Single cell RNA sequencing confirms retinal microglia activation associated with early onset retinal degeneration.

Mutations in the Membrane-type frizzled related protein (Mfrp) gene results in an early-onset retinal degeneration associated with retinitis pigmentosa, microphthalmia, optic disc drusen and foveal schisis. In the current study, a previously characterized mouse model of human retinal degeneration carrying homozygous c.498_499insC mutations in Mfrp (MfrpKI/KI) was used. Patients carrying this mutation have retinal degeneration at an early age. The model demonstrates subretinal deposits and develops early-onset photoreceptor degeneration. We observed large subretinal deposits in MfrpKI/KI mice which were strongly CD68 positive and co-localized with autofluorescent spots. Single cell RNA sequencing of MfrpKI/KI mice retinal microglia showed a significantly higher number of pan-macrophage marker Iba-1 and F4/80 positive cells with increased expression of activation marker (CD68) and lowered microglial homeostatic markers (TMEM119, P2ry13, P2ry13, Siglech) compared with wild type mice confirming microglial activation as observed in retinal immunostaining showing microglia activation in subretinal region. Trajectory analysis identified a small cluster of microglial cells with activation transcriptomic signatures that could represent a subretinal microglia population in MfrpKI/KI mice expressing higher levels of APOE. We validated these findings using immunofluorescence staining of retinal cryosections and found a significantly higher number of subretinal Iba-1/ApoE positive microglia in MfrpKI/KI mice with some subretinal microglia also expressing lowered levels of microglial homeostatic marker TMEM119, confirming microglial origin. In summary, we confirm that MfrpKI/KI mice carrying the c.498_499insC mutation had a significantly higher population of activated microglia in their retina with distinct subsets of subretinal microglia. Further, studies are required to confirm whether the association of increased subretinal microglia in MfrpKI/KI mice are causal in degeneration.

Fast and nonuniform dynamics of perisaccadic vision in the central fovea.

Humans use rapid eye movements (saccades) to inspect stimuli with the foveola, the region of the retina where receptors are most densely packed. It is well established that visual sensitivity is generally attenuated during these movements, a phenomenon known as saccadic suppression. This effect is commonly studied with large, often peripheral, stimuli presented during instructed saccades. However, little is known about how saccades modulate the foveola and how the resulting dynamics unfold during natural visual exploration. Here we measured the foveal dynamics of saccadic suppression in a naturalistic high-acuity task, a task designed after primates' social grooming, which-like most explorations of fine patterns-primarily elicits minute saccades (microsaccades). Leveraging on recent advances in gaze-contingent display control, we were able to systematically map the perisaccadic time course of sensitivity across the foveola. We show that contrast sensitivity is not uniform across this region and that both the extent and dynamics of saccadic suppression vary within the foveola. Suppression is stronger and faster in the most central portion, where sensitivity is generally higher and selectively rebounds at the onset of a new fixation. These results shed light on the modulations experienced by foveal vision during the saccade-fixation cycle and explain some of the benefits of microsaccades.

Genotype-Phenotype of Isolated Foveal Hypoplasia in a Large Cohort: Minor Iris Changes as an Indicator of PAX6 Involvement.

Purpose: Foveal hypoplasia (FVH) is defined as the lack of fovea with a relatively preserved neuroretina, occurring either as an isolated FVH (IFVH) condition or associated with other diseases. This study aimed to systemically molecularly characterize IFVH. Methods: Genetic defects in 33 families with IFVH were analyzed by exome sequencing. Variants in three genes (PAX6, SLC38A8, and AHR) were selected and evaluated with multistep bioinformatic tools. Results: Mutations in the three genes were identified in 69.7% (23/33) of families with IFVH and infantile nystagmus, including 18 families with PAX6 mutations, 5 with SLC38A8 mutations, but none with AHR mutations. Clinical data from 32 patients in the 23 families showed FVH, infantile nystagmus, and full iris. Careful follow-up visits revealed subtle changes in iris in 9 of 14 patients with PAX6 variants. The PAX6 variants of the 18 families (15 missense and one stop-loss) were mostly located in the C-terminal region of the paired box domain. Variants in AHR, SLC38A8, and PAX6 contributed to IFVH in one (2%), 25 (45%), and 30 (53%) families with identified genetic defects (23 families in this study and 33 reported previously), respectively. Conclusions: PAX6 and SLC38A8 mutations are the main cause of IFVH based on our data and a systematic review. IFVH-associated PAX6 variants are mostly missense with a specific location, indicating a specific correlation of these variants with IFVH but not with typical aniridia. Full iris with subtle structural abnormalities is more common in patients with PAX6-associated IFVH, suggesting a potential diagnostic indicator.

The potential role of Arhgef33 RhoGEF in foveal development in the zebra finch retina.

The fovea is a pit formed in the center of the retina that enables high-acuity vision in certain vertebrate species. While formation of the fovea fascinates many researchers, the molecular mechanisms underlying foveal development are poorly understood. In the current study, we histologically investigated foveal development in zebra finch (Taeniopygia guttata) and found that foveal pit formation begins just before post-hatch day 14 (P14). We next performed RNA-seq analysis to compare gene expression profiles between the central (foveal and parafoveal) and peripheral retina in zebra finch at P14. We found that the Arhgef33 expression is enriched in the middle layer of the inner nuclear layer at the parafovea, suggesting that Arhgef33 is dominantly expressed in Müller glial cells in the developing parafovea. We then performed a pull-down assay using Rhotekin-RBD and observed GEF activity of Arhgef33 against RhoA. We found that overexpression of Arhgef33 in HEK293 cells induces cell contraction and that Arhgef33 expression inhibits neurite extension in Neuro 2A cells, which is partially recovered by a Rho-kinase (ROCK) inhibitor. Taken together, we used zebra finch as a model animal to investigate foveal development and identified Arhgef33 as a candidate protein possibly involved in foveal development through modulating RhoA activity.

Elevated energy requirement of cone photoreceptors.

We have used recent measurements of mammalian cone light responses and voltage-gated currents to calculate cone ATP utilization and compare it to that of rods. The largest expenditure of ATP results from ion transport, particularly from removal of Na+ entering outer segment light-dependent channels and inner segment hyperpolarization-activated cyclic nucleotide-gated channels, and from ATP-dependent pumping of Ca2+ entering voltage-gated channels at the synaptic terminal. Single cones expend nearly twice as much energy as single rods in darkness, largely because they make more synapses with second-order retinal cells and thus must extrude more Ca2+ In daylight, cone ATP utilization per cell remains high because cones never remain saturated and must continue to export Na+ and synaptic Ca2+ even in bright illumination. In mouse and human retina, rods greatly outnumber cones and consume more energy overall even in background light. In primates, however, the high density of cones in the fovea produces a pronounced peak of ATP utilization, which becomes particularly prominent in daylight and may make this part of the retina especially sensitive to changes in energy availability.

Why is Zeaxanthin the Most Concentrated Xanthophyll in the Central Fovea?

Diet-based xanthophylls (zeaxanthin and lutein) are conditionally essential polar carotenoids preferentially accreted in high concentrations (1 mM) to the central retina, where they have the capacity to impart unique physiologically significant biophysical biochemical properties implicated in cell function, rescue, and survival. Macular xanthophylls interact with membrane-bound proteins and lipids to absorb/attenuate light energy, modulate oxidative stress and redox balance, and influence signal transduction cascades implicated in the pathophysiology of age-related macular degeneration. There is exclusive transport, sequestration, and appreciable bioamplification of macular xanthophylls from the circulating carotenoid pool to the retina and within the retina to regions required for high-resolution sensory processing. The distribution of diet-based macular xanthophylls and the lutein metabolite meso-zeaxanthin varies considerably by retinal eccentricity. Zeaxanthin concentrations are 2.5-fold higher than lutein in the cone-dense central fovea. This is an ~20-fold increase in the molar ratio relative to eccentric retinal regions with biochemically detectable macular xanthophylls. In this review, we discuss how the differences in the specific properties of lutein and zeaxanthin could help explain the preferential accumulation of zeaxanthin in the most vulnerable region of the macula.

Optical coherence tomography-angiographic vascular densities in Familial Mediterranean Fever (FMF) Patients with M694V Mutations.

Familial Mediterranean fever (FMF) is a hereditary auto-inflammatory disease with accompanying findings of amyloidosis and vasculitis. M694V is one of the most common mutations associated with amyloidosis. This study compared the macular optical coherence tomography angiography measurements in FMF patients who were genetically verified to carry the M694V mutation of the MEFV gene to those in healthy controls. The vessel densities (VDs) of superficial (SVP) and deep vascular plexus (DVP) of the retina, and choriocapillaris, foveal avascular zone (FAZ) perimetry, foveal VD 300µ around the FAZ (FD-300), acirculatory index (AI) and non-flow area were measured with  optical coherence tomography angiography (OCT-A). The FMF and control groups were matched for age and gender. Compound heterozygous pathogenic variants were excluded. Thirty-eight FMF patients with M694V mutations (28 heterozygous and 10 homozygous) and 40 healthy controls were included. The two groups were similar with the regard to age and gender (P=0.88 and P=0.49, respectively). None of the investigated parameters, including the vessel densities of the SVP and DVP, and choriocapillaris, FAZ perimetry, FD-300, AI, and non-flow area showed a statistically significant difference between the FMF and control groups. The macular vessel density measurements and FAZ parameters of FMF patients with M694V mutations do not differ from age- and sex-matched healthy controls.

Glial cells of the human fovea.

Purpose: The exact cellular types that form the human fovea remain a subject of debate, and few studies have been conducted on human macula to solve this question. The purpose of this study was to perform immunohistochemistry on fresh human samples to characterize the glial cells that form the human fovea. Methods: Immunohistochemistry was performed using antibodies against proteins expressed in astrocytes or in retinal Müller glial cells or both types of cells on six human macula obtained from eyes enucleated for peripheral intraocular tumors and on two postmortem eyes from healthy donors. The posterior poles of the enucleated eyes were cryosectioned and stained with antibodies against the glial proteins GFAP, vimentin, CRALBP, glutamine synthetase, and connexin 43. Results: A population of cells positive for GFAP and negative for glutamine synthetase and CRALBP that express connexin 43 were identified at the roof of the foveal pit. These cells are distinct from the Müller cone cells described by Yamada and Gass, suggesting that another type of foveal glial cells, most likely astrocytes, are present in the human fovea. Conclusions: This study showed that in humans, astrocytic glial cells cover the foveal pit. Their roles in macula homeostasis and mechanisms of macular diseases disease remain to be determined.

Relationship between Size of the Foveal Avascular Zone and Carbohydrate Metabolic Disorders during Pregnancy.

AIM: To determine whether the area of the foveal avascular zone (FAZ), as a morphological indicator of the microcirculation of the perifoveal capillary network, changes in the carbohydrate metabolism disorders during pregnancy (the gestational age of patients with gestational diabetes mellitus (GDM) and preexisting diabetes (PexD)). METHODS: Ten normal individuals and 41 eyes of 41 patients, 28 with GDM and 13 with PexD, were studied. A 3 × 3 mm area of the FAZ of the superficial capillary plexus layer (SCP) and the deep capillary plexus layer (DCP) was determined by optical coherence tomography angiography (OCTA; RS-3000 Advance, NIDEK). The significance of the correlation between the size of the FAZ and the weeks of pregnancy was determined. RESULTS: The area of the FAZ of the SCP was 0.38 ± 0.11 mm2 (normal eyes), 0.41 ± 0.16 mm2 (GDM), and 0.43 ± 0.10 mm2 (PexD). The area of the FAZ of the DCP was 0.78 ± 0.23 mm2 (normal eyes), 0.69 ± 0.16 mm2 (GDM), and 0.79 ± 0.25 mm2 (PexD). No significant difference in the FAZ sizes was observed between the groups. The average number of weeks of pregnancy was 24.1 ± 8.2 weeks in the eyes with GDM and 23.3 ± 11.4 weeks in the eyes with PexD (P > 0.05). Significant correlations were found between the size of the FAZ of the SCP and the number of weeks (r = 0.37, P=0.04 for GDM, and r = 0.49, P=0.04 for PexD, Spearman's rank-order correlation coefficient). CONCLUSIONS: For GDM and PexD under established glycemic control, the area of the FAZ is not affected, but vascular changes occurred at the early phase of pregnancy.

Influence of the foveal curvature on myopic macular complications.

In this cross-sectional study, we investigated whether a foveal curvature affects the development of two major myopic macular complications, myopic traction maculopathy (MTM) and myopic choroidal neovascularization (mCNV). In high myopic eyes (axial length ≥ 26.5 mm, refractive error ≤ -6 diopters) with posterior staphyloma, three different parameters of foveal curvature (staphyloma height, coefficient a, and curvature index) calculated based on the retinal pigment epithelium hyperreflective line in spectral domain optical coherence tomography image were compared among the MTM (72 eyes), mCNV (58 eyes), and control (69 eyes) group. The three curvature parameters showed a significant correlation with each other (all P's < 0.001). The axial length, refractive error, and staphyloma types were comparable among the groups, but the means of all three curvature parameters were significantly greater in the MTM group compared to the mCNV and control groups (all P's < 0.001). Furthermore, the curvature parameters had a significant correlation with myopic severity in the MTM group, but not in the other groups. These results suggest that a steeper change of foveal curvature plays a role in the development of MTM but not mCNV in high myopes.

Posterior microphthalmos, retinitis pigmentosa, and foveoschisis caused by a mutation in the MFRP gene: a familial study.

Background: To characterize the phenotype and genotype of a rare syndrome associating posterior microphthalmos (PM), retinitis pigmentosa (RP), and foveoschisis in a consanguineous Spanish family. Methods: The study involved five family members, consisting of three siblings and their parents. All members underwent comprehensive eye examinations for best corrected visual acuity, axial length and refractive error, electroretinography (ERG), fundus photography, retinal fluorescein angiography (FA), and optical coherence tomography (OCT). Clinical exome sequencing of more than 6,000 clinically relevant genes (SureSelect Focused Exome, Agilent) was performed using the Illumina HiSeq 3000 system. Candidate variants were validated and segregated by Sanger sequencing. Results: The affected siblings had bilateral shortening of the posterior ocular segment and normal anterior segment dimensions. The fundoscopy, ERG, and FA results were compatible with RP. Macular OCT analysis revealed schisis of the outer retinal layer. Our data analysis pipeline identified a homozygous frameshift mutation in exon 5 of the membrane frizzled-related protein (MFRP) gene (c.498delC; p.Asn167Thrfs*25). Conclusion: Our study confirmed the association of PM with RP as an autosomal recessive syndrome. Although this has previously been described, it seems that there are some constant (i.e., PM and RP) and some variable features (i.e., optic nerve drusen and foveoschisis). The MFRP mutation has also been detected in other studies associating PM with RP. Analysis of a larger series of cases at the clinical and genetic levels would certainly help us to better understand the phenotype-genotype correlations of this syndrome.

Influence of submacular fluid on recovery of retinal function and structure after successful rhegmatogenous retinal reattachment.

PURPOSE: To determine the influence of residual submacular fluid (SMF) on the recovery of function and structure of the retina after successful rhegmatogenous retinal detachment (RRD) reattachment. METHODS: We reviewed the medical records of all patients who had undergone successful RRD repair by scleral buckling (SB) surgery or by pars plana vitrectomy (PPV) from March 2011 to August 2014. Spectral-domain optical coherence tomographic images of the macular regions were used at 1, 2, 3, 6, 9, and 12 months following the surgery. The best-corrected visual acuities (BCVA) were evaluated at the same times. RESULTS: The eyes with a macula-off RRD that were treated by SB surgery had a significant higher incidence of residual SMF (52%) than those treated by PPV (6.8%; P <0.001). Nevertheless, the postoperative BCVA was significantly improved in the eyes that had undergone SB surgery (P = 0.007). The postoperative BCVAs were not significantly different between the groups in which the SMF was absorbed (12 eyes) and not absorbed (13 eyes) within 1 month after the SB surgery. The photoreceptor outer segment length and the presence of a foveal bulge were not significantly different between these two groups at 12 months. Multiple regression analyses showed that the presence of a foveal bulge (ß = 0.531, P = 0.001) and the duration of the retinal detachment before surgery (ß = 0.465, P = 0.002) but not the duration of the SMF were independent factors significantly correlated with the final BCVA. CONCLUSIONS: These results suggest that the postoperative residual SMF does not significantly disrupt the functional and structural recovery of eyes with macula-off RRD treated by SB surgery.

Investigating the potential of Zernike polynomials to characterise spatial distribution of macular pigment.

It has been postulated that particular patterns of macular pigment (MP) distribution may be associated with the risk for eye diseases such as age-related macular degeneration (AMD). This work investigates the potential of Zernike polynomials (ZP) to characterise the level and distribution of MP, and their suitability as a representation for analysis of the effects of age and AMD on MP patterns. As the case study, MP distribution maps computed using an experimental method based on fundus reflectance (MRIA) were obtained for ninety volunteers representing three groups: under-fifty without AMD, fifty and over without AMD, and fifty and over with AMD. ZP with 105 coefficients were fitted to the maps using least-squares optimisation and found to represent MP maps accurately (RMSE<10-1). One-way MANOVA analysis carried out on ZP representations showed that the three subject groups have significantly different means (Wilk's Lambda 0.125, p<0.0001). Linear discriminant analysis with leave-one-out scheme resulted in accuracy, sensitivity and specificity of classification according to, respectively, disease status regardless of age (81% all); disease status in the age-matched groups (87%, 88%, 86%); age irrespective of disease status (81%, 83%, 73%); and age for subjects without AMD (83%, 88%, 80%). Mean MP distributions computed from ZP coefficients for the three groups showed more elevated and more peaked MP for the healthy under-fifty group; more irregular and more elevated peripheral levels in over-fifty AMD group than in over-fifty non-AMD group; and moderate radial asymmetry in non-AMD over-50 group. The results suggest that ZP coefficients are capable of accurately representing MP in a way that captures certain spatial patterns of its distribution. Using the ZP representation MP maps could be classified according to both age and disease status with accuracy significantly greater than chance, with peak elevation, pattern irregularity and radial asymmetry identified as important features.

Molecular characterization of foveal versus peripheral human retina by single-cell RNA sequencing.

The human retina is a complex tissue responsible for detecting photons of light and converting information from these photons into the neurochemical signals interpreted as vision. Such visual signaling not only requires sophisticated interactions between multiple classes of neurons, but also spatially-dependent molecular specialization of individual cell types. In this study, we performed single-cell RNA sequencing on neural retina isolated from both the fovea and peripheral retina in three human donors. We recovered a total of 8,217 cells, with 3,578 cells originating from the fovea and 4,639 cells originating from the periphery. Expression profiles for all major retinal cell types were compiled, and differential expression analysis was performed between cells of foveal versus peripheral origin. Globally, mRNA for the serum iron binding protein transferrin (TF), which has been associated with age-related macular degeneration pathogenesis, was enriched in peripheral samples. Cone photoreceptor cells were of particular interest and formed two predominant clusters based on gene expression. One cone cluster had 96% of cells originating from foveal samples, while the second cone cluster consisted exclusively of peripherally isolated cells. A total of 148 genes were differentially expressed between cones from the fovea versus periphery. Interestingly, peripheral cones were enriched for the gene encoding Beta-Carotene Oxygenase 2 (BCO2). A relative deficiency of this enzyme may account for the accumulation of carotenoids responsible for yellow pigment deposition within the macula. Overall, this data set provides rich expression profiles of the major human retinal cell types and highlights transcriptomic features that distinguish foveal and peripheral cells.

Multi-platform imaging in ABCA4-Associated Disease.

Fundus autofluorescence (FAF) imaging is crucial to the diagnosis and monitoring of recessive Stargardt disease (STGD1). In a retrospective cohort study of 34 patients, we compared FAF imaging platforms varying in field size (30° and 55°: blue/SW-AF and NIR-AF; 200°: ultrawide-field, UWF-AF), excitation wavelength (488 nm, blue/SW-AF; 532 nm, UWF-AF and 787 nm, NIR-AF) and image processing. Due to reduced absorption of 532 nm and 787 nm light by macular pigment, foveal sparing was more readily demonstrable by green/UWF-AF and NIR-AF imaging. Prominent in green/UWF-AF images is a central zone of relatively elevated AF that is continuous inferonasal with a demarcation line bordering lower AF nasally and higher AF temporally. This zone and border are more visible in STGD1 than in healthy eyes and more visible with green/UWF-AF. With the development of AF flecks, inferonasal retina is initially spared. Central atrophic areas were larger in NIR-AF images than in blue/SW-AF and green/UWF-AF images and the presence of a contiguous hyperAF ring varied with imaging modality. Flecks visible as hyperAF foci in blue/SW-AF images were also visible in green/UWF-AF but were often hypoAF in NIR-AF. Since disease in STGD1 often extends beyond the 30° and 55° fields, green/UWF-AF has advantages including for pediatric patients. The imaging platforms examined provided complementary information.

Immunohistological Study of Monkey Foveal Retina.

The fovea centralis, an anatomically concave pit located at the center of the macula, is avascular, hypoxic, and characteristic of stem-cell niches of other tissues. We hypothesized that in the fovea, undifferentiated retinal-stem-cell-like cells may exist, and that neurogenesis may occur. Hence, we performed an immunohistological study using cynomolgus monkey retinas. After preparing frozen tissue sections of the retina including the foveal pit, immunostaining was performed for glial fibrillary acidic protein (GFAP), nestin, vimentin, neuron-specific class III ß-tubulin (Tuj-1), arrestin 4, neurofilament, CD117, CD44, Ki67, and cellular retinaldehyde-binding protein (CRALBP), followed by fluorescence and/or confocal microscopy examinations. Immunostaining of the tissue sections enabled clear observation of strongly GFAP-positive cells that corresponded to the inner-half layer of the foveolar Müller cell cone. The surface layer of the foveal slope was partially costained with GFAP and vimentin. Tuj-1-positive cells were observed in the innermost layer of the foveolar retina, which spanned to the surrounding ganglion cell layer. Moreover, colocalization of Tuj-1 and GFAP was observed at the foveal pit. The coexpression of CD117 and CD44 was found in the interphotoreceptor matrix of the fovea. The foveolar cone stained positive for both nestin and arrestin 4, however, the photoreceptor layer outside of the foveola displayed weak staining for nestin. Colocalization of nestin and vimentin was observed in the inner half of the Henle layer, while colocalization of nestin and neurofilament was observed in the outer half, predominantly. Scattered Ki67-positive cells were observed in the cellular processes of the outer plexiform layer and the ganglion cell layer around the foveola. Immunostaining for CRALBP was negative in most parts of the GFAP-positive area. The Müller cell cone was divided into GFAP-strongly positive cells, presumably astrocytes, in the inner layer and nestin-positive/GFAP-weakly positive radial glia-like cells in the outer layer. These findings indicated that groups of such undifferentiated cells in the foveola might be involved in maintaining morphology and regeneration.

An investigation of parafoveal masks with the incremental boundary paradigm.

Most of what we know about parafoveal preprocessing during reading is based on the boundary paradigm in combination with parafoveal masks as a presumably neutral baseline condition. Recent evidence questions the neutrality of the baseline condition by showing that parafoveal masks inflict preview costs. Using a novel, incremental boundary paradigm we studied the effect of parafoveal masks. Manipulating the salience of parafoveal previews, we found that increasing salience of the masks resulted in increasingly longer fixation times on target words, but also on pretarget words-suggesting preview costs. We conclude that the hidden preview costs of parafoveal masks in the classical boundary paradigm inflate the processing times for the baseline condition and hence lead to an overestimation of the preview benefit. Thus, the present study questions the validity of some of the conclusions drawn on the basis of the classical boundary paradigm.

Functional architecture of the foveola revealed in the living primate.

The primate foveola, with its high cone density and magnified cortical representation, is exquisitely specialized for high-resolution spatial vision. However, uncovering the wiring of retinal circuitry responsible for this performance has been challenging due to the difficulty in recording receptive fields of foveal retinal ganglion cells (RGCs) in vivo. In this study, we use adaptive optics scanning laser ophthalmoscopy (AOSLO) to image the calcium responses of RGCs in the living primate, with a stable, high precision visual stimulus that allowed us to localize the receptive fields of hundreds of foveal ganglion cells. This approach revealed a precisely radial organization of foveal RGCs, despite the many distortions possible during the extended developmental migration of foveal cells. By back projecting the line connecting RGC somas to their receptive fields, we have been able to define the 'physiological center' of the foveola, locating the vertical meridian separating left and right hemifields in vivo.

Human L- and M-opsins restore M-cone function in a mouse model for human blue cone monochromacy.

Purpose: Blue cone monochromacy (BCM) is an X-linked congenital vision disorder characterized by complete loss or severely reduced L- and M-cone function. Patients with BCM display poor visual acuity, severely impaired color discrimination, myopia, nystagmus, and minimally detectable cone-mediated electroretinogram. Recent studies of patients with BCM with adaptive optics scanning laser ophthalmoscopy (AOSLO) showed that they have a disrupted cone mosaic with reduced numbers of cones in the fovea that is normally dominated by L- and M-cones. The remaining cones in the fovea have significantly shortened outer segments but retain sufficient structural integrity to serve as potential gene therapy targets. In this study, we tested whether exogenously expressed human L- and M-opsins can rescue M-cone function in an M-opsin knockout (Opn1mw-/- ) mouse model for BCM. Methods: Adeno-associated virus type 5 (AAV5) vectors expressing OPN1LW, OPN1MW, or C-terminal tagged OPN1LW-Myc, or OPN1MW-HA driven by a cone-specific promoter were injected subretinally into one eye of Opn1mw-/- mice, while the contralateral eye served as the uninjected control. Expression of cone pigments was determined with western blotting and their cellular localization identified with immunohistochemistry. M-cone function was analyzed with electroretinogram (ERG). Antibodies against cone phototransduction proteins were used to study cone outer segment (OS) morphology in untreated and treated Opn1mw-/- eyes. Results: We showed that cones in the dorsal retina of the Opn1mw-/- mouse do not form outer segments, resembling cones that lack outer segments in the human BCM fovea. We further showed that AAV5-mediated expression of either human M- or L-opsin individually or combined promotes regrowth of cone outer segments and rescues M-cone function in the treated Opn1mw-/- dorsal retina. Conclusions: Exogenously expressed human opsins can regenerate cone outer segments and rescue M-cone function in Opn1mw-/- mice, thus providing a proof-of-concept gene therapy in an animal model of BCM.