1−/− mice, revealing a shunt in the classical RPE visual cycle that bypasses CRALBP and allows partial but unexpectedly rapid cone dark adaptation.

Rods and cones use intracellular Ca2+ to regulate many functions, including phototransduction and neurotransmission. The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca2+ entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca2+ uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu-/- zebrafish and a rod photoreceptor-specific Mcu-/- mouse. Using genetically encoded Ca2+ sensors to directly examine Ca2+ uptake in zebrafish cone mitochondria, we found that loss of MCU reduces but does not eliminate mitochondrial Ca2+ uptake. Loss of MCU does not lead to photoreceptor degeneration, mildly affects mitochondrial metabolism, and does not alter physiological responses to light, even in the absence of the Na+/Ca2+, K+ exchanger. Our results reveal that MCU is dispensable for vertebrate photoreceptor function, consistent with its low expression and the presence of an alternative pathway for Ca2+ uptake into photoreceptor mitochondria.

Thyroid hormone (TH) signaling plays an important role in the regulation of long-wavelength vision in vertebrates. In the retina, thyroid hormone receptor β ( thrb ) is required for expression of long-wavelength-sensitive opsin ( lws ) in red cone photoreceptors, while in retinal pigment epithelium (RPE), TH regulates expression of a cytochrome P450 enzyme, cyp27c1 , that converts vitamin A 1 into vitamin A 2 to produce a red-shifted chromophore. To better understand how TH controls these processes, we analyzed the phenotype of zebrafish with mutations in the three known TH nuclear receptor transcription factors ( thraa , thrab , and thrb ). We found that no single TH nuclear receptor is required for TH-mediated induction of cyp27c1 but that deletion of all three ( thraa −/− ;thrab −/− ;thrb −/− ) completely abrogates its induction and the resulting conversion of A 1 - to A 2 -based retinoids. In the retina, loss of thrb resulted in an absence of red cones at both larval and adult stages without disruption of the underlying cone mosaic. RNA-sequencing analysis revealed significant down-regulation of only five genes in adult thrb −/− retina, of which three ( lws1 , lws2 , and miR-726 ) occur in a single syntenic cluster. In the thrb −/− retina, retinal progenitors destined to become red cones were transfated into ultraviolet (UV) cones and horizontal cells. Taken together, our findings demonstrate cooperative regulation of cyp27c1 by TH receptors and a requirement for thrb in red cone fate determination. Thus, TH signaling coordinately regulates both spectral sensitivity and sensory plasticity.

The nuclei of cone photoreceptors are located on the apical side of the outer nuclear layer (ONL) in vertebrate retinas. However, the functional role of this evolutionarily conserved localization of cone nuclei is unknown. We previously showed that Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes) are essential for the apical migration of cone nuclei during development. Here, we developed an efficient genetic strategy to disrupt cone LINC complexes in mice. Experiments with animals from both sexes revealed that disrupting cone LINC complexes resulted in mislocalization of cone nuclei to the basal side of ONL in mouse retina. This, in turn, disrupted cone pedicle morphology, and appeared to reduce the efficiency of synaptic transmission from cones to bipolar cells. Although we did not observe other developmental or phototransduction defects in cones with mislocalized nuclei, their dark adaptation was impaired, consistent with a deficiency in chromophore recycling. These findings demonstrate that the apical localization of cone nuclei in the ONL is required for the timely dark adaptation and efficient synaptic transmission in cone photoreceptors.

Light adaptation of photoreceptor cells is mediated by Ca(2+)-dependent mechanisms. In darkness, Ca(2+) influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca(2+) extrusion via Na(+)/Ca(2+), K(+) exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca(2+) levels in photoreceptor outer segment because of continuing Ca(2+) extrusion by NCKXs. Guanylate cyclase–activating proteins (GCAPs) then up-regulate cGMP synthesis by activating retinal membrane guanylate cyclases (RetGCs) in low Ca(2+). This activation of RetGC accelerates photoresponse recovery and critically contributes to light adaptation of the nighttime rod and daytime cone photoreceptors. In mouse rod photoreceptors, GCAP1 and GCAP2 both contribute to the Ca(2+)-feedback mechanism. In contrast, only GCAP1 appears to modulate RetGC activity in mouse cones because evidence of GCAP2 expression in cones is lacking. Surprisingly, we found that GCAP2 is expressed in cones and can regulate light sensitivity and response kinetics as well as light adaptation of GCAP1-deficient mouse cones. Furthermore, we show that GCAP2 promotes cGMP synthesis and cGMP-gated channel opening in mouse cones exposed to low Ca(2+). Our biochemical model and experiments indicate that GCAP2 significantly contributes to the activation of RetGC1 at low Ca(2+) when GCAP1 is not present. Of note, in WT mouse cones, GCAP1 dominates the regulation of cGMP synthesis. We conclude that, under normal physiological conditions, GCAP1 dominates the regulation of cGMP synthesis in mouse cones, but if its function becomes compromised, GCAP2 contributes to the regulation of phototransduction and light adaptation of cones.

SUMMARY Cone photoreceptors scale dynamically the sensitivity of responses to maintain responsiveness across wide range of changes in luminance. Synaptic changes contribute to this adaptation, but how this process is coordinated at the molecular level is poorly understood. Here, we report that a cell adhesion-like molecule, LRIT1, is enriched selectively at cone photoreceptor synapses where it engages in a trans-synaptic interaction with mGluR6, the principal receptor in postsynaptic ON-bipolar cells. The levels of LRIT1 are regulated by the neurotransmitter release apparatus that controls photoreceptor output. Knockout of LRIT1 in mice increases the sensitivity of cone synaptic signaling while impairing its ability to adapt to background light without overtly influencing the morphology or molecular composition of photoreceptor synapses. Accordingly, mice lacking LRIT1 show visual deficits under conditions requiring temporally challenging discrimination of visual signals in steady background light. These observations reveal molecular mechanisms involved in scaling synaptic communication in the retina., In Brief Sarria et al. show LRIT1 accumulates at photoreceptors synapses. It forms a complex with a key receptor involved in processing neurotransmitter output of photoreceptors. They show that this protein plays an essential role in synaptic communication of cone photoreceptors and impacts daylight vision.

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 inR838S+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 inGCAP1,2+/+orGCAP1,2+/−backgrounds, was prevented inGCAP1,2−/−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 STATEMENTIn 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 ofGUCY2DCORD6-linked mutation on photoreceptor physiologyin vivo. It also identifies the abnormal regulation of the cyclase by calcium-sensor proteins as the main trigger for the photoreceptor death.

Key points This study explores the nature of the cis retinol that Muller cells in the retina provide to cones for the regeneration of their visual pigment. We report that the retina visual cycle provides cones exclusively with 11-cis chromophore in both salamander and mouse and show that this selectivity is dependent on the 11-cis-specific cellular retinaldehyde binding protein (CRALBP) present in Muller cells. Even though salamander blue cones and green rods share the same visual pigment, only blue cones but not green rods are able to dark-adapt in the retina following a bleach and to use exogenous 9-cis retinol for pigment regeneration, suggesting that access to the retina visual cycle is cone-specific and pigment-independent. Our results show that the retina produces 11-cis retinol that can be oxidized and used for pigment regeneration and dark adaptation selectively in cones and not in rods. Abstract Chromophore supply by the retinal Muller cells (retina visual cycle) supports the efficient pigment regeneration required for cone photoreceptor function in bright light. Surprisingly, a large fraction of the chromophore produced by dihydroceramide desaturase-1, the putative all-trans retinol isomerase in Muller cells, appears to be 9-cis retinol. In contrast, the canonical retinal pigment epithelium (RPE) visual cycle produces exclusively 11-cis retinal. Here, we used the different absorption spectra of 9-cis and 11-cis pigments to identify the isoform of the chromophore produced by the visual cycle of the intact retina. We found that the spectral sensitivity of salamander and mouse cones dark-adapted in the isolated retina (with only the retina visual cycle) was similar to that of cones dark-adapted in the intact eye (with both the RPE and retina visual cycles) and consistent with pure 11-cis pigment composition. However, in mice lacking the cellular retinaldehyde binding protein (CRALBP), cone spectral sensitivity contained a substantial 9-cis component. Thus, the retina visual cycle provides cones exclusively with 11-cis chromophore and this process is mediated by the 11-cis selective CRALBP in Muller cells. Finally, despite sharing the same pigment, salamander blue cones, but not green rods, recovered their sensitivity in the isolated retina. Exogenous 9-cis retinol produced robust sensitivity recovery in bleached red and blue cones but not in red and green rods, suggesting that cis retinol oxidation restricts access to the retina visual cycle to cones.

Key points This study explores the molecular mechanisms that regulate the recycling of chromophore required for pigment regeneration in mammalian cones. We report that two chromophore binding proteins, retinol dehydrogenase 8 (RDH8) and photoreceptor-specific ATP-binding cassette transporter (ABCA4) accelerate the dark adaptation of cones, first, directly, by facilitating the processing of chromophore in cones, and second, indirectly, by accelerating the turnover of chromophore in rods, which is then recycled and delivered to both rods and cones. Preventing competition with the rods by knocking out rhodopsin accelerated cone dark adaptation, demonstrating the interplay between rod and cone pigment regeneration driven by the retinal pigment epithelium (RPE). This novel interdependence of rod and cone pigment regeneration should be considered when developing therapies targeting the recycling of chromophore for rods, and evaluating residual cone function should be a critical test for such regimens targeting the RPE. Abstract Rapid recycling of visual chromophore and regeneration of the visual pigment are critical for the continuous function of mammalian cone photoreceptors in daylight vision. However, the molecular mechanisms modulating the supply of visual chromophore to cones have remained unclear. Here we explored the roles of two chromophore-binding proteins, retinol dehydrogenase 8 (RDH8) and photoreceptor-specific ATP-binding cassette transporter 4 (ABCA4), in dark adaptation of mammalian cones. We report that young adult RDH8/ABCA4-deficient mice have normal M-cone morphology but reduced visual acuity and photoresponse amplitudes. Notably, the deletion of RDH8 and ABCA4 suppressed the dark adaptation of M-cones driven by both the intraretinal visual cycle and the retinal pigmented epithelium (RPE) visual cycle. This delay can be caused by two separate mechanisms: direct involvement of RDH8 and ABCA4 in cone chromophore processing, and an indirect effect from the delayed recycling of chromophore by the RPE due to its slow release from RDH8/ABCA4-deficient rods. Intriguingly, our data suggest that RDH8 could also contribute to the oxidation of cis-retinoids in cones, a key reaction of the retina visual cycle. Finally, we dissected the roles of rod photoreceptors and RPE for dark adaptation of M-cones. We found that rods suppress, whereas RPE promotes, cone dark adaptation. Thus, therapeutic approaches targeting the RPE visual cycle could have adverse effects on the function of cones, making the evaluation of residual cone function a critical test for regimens targeting the RPE.

Cones comprise only a small portion of the photoreceptors in mammalian retinas. However, cones are vital for color vision and visual perception, and their loss severely diminishes the quality of life for patients with retinal degenerative diseases. Cones function in bright light and have higher demand for energy than rods; yet, the mechanisms that support the energy requirements of cones are poorly understood. One such pathway that potentially could sustain cones under basal and stress conditions is macroautophagy. We addressed the role of macroautophagy in cones by examining how the genetic block of this pathway affects the structural integrity, survival, and function of these neurons. We found that macroautophagy was not detectable in cones under normal conditions but was readily observed following 24 h of fasting. Consistent with this, starvation induced phosphorylation of AMPK specifically in cones indicating cellular starvation. Inhibiting macroautophagy in cones by deleting the essential macroautophagy gene Atg5 led to reduced cone function following starvation suggesting that cones are sensitive to systemic changes in nutrients and activate macroautophagy to maintain their function. ATG5-deficiency rendered cones susceptible to light-induced damage and caused accumulation of damaged mitochondria in the inner segments, shortening of the outer segments, and degeneration of all cone types, revealing the importance of mitophagy in supporting cone metabolic needs. Our results demonstrate that macroautophagy supports the function and long-term survival of cones providing for their unique metabolic requirements and resistance to stress. Targeting macroautophagy has the potential to preserve cone-mediated vision during retinal degenerative diseases.

Illumination of the retina is a major determinant of energy expenditure by its neurons. However, it remains unclear whether light exposure significantly contributes to the pathophysiology of common retinal disease. Driven by the premise that light exposure reduces the metabolic demand of the retina, recent clinical trials failed to demonstrate a benefit for constant illumination in the treatment of diabetic retinopathy. Here, we instead ask whether light deprivation or blockade of visual transduction could modulate the severity of this common cause of blindness. We randomized adult mice with two different models of diabetic retinopathy to 1-3 months of complete dark housing. Unexpectedly, we find that diabetic mice exposed to short or prolonged light deprivation have reduced diabetes-induced retinal pathology, using measures of visual function, compared to control animals in standard lighting conditions. To corroborate these results, we performed assays of retinal vascular health in diabetic Gnat1−/− and Rpe65−/− mice, which lack phototransduction. Both mutants displayed less diabetes-associated retinal vascular disease compared to respective wild-type controls. Collectively, these results suggest that light-induced visual transduction promotes the development of diabetic retinopathy and implicate photoreceptors as an early source of visual pathology in diabetes.

Calcium (Ca2+) plays an important role in the function and health of neurons. In vertebrate cone photoreceptors, Ca2+ controls photoresponse sensitivity, kinetics, and light adaptation. Despite the critical role of Ca2+ in supporting the function and survival of cones, the mechanism for its extrusion from cone outer segments is not well understood. Here, we show that the Na+/Ca2+, K+ exchanger NCKX4 is expressed in zebrafish, mouse, and primate cones. Functional analysis of NCKX4-deficient mouse cones revealed that this exchanger is essential for the wide operating range and high temporal resolution of cone-mediated vision. We show that NCKX4 shapes the cone photoresponse together with the cone-specific NCKX2: NCKX4 acts early to limit response amplitude, while NCKX2 acts late to further accelerate response recovery. The regulation of Ca2+ by NCKX4 in cones is a novel mechanism that supports their ability to function as daytime photoreceptors and promotes their survival. DOI: http://dx.doi.org/10.7554/eLife.24550.001, eLife digest Cells known as photoreceptors sense light in the eye. Light activates signaling pathways inside the photoreceptors that relay visual information to nerve cells, which carry the information to the brain. Photoreceptors called cone cells allow us to distinguish different colors of light and therefore play an important role in daytime vision. Over the course of the day, the overall levels of light in the environment can vary widely and so photoreceptors need to be able to adjust their signaling pathways so that they can still respond to light stimuli. Calcium ions modulate the signaling pathways inside cone cells to help them adjust to changing light levels. These ions also play other important roles in the health and activity of photoreceptors, so the cells need to carefully control how many calcium ions they contain. Cone cells contain a structure known as the outer segment, which is responsible for detecting light stimuli. It is believed that cones control the levels of calcium ions in the outer segment by balancing the flow of calcium ions into and out of the segment. The calcium ions enter the outer segment via channels that sit in the membrane surrounding the cell. A transporter protein known as NCKX2, which is only found in cone cells, was thought to pump the calcium ions out of the cell. However, recent data has challenged this idea by demonstrating that NCKX2 only plays a minor role in this process. Vinberg et al. investigated how calcium ions leave the outer segments of cone cells in several different animals. The experiments show that a transporter protein called NCKX4 – which belongs to the same protein family as NCKX2 – is the main transporter involved in removing calcium ions from the cone cells of mice. Loss of NCKX4 from mouse cones reduced the ability of these cells to respond to fast and rapidly changing light stimuli, and to operate in bright light. Further experiments show that NCKX4 is also found in the outer segments of zebrafish and monkey cone cells. The next challenges will be to find out if NCKX4 is also present in human cones and whether it plays a role in regulating our daytime vision. DOI: http://dx.doi.org/10.7554/eLife.24550.002

Pigment regeneration is critical for the function of cone photoreceptors in bright and rapidly-changing light conditions. This process is facilitated by the recently-characterized retina visual cycle, in which Müller cells recycle spent all-trans-retinol visual chromophore back to 11-cis-retinol. This 11-cis-retinol is oxidized selectively in cones to the 11-cis-retinal used for pigment regeneration. However, the enzyme responsible for the oxidation of 11-cis-retinol remains unknown. Here, we sought to determine whether retinol dehydrogenase 10 (RDH10), upregulated in rod/cone hybrid retinas and expressed abundantly in Müller cells, is the enzyme that drives this reaction. We created mice lacking RDH10 either in cone photoreceptors, Müller cells, or the entire retina. In vivo electroretinography and transretinal recordings revealed normal cone photoresponses in all RDH10-deficient mouse lines. Notably, their cone-driven dark adaptation both in vivo and in isolated retina was unaffected, indicating that RDH10 is not required for the function of the retina visual cycle. We also generated transgenic mice expressing RDH10 ectopically in rod cells. However, rod dark adaptation was unaffected by the expression of RDH10 and transgenic rods were unable to use cis-retinol for pigment regeneration. We conclude that RDH10 is not the dominant retina 11-cis-RDH, leaving its primary function in the retina unknown.

RPE65 is a retinoid isomerase essential for rod function, but its contribution to cone vision is enigmatic. Using selective RPE65 inhibitors, Kiser et al. demonstrate that cone function depends only partially on continuous RPE65 activity, providing support for cone-specific regeneration mechanisms., Visual function in vertebrates critically depends on the continuous regeneration of visual pigments in rod and cone photoreceptors. RPE65 is a well-established retinoid isomerase in the pigment epithelium that regenerates rhodopsin during the rod visual cycle; however, its contribution to the regeneration of cone pigments remains obscure. In this study, we use potent and selective RPE65 inhibitors in rod- and cone-dominant animal models to discern the role of this enzyme in cone-mediated vision. We confirm that retinylamine and emixustat-family compounds selectively inhibit RPE65 over DES1, the putative retinoid isomerase of the intraretinal visual cycle. In vivo and ex vivo electroretinography experiments in Gnat1−/− mice demonstrate that acute administration of RPE65 inhibitors after a bleach suppresses the late, slow phase of cone dark adaptation without affecting the initial rapid portion, which reflects intraretinal visual cycle function. Acute administration of these compounds does not affect the light sensitivity of cone photoreceptors in mice during extended exposure to background light, but does slow all phases of subsequent dark recovery. We also show that cone function is only partially suppressed in cone-dominant ground squirrels and wild-type mice by multiday administration of an RPE65 inhibitor despite profound blockade of RPE65 activity. Complementary experiments in these animal models using the DES1 inhibitor fenretinide show more modest effects on cone recovery. Collectively, these studies demonstrate a role for continuous RPE65 activity in mammalian cone pigment regeneration and provide further evidence for RPE65-independent regeneration mechanisms.

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 Function of the retina and effects of drugs on it can be assessed by recording transretinal voltage across isolated retina that is perfused with physiological medium. However, building ex vivo ERG apparatus requires substantial amount of time, resources and expertise. Here we adapted a commercial in vivo ERG system for transretinal ERG recordings from rod and cone photoreceptors and compared rod and cone signaling between ex vivo and in vivo environments. We found that the rod and cone a- and b-waves recorded with the transretinal ERG adapter and a standard in vivo ERG system are comparable to those obtained from live anesthetized animals. However, ex vivo responses are somewhat slower and their oscillatory potentials are suppressed as compared to those recorded in vivo. We found that rod amplification constant (A) was comparable between ex vivo and in vivo conditions, ∼10–30s−2 depending on the choice of response normalization. We estimate that the A in cones is between 3 and 6s−2 in ex vivo conditions and by assuming equal A in vivo we arrive to light funnelling factor of 3 for cones in the mouse retina. The ex vivo ERG adapter provides a simple and affordable alternative to designing a custom-built transretinal recordings setup for the study of photoreceptors. Our results provide a roadmap to the rigorous quantitative analysis of rod and cone responses made possible with such a system.

Photoreceptor cell death is the proximal cause of blindness in many retinal degenerative disorders; hence, understanding the gene regulatory networks that promote photoreceptor survival is at the forefront of efforts to combat blindness. Down-regulation of the microRNA (miRNA)-processing enzyme DICER1 in the retinal pigmented epithelium has been implicated in geographic atrophy, an advanced form of age-related macular degeneration (AMD). However, little is known about the function of DICER1 in mature rod photoreceptor cells, another retinal cell type that is severely affected in AMD. Using a conditional-knockout (cKO) mouse model, we report that loss of DICER1 in mature postmitotic rods leads to robust retinal degeneration accompanied by loss of visual function. At 14 wk of age, cKO mice exhibit a 90% reduction in photoreceptor nuclei and a 97% reduction in visual chromophore compared with those in control littermates. Before degeneration, cKO mice do not exhibit significant defects in either phototransduction or the visual cycle, suggesting that miRNAs play a primary role in rod photoreceptor survival. Using comparative small RNA sequencing analysis, we identified rod photoreceptor miRNAs of the miR-22, miR-26, miR-30, miR-92, miR-124, and let-7 families as potential factors involved in regulating the survival of rods.—Sundermeier, T. R., Zhang, N., Vinberg, F., Mustafi, D., Kohno, H., Golczak, M., Bai, X., Maeda, A., Kefalov, V. J., Palczewski, K. DICER1 is essential for survival of postmitotic rod photoreceptor cells in mice.

Purpose The purpose of this study was to test the extent of light damage in different models of night blindness and apply these paradigms in testing the therapeutic efficacy of combination therapy by drugs acting on the Gi, Gs, and Gq protein-coupled receptors. Methods Acute bright light exposure was used to test susceptibility to light damage in mice lacking the following crucial phototransduction proteins: rod transducin (GNAT1), cone transducin (GNAT2), visual arrestin 1 (ARR1), and rhodopsin kinase 1 (GRK1). Mice were intraperitoneally injected with either vehicle or drug combination consisting of metoprolol (β1-receptor antagonist), bromocriptine (dopamine family-2 receptor agonist) and tamsulosin (α1-receptor antagonist) before bright light exposure. Light damage was primarily assessed with optical coherence tomography and inspection of cone population in retinal whole mounts. Retinal inflammation was assessed in a subset of experiments using autofluorescence imaging by scanning laser ophthalmoscopy and by postmortem inspection of microglia and astrocyte activity. Results The Gnat1-/- mice showed slightly increased susceptibility to rod light damage, whereas the Gnat2-/- mice were very resistant. The Arr1-/- and Grk1-/- mice were sensitive for both rod and cone light damage and showed robust retinal inflammation 7 days after bright light exposure. Pretreatment with metoprolol + bromocriptine + tamsulosin rescued the retina in all genetic backgrounds, starting at doses of 0.2 mg/kg metoprolol, 0.02 mg/kg bromocriptine, and 0.01 mg/kg tamsulosin in the Gnat1-/- mice. The therapeutic drug doses increased in parallel with light-damage severity. Conclusions Our results suggest that congenital stationary night blindness and Oguchi disease patients can be at an elevated risk of the toxic effects of bright light. Furthermore, systems pharmacology drug regimens that stimulate Gi signaling and attenuate Gs and Gq signaling present a promising disease-modifying therapy for photoreceptor degenerative diseases.

G-protein β subunits perform essential neuronal functions as part of G-protein βγ and Gβ5-regulators of G-protein signaling (RGS) complexes. Both Gβγ and Gβ5-RGS are obligate dimers that are thought to require the assistance of the cytosolic chaperonin CCT and a cochaperone, phosducin-like protein 1 (PhLP1) for dimer formation. To test this hypothesisin vivo, we deleted thePhlp1gene in mouse (Mus musculus) retinal rod photoreceptor cells and measured the effects on G-protein biogenesis and visual signal transduction. In the PhLP1-depleted rods, Gβγ dimer formation was decreased 50-fold, resulting in a >10-fold decrease in light sensitivity. Moreover, a 20-fold reduction in Gβ5and RGS9–1 expression was also observed, causing a 15-fold delay in the shutoff of light responses. These findings conclusively demonstratein vivothat PhLP1 is required for the folding and assembly of both Gβγ and Gβ5-RGS9.

A prime goal of regenerative medicine is to direct cell fates in a therapeutically useful manner. Retinitis pigmentosa is one of the most common degenerative diseases of the eye and is associated with early rod photoreceptor death followed by secondary cone degeneration. We hypothesized that converting adult rods into cones, via knockdown of the rod photoreceptor determinant Nrl , could make the cells resistant to the effects of mutations in rod-specific genes, thereby preventing secondary cone loss. To test this idea, we engineered a tamoxifen-inducible allele of Nrl to acutely inactivate the gene in adult rods. This manipulation resulted in reprogramming of rods into cells with a variety of cone-like molecular, histologic, and functional properties. Moreover, reprogramming of adult rods achieved cellular and functional rescue of retinal degeneration in a mouse model of retinitis pigmentosa. These findings suggest that elimination of Nrl in adult rods may represent a unique therapy for retinal degeneration.

The pathophysiology of the E150K mutation in the rod opsin gene associated with autosomal recessive retinitis pigmentosa (arRP) has yet to be determined. We generated knock-in mice carrying a single nucleotide change in exon 2 of the rod opsin gene resulting in the E150K mutation. This novel mouse model displayed severe retinal degeneration affecting rhodopsin's stabilization of rod outer segments (ROS). Homozygous E150K (KK) mice exhibited early-onset retinal degeneration, with disorganized ROS structures, autofluorescent deposits in the subretinal space, and aberrant photoreceptor phagocytosis. Heterozygous (EK) mice displayed a delayed-onset milder retinal degeneration. Further, mutant receptors were mislocalized to the inner segments and perinuclear region. Though KK mouse rods displayed markedly decreased phototransduction, biochemical studies of the mutant rhodopsin revealed only minimally affected chromophore binding and G protein activation. Ablation of the chromophore by crossing KK mice with mice lacking the critical visual cycle protein LRAT slowed retinal degeneration, whereas blocking phototransduction by crossing KK mice with GNAT1-deficient mice slightly accelerated this process. This study highlights the importance of proper higher-order organization of rhodopsin in the native tissue and provides information about the signaling properties of this mutant rhodopsin. Additionally, these results suggest that patients heterozygous for the E150K mutation should be periodically reevaluated for delayed-onset retinal degeneration.

Calcium ions (Ca2+) modulate the phototransduction cascade of vertebrate cone photoreceptors to tune gain, inactivation, and light adaptation. In darkness, the continuous current entering the cone outer segment through cGMP-gated (CNG) channels is carried in part by Ca2+, which is then extruded back to the extracellular space. The mechanism of Ca2+ extrusion from mammalian cones is not understood. The dominant view has been that the cone-specific isoform of the Na+/Ca2+, K+ exchanger, NCKX2, is responsible for removing Ca2+ from their outer segments. However, indirect evaluation of cone function in NCKX2-deficient (Nckx2−/−) mice by electroretinogram recordings revealed normal photopic b-wave responses. This unexpected result suggested that NCKX2 may not be involved in the Ca2+ homeostasis of mammalian cones. To address this controversy, we examined the expression of NCKX2 in mouse cones and performed transretinal recordings from Nckx2−/− mice to determine the effect of NCKX2 deletion on cone function directly. We found that Nckx2−/− cones exhibit compromised phototransduction inactivation, slower response recovery and delayed background adaptation. We conclude that NCKX2 is required for the maintenance of efficient Ca2+ extrusion from mouse cones. However, surprisingly, Nckx2−/− cones adapted normally in steady background light, indicating the existence of additional Ca2+-extruding mechanisms in mammalian cones.

During retinogenesis, lamin B1 is critical to maintaining the nuclear integrity of embryonic retinal neurons, whereas lamin B2 is not. The latter is required for postnatal retinal lamination and synaptogenesis. In adult photoreceptors, lamin B1 and lamin B2 have very long half-lives but are dispensable for cone photoreceptor survival and function., Lamin B1 and lamin B2 are essential building blocks of the nuclear lamina, a filamentous meshwork lining the nucleoplasmic side of the inner nuclear membrane. Deficiencies in lamin B1 and lamin B2 impair neurodevelopment, but distinct functions for the two proteins in the development and homeostasis of the CNS have been elusive. Here we show that embryonic depletion of lamin B1 in retinal progenitors and postmitotic neurons affects nuclear integrity, leads to the collapse of the laminB2 meshwork, impairs neuronal survival, and markedly reduces the cellularity of adult retinas. In stark contrast, a deficiency of lamin B2 in the embryonic retina has no obvious effect on lamin B1 localization or nuclear integrity in embryonic retinas, suggesting that lamin B1, but not lamin B2, is strictly required for nucleokinesis during embryonic neurogenesis. However, the absence of lamin B2 prevents proper lamination of adult retinal neurons, impairs synaptogenesis, and reduces cone photoreceptor survival. We also show that lamin B1 and lamin B2 are extremely long-lived proteins in rod and cone photoreceptors. OF interest, a complete absence of both proteins during postnatal life has little or no effect on the survival and function of cone photoreceptors.

Cone photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in cone phototransduction, which is a process essential for daylight vision, color vision, and visual acuity. Mutations in the cone channel subunits CNGA3 and CNGB3 are associated with human cone diseases, including achromatopsia, cone dystrophies, and early onset macular degeneration. Mutations in CNGB3 alone account for 50% of reported cases of achromatopsia. This work investigated the role of CNGB3 in cone light response and cone channel structural stability. As cones comprise only 2-3% of the total photoreceptor population in the wild-type mouse retina, we used Cngb3(-/-)/Nrl(-/-) mice with CNGB3 deficiency on a cone-dominant background in our study. We found that, in the absence of CNGB3, CNGA3 was able to travel to the outer segments, co-localize with cone opsin, and form tetrameric complexes. Electroretinogram analyses revealed reduced cone light response amplitude/sensitivity and slower response recovery in Cngb3(-/-)/Nrl(-/-) mice compared with Nrl(-/-) mice. Absence of CNGB3 expression altered the adaptation capacity of cones and severely compromised function in bright light. Biochemical analysis demonstrated that CNGA3 channels lacking CNGB3 were more resilient to proteolysis than CNGA3/CNGB3 channels, suggesting a hindered structural flexibility. Thus, CNGB3 regulates cone light response kinetics and the channel structural flexibility. This work advances our understanding of the biochemical and functional role of CNGB3 in cone photoreceptors.

Exposure to daylight closes cyclic nucleotide-gated (CNG) and voltage-operated Ca 2+ -permeable channels in mammalian rods. The consequent lowering of the cytosolic calcium concentration ((Ca 2+ )i), if protracted, can contribute to light-induced damage and apoptosis in thesecells.Weherereportthatmouserodsareprotectedagainstprolongedloweringof(Ca 2+ )i by store-operated Ca 2+ entry (SOCE). Ca 2+ stores were depleted in Ca 2+ -free saline supplemented with the endoplasmic reticulum (ER) sequestration blocker cyclopiazonic acid. Store depletion elicited(Ca 2+ )isignalsthatexceededbaseline(Ca 2+ )iby5.9 ±0.7-foldandwereantagonizedbyan inhibitorycocktailcontaining2-APB,SKF96365andGd 3+ .CationinfluxthroughSOCEchannels wassufficienttoelicitasecondaryactivationofL-typevoltage-operatedCa 2+ entry.Wealsofound that TRPC1, the type 1 canonical mammalian homologue of the Drosophila photoreceptor TRP channel, is predominantly expressed within the outer nuclear layer of the retina. Rod loss in Pde6b rd1 (rd1), Chx10/Kip1 −/−rd1 and Elovl4 TG2 dystrophic models was associated with ∼70% reduction in Trpc1 mRNA content whereasTrpc1 mRNA levels in rodless cone-fullNrl −/− retinas were decreased by ∼50%. Genetic ablation of TRPC1 channels, however, had no effect on SOCE

Continuous visual perception and the dark adaptation of vertebrate photoreceptors after bright light exposure require recycling of their visual chromophore through a series of reactions in the retinal pigmented epithelium (RPE visual cycle). Light-driven chromophore consumption by photoreceptors is greater in daytime vs. nighttime, suggesting that correspondingly higher activity of the visual cycle may be required. However, as rod photoreceptors are saturated in bright light, the continuous turnover of their chromophore by the visual cycle throughout the day would not contribute to vision. Whether the recycling of chromophore that drives rod dark adaptation is regulated by the circadian clock and light exposure is unknown. Here, we demonstrate that mouse rod dark adaptation is slower during the day or after light pre-exposure. This surprising daytime suppression of the RPE visual cycle was accompanied by light-driven reduction in expression of Rpe65, a key enzyme of the RPE visual cycle. Notably, only rods in melatonin-proficient mice were affected by this daily visual cycle modulation. Our results demonstrate that the circadian clock and light exposure regulate the recycling of chromophore in the RPE visual cycle. This daily melatonin-driven modulation of rod dark adaptation could potentially protect the retina from light-induced damage during the day.

Rapid regeneration of the visual pigment following its photoactivation is critical for the function of cone photoreceptors throughout the day. Though the reactions of the visual cycle in the retinal pigment epithelium (RPE) that recycle chromophore for rod pigment regeneration are well characterized, the corresponding mechanisms that enable rapid regeneration of cone pigment are poorly understood. A key remaining question is the relative contribution of the recently discovered cone-specific retina visual cycle and the classic RPE-dependent visual cycle to mammalian cone pigment regeneration. In addition, it is not clear what role, if any, the abundant interphotoreceptor retinoid-binding protein (IRBP) presumed to facilitate the traffic of chromophore, plays in accelerating mammalian cone pigment regeneration. To address these issues, we used transretinal recordings to evaluate M/L-cone pigment regeneration in isolated retinas and eyecups from control and IRBP-deficient mice. Remarkably, the mouse retina promoted M/L-cone dark adaptation eightfold faster than the RPE. However, complete cone recovery required both visual cycles. We conclude that the retina visual cycle is critical for the initial rapid regeneration of mouse M/L-cone pigment during dark adaptation, whereas the slower RPE visual cycle is required to complete the process. While the deletion of IRBP reduced the amplitude and slowed the kinetics of mouse M/L-cone photoresponses, cone adaptation in bright, steady light and the kinetics of cone dark adaptation were not affected in isolated retina or in intact eyecup. Thus, IRBP does not accelerate cone pigment regeneration and is not critical for the function of mouse M/L-cones in bright light.

Cones function in constant light and are responsible for mediating daytime human vision. Like rods, cones use the photosensitive molecule 11-cis-retinal to detect light, and in constant illumination, a continuous supply of 11-cis-retinal is needed. A retina visual cycle is thought to provide a privileged supply of 11-cis-retinal to cones by using 11-cis-retinol generated in Müller cells. In the cycle, 11-cis-retinol is transported from Müller cells to cone inner segments, where it is oxidized to 11-cis-retinal. This oxidation step is only performed in cones, thus rendering the cycle cone-specific. Interphotoreceptor retinoid-binding protein (IRBP) is a retinoid-binding protein in the subretinal space that binds 11-cis-retinol endogenously. Cones inIrbp−/−mice are retinoid-deficient under photopic conditions, and it is possible that 11-cis-retinol supplies are disrupted in the absence of IRBP. We tested the hypothesis that IRBP facilitates the delivery of 11-cis-retinol to cones by preserving the isomeric state of 11-cis-retinol in light. With electrophysiology, we show that the cone-like photoreceptors ofNrl−/−mice use the cone visual cycle similarly to wild-type cones. Then, using oxidation assays in isolatedNrl−/−Rpe65−/−retinas, we show that IRBP delivers 11-cis-retinol for oxidation in cones and improves the efficiency of the oxidation reaction. Finally, we show that IRBP protects the isomeric state of 11-cis-retinol in the presence of light. Together, these findings suggest that IRBP plays an important role in the delivery of 11-cis-retinol to cones and can facilitate cone function in the presence of light.

Cone photoreceptors mediate our daytime vision and function under bright and rapidly-changing light conditions. As their visual pigment is destroyed in the process of photoactivation, the continuous function of cones imposes the need for rapid recycling of their chromophore and regeneration of their pigment. The canonical retinoid visual cycle through the retinal pigment epithelium cells recycles chromophore and supplies it to both rods and cones. However, shortcomings of this pathway, including its slow rate and competition with rods for chromophore, have led to the suggestion that cones might use a separate mechanism for recycling of chromophore. In the past four decades biochemical studies have identified enzymatic activities consistent with recycling chromophore in the retinas of cone-dominant animals, such as chicken and ground squirrel. These studies have led to the hypothesis of a cone-specific retina visual cycle. The physiological relevance of these studies was controversial for a long time and evidence for the function of this visual cycle emerged only in very recent studies and will be the focus of this review. The retina visual cycle supplies chromophore and promotes pigment regeneration only in cones but not in rods. This pathway is independent of the pigment epithelium and instead involves the Müller cells in the retina, where chromophore is recycled and supplied selectively to cones. The rapid supply of chromophore through the retina visual cycle is critical for extending the dynamic range of cones to bright light and for their rapid dark adaptation following exposure to light. The importance of the retina visual cycle is emphasized also by its preservation through evolution as its function has now been demonstrated in species ranging from salamander to zebrafish, mouse, primate, and human.

Even in healthy individuals, aging leads to deterioration in visual acuity, contrast sensitivity, visual field, and dark adaptation. Little is known about the neural mechanisms that drive the age-related changes of the retina and, more specifically, photoreceptors. According to one hypothesis, the age-related deterioration in rod function is due to the limited availability of 11-cis-retinal for rod pigment formation. To determine how aging affects rod photoreceptors and to test the retinoid-deficiency hypothesis, we compared the morphological and functional properties of rods of adult and aged B6D2F1/J mice. We found that the number of rods and the length of their outer segments were significantly reduced in 2.5-year-old mice compared with 4-month-old animals. Aging also resulted in a twofold reduction in the total level of opsin in the retina. Behavioral tests revealed that scotopic visual acuity and contrast sensitivity were decreased by twofold in aged mice, and rod ERG recordings demonstrated reduced amplitudes of both a- and b-waves. Sensitivity of aged rods determined from single-cell recordings was also decreased by 1.5-fold, corresponding to not more than 1% free opsin in these photoreceptors, and kinetic parameters of dim flash response were not altered. Notably, the rate of rod dark adaptation was unaffected by age. Thus, our results argue against age-related deficiency of 11-cis-retinal in the B6D2F1/J mouse rod visual cycle. Surprisingly, the level of cellular dark noise was increased in aged rods, providing an alternative mechanism for their desensitization.

Rpe65−/−mice are unable to produce 11-cis-retinal, the chromophore of visual pigments. Consequently, the pigment is present as the apoprotein opsin with a minute level of pigment containing 9-cis-retinal as chromophore. Notably, a 10–20% fraction of this opsin is mono-phosphorylated independently of light conditions. To determine the role of rhodopsin kinase (GRK1) in phosphorylating this opsin and to test whether eliminating this phosphorylation would accelerate photoreceptor degeneration, we generated theRpe65−/−Grk1−/−mouse. The retinae ofRpe65−/−Grk1−/−mice had negligible opsin phosphorylation, extensive degeneration with decreased opsin levels, and diminished light-evoked rod responses relative toRpe65−/−mice. These data show that opsin phosphorylation in theRpe65−/−mouse is due to the action of GRK1 and is neuroprotective. However, despite the higher activity of unphosphorylated opsin, the severe loss of opsin in the rapidly degeneratingRpe65−/−Grk1−/−mice resulted in lower overall opsin activity and in higher rod sensitivity compared withRpe65−/−mice. InRpe65−/−Grk1−/−Gnat1−/−mice where transduction activation was blocked, degeneration was only partially prevented. Therefore, increased opsin activity in the absence of phosphorylation was not the only mechanism for the accelerated retinal degeneration. Finally, the deletion of GRK1 triggered retinal degeneration inGrk1−/−mice after 1 month, even in the absence of apo-opsin. This degeneration was independent of light conditions and occurred even in the absence of transducin inGrk1−/−Gnat1−/−mice. Taken together, our results demonstrate a light-independent mechanism for retinal degeneration in the absence of GRK1, suggesting a second, not previously recognized role for that kinase.

Rod and cone photoreceptors use similar but distinct sets of phototransduction proteins to achieve different functional properties, suitable for their role as dim and bright light receptors, respectively. For example, rod and cone visual pigments couple to distinct variants of the heterotrimeric G protein transducin. However, the role of the structural differences between rod and cone transducin α subunits (Tα) in determining the functional differences between rods and cones is unknown. To address this question, we studied the translocation and signaling properties of rod Tα expressed in cones and cone Tα expressed in rods in three mouse strains: rod Tα knockout, cone Tα GNAT2 cpfl3 mutant, and rod and cone Tα double mutant rd17 mouse. Surprisingly, although the rod/cone Tα are only 79% identical, exogenously expressed rod or cone Tα localized and translocated identically to endogenous Tα in each photoreceptor type. Moreover, exogenously expressed rod or cone Tα rescued electroretinogram responses (ERGs) in mice lacking functional cone or rod Tα, respectively. Ex vivo transretinal ERG and single-cell recordings from rd17 retinas treated with rod or cone Tα showed comparable rod sensitivity and response kinetics. These results demonstrate that cone Tα forms a functional heterotrimeric G protein complex in rods and that rod and cone Tα couple equally well to the rod phototransduction cascade. Thus, rod and cone transducin α-subunits are functionally interchangeable and their signaling properties do not contribute to the intrinsic light sensitivity differences between rods and cones. Additionally, the technology used here could be adapted for any such homologue swap desired.

Retinitis pigmentosa 1 (RP1) is a common inherited retinopathy with variable onset and severity. TheRP1gene encodes a photoreceptor-specific, microtubule-associated ciliary protein containing the doublecortin (DCX) domain. Here we show that another photoreceptor-specific Rp1-like protein (Rp1L1) in mice is also localized to the axoneme of outer segments (OSs) and connecting cilia in rod photoreceptors, overlapping with Rp1.Rp1L1−/− mice display scattered OS disorganization, reduced electroretinogram amplitudes, and progressive photoreceptor degeneration, less severe and slower than inRp1−/− mice. In single rods ofRp1L1−/−, photosensitivity is reduced, similar to that ofRp1−/−. While individual heterozygotes are normal, double heterozygotes ofRp1andRp1L1exhibit abnormal OS morphology and reduced single rod photosensitivity and dark currents. The electroretinogram amplitudes of double heterozygotes are more reduced than those of individual heterozygotes combined. In support, Rp1L1 interacts with Rp1 in transfected cells and in retina pull-down experiments. Interestingly, phototransduction kinetics are normal in single rods and whole retinas of individual or doubleRp1andRp1L1mutant mice. Together, Rp1 and Rp1L1 play essential and synergistic roles in affecting photosensitivity and OS morphogenesis of rod photoreceptors. Our findings suggest that mutations inRP1L1could underlie retinopathy or modify RP1 disease expression in humans.

Summary Daytime vision is mediated by retinal cones which, unlike rods, remain functional even in bright light and dark-adapt rapidly. These cone properties are enabled by rapid regeneration of their pigment. This in turn requires rapid chromophore recycling which may not be achieved by the canonical retinal pigment epithelium visual cycle. Recent biochemical studies have suggested the presence of a second, cone-specific visual cycle, although its physiological function remains to be established. Here we report that the Müller cells within the salamander neural retina promote cone-specific pigment regeneration and dark adaptation that are independent of the pigment epithelium. Without this pathway, dark adaptation of cones is slow and incomplete. Interestingly, the rates of cone pigment regeneration by the retina and pigment epithelium visual cycles are essentially identical suggesting a possible common rate-limiting step. Finally, we also observed cone dark adaptation in the isolated mouse retina.

The rod pigment, rhodopsin, shows spontaneous isomerization activity. This quantal noise produces a dark light of approximately 0.01 photons s(-1) rod(-1) in human, setting the threshold for rod vision. The spontaneous isomerization activity of human cone pigments has long remained a mystery because the effect of a single isomerized pigment molecule in cones, unlike that in rods, is small and beyond measurement. We have now overcome this problem by expressing human red cone pigment transgenically in mouse rods in order to exploit their large single-photon response, especially after genetic removal of a key negative-feedback regulation. Extrapolating the measured quantal noise of transgenic cone pigment to native human red cones, we obtained a dark rate of approximately 10 false events s(-1) cone(-1), almost 10(3)-fold lower than the overall dark transduction noise previously reported in primate cones. Our measurements provide a rationale for why mammalian red, green and blue cones have comparable sensitivities, unlike their amphibian counterparts.

Background Mutations in the cone-rod-homeobox protein CRX are typically associated with dominant blinding retinopathies with variable age of onset and severity. Five well-characterized mouse models carrying different Crx mutations show a wide range of disease phenotypes. To determine if the phenotype variability correlates with distinct changes in CRX target gene expression, we perform RNA-seq analyses on three of these models and compare the results with published data. Results Despite dramatic phenotypic differences between the three models tested, graded expression changes in shared sets of genes are detected. Phenotype severity correlates with the down-regulation of genes encoding key rod and cone phototransduction proteins. Interestingly, in increasingly severe mouse models, the transcription of many rod-enriched genes decreases decrementally, whereas that of cone-enriched genes increases incrementally. Unlike down-regulated genes, which show a high degree of CRX binding and dynamic epigenetic profiles in normal retinas, the up-regulated cone-enriched genes do not correlate with direct activity of CRX, but instead likely reflect a change in rod cell-fate integrity. Furthermore, these analyses describe the impact of minor gene expression changes on the phenotype, as two mutants showed marginally distinguishable expression patterns but huge phenotypic differences, including distinct mechanisms of retinal degeneration. Conclusions Our results implicate a threshold effect of gene expression level on photoreceptor function and survival, highlight the importance of CRX in photoreceptor subtype development and maintenance, and provide a molecular basis for phenotype variability in CRX-associated retinopathies. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0732-z) contains supplementary material, which is available to authorized users.

Mutations that affect calcium homeostasis (Ca(2+)) in rod photoreceptors are linked to retinal degeneration and visual disorders such as retinitis pigmentosa and congenital stationary night blindness (CSNB). It is thought that the concentration of Ca(2+) in rod outer segments is controlled by a dynamic balance between influx via cGMP-gated (CNG) channels and extrusion via Na(+)/Ca(2+), K(+) exchangers (NCKX1). The extrusion-driven lowering of rod [Ca(2+)]i following light exposure controls their light adaptation and response termination. Mutant NCKX1 has been linked to autosomal-recessive stationary night blindness. However, whether NCKX1 contributes to light adaptation has not been directly tested and the mechanisms by which human NCKX1 mutations cause night blindness are not understood. Here, we report that the deletion of NCKX1 in mice results in malformed outer segment disks, suppressed expression and function of rod CNG channels and a subsequent 100-fold reduction in rod responses, while preserving normal cone responses. The compensating loss of CNG channel function in the absence of NCKX1-mediated Ca(2+) extrusion may prevent toxic Ca(2+) buildup and provides an explanation for the stationary nature of the associated disorder in humans. Surprisingly, the lack of NCKX1 did not compromise rod background light adaptation, suggesting additional Ca(2+)-extruding mechanisms exist in these cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

In the retina, synaptic transmission between photoreceptors and downstream ON-bipolar neurons (ON-BCs) is mediated by a GPCR pathway, which plays an essential role in vision. However, the mechanisms that control signal transmission at this synapse and its relevance to behavior remain poorly understood. In this study we used a genetic system to titrate the rate of GPCR signaling in ON-BC dendrites by varying the concentration of key RGS proteins and measuring the impact on transmission of signal between photoreceptors and ON-BC neurons using electroretinography and single cell recordings. We found that sensitivity, onset timing, and the maximal amplitude of light-evoked responses in rod- and cone-driven ON-BCs are determined by different RGS concentrations. We further show that changes in RGS concentration differentially impact visually guided-behavior mediated by rod and cone ON pathways. These findings illustrate that neuronal circuit properties can be modulated by adjusting parameters of GPCR-based neurotransmission at individual synapses. DOI: http://dx.doi.org/10.7554/eLife.06358.001, eLife digest At the back of the eye, a structure called the retina contains several types of cell that convert light into the electrical signals that the brain interprets to produce vision. Cells called rods and cones detect the light, and then signal to other neurons in the retina that relay this information to the brain. Rods and cones are specialized to respond best to different visual features: cones detect color and can track rapid movement; whereas rods are more sensitive to low light levels and so enable night vision. All rods and cones communicate with particular types of neuron called an ‘ON bipolar cell’: rods send their information to rod-specific ON bipolar cells and cones to cone ON-bipolar cells. To maintain the differences in how visual features are detected, the signals sent by the rod or cone cells need to be tuned separately. Previous studies showed that bipolar cells rely on the action of proteins called RGSs to control how information is passed from rods and cones to ON bipolar cells. However, how the RGS proteins produce their effects is not well understood, and neither is their impact on vision or behavior. Sarria et al. used a genetic approach to create mice that progressively lost RGS proteins from their retina over the course of several weeks. Recording the nerve impulses produced by the bipolar cells as light shone on the retina revealed that RGS depletion affects these neurons in three ways: how sensitive they are to the signals sent by the rod and cone cells, how quickly they respond to a signal, and the size of the electrical response that they produce. Sarria et al. then investigated how these changes affected the behavior of the mice. To test the response of the rod cells, the mice performed tasks in dim light. This revealed that it was only when the sensitivity of the bipolar cells decreased that the mice performed worse. However, in a task involving fast-moving objects that investigated the response of cone cells, only changes to the speed of the response affected vision. Therefore, the RGS protein has different effects on the signals from rod cells and cone cells. These findings will be useful for understanding how different light sensitive cells in the retina communicate their signals to extract important visual features, allowing us to both see well at night and track rapid changes in scenery on a bright sunny day. DOI: http://dx.doi.org/10.7554/eLife.06358.002

Mutations in the cellular retinaldehyde-binding protein (CRALBP, encoded by RLBP1) can lead to severe cone photoreceptor-mediated vision loss in patients. It is not known how CRALBP supports cone function or how altered CRALBP leads to cone dysfunction. Here, we determined that deletion of Rlbp1 in mice impairs the retinal visual cycle. Mice lacking CRALBP exhibited M-opsin mislocalization, M-cone loss, and impaired cone-driven visual behavior and light responses. Additionally, M-cone dark adaptation was largely suppressed in CRALBP-deficient animals. While rearing CRALBP-deficient mice in the dark prevented the deterioration of cone function, it did not rescue cone dark adaptation. Adeno-associated virus-mediated restoration of CRALBP expression specifically in Müller cells, but not retinal pigment epithelial (RPE) cells, rescued the retinal visual cycle and M-cone sensitivity in knockout mice. Our results identify Müller cell CRALBP as a key component of the retinal visual cycle and demonstrate that this pathway is important for maintaining normal cone-driven vision and accelerating cone dark adaptation.

Mutations in the ABCA4 gene are a common cause of autosomal recessive retinal degeneration. All mouse models to date are based on knockouts of Abca4, even though the disease is often caused by missense mutations such as the complex allele L541P;A1038V (PV). We now show that the PV mutation causes severe human disease whereas the V mutation alone causes mild disease. Mutant ABCA4 proteins expressed heterologously in mammalian cells retained normal cellular localization. However, basal and all-trans-retinal-stimulated ATPase activities were reduced substantially for P and PV but only mildly for V. Electron microscopy revealed marked structural changes and misfolding for the P and PV mutants but few changes for the V mutant, consistent with the disease severity difference in patients. We generated Abca4(PV/PV) knock-in mice homozygous for the complex PV allele to investigate the effects of this misfolding mutation in vivo. Mutant ABCA4 RNA levels approximated WT ABCA4 RNA levels but, surprisingly, only trace amounts of mutant ABCA4 protein were noted in the retina. RNA sequencing of WT, Abca4(-/-) and Abca4(PV/PV) mice revealed mild gene expression alterations in the retina and RPE. Similar to Abca4(-/-) mice, Abca4(PV/PV) mice showed substantial A2E and lipofuscin accumulation in their RPE cells but no retinal degeneration up to 12 months of age. Thus, rapid degradation of this large misfolded mutant protein in mouse retina caused little detectable photoreceptor degeneration. These findings suggest likely differences in the unfolded protein response between murine and human photoreceptors and support development of therapies directed at increasing this capability in patients.