Possible retinotoxicity of long-term vardenafil treatment.

Phosphodiesterase (PDE) inhibitors - such as vardenafil - are used primarily for treating erectile dysfunction via increasing cyclic guanosine monophosphate (cGMP) levels. Recent studies have also demonstrated their significant cardioprotective effects in several diseases, including diabetes, upon long-term, continuous application. However, PDE inhibitors are not specific for PDE5 and also inhibit the retinal isoform. A sustained rise in cGMP in photoreceptors is known to be toxic; therefore, we hypothesized that long-term vardenafil treatment might result in retinotoxicity. The hypothesis was tested in a clinically relevant animal model of type 2 diabetes mellitus. Histological experiments were performed on lean and diabetic Zucker Diabetic Fatty rats. Half of the animals were treated with vardenafil for six months, and the retinal effects were evaluated. Vardenafil treatment alleviated rod outer segment degeneration but decreased rod numbers in some positions and induced changes in the interphotoreceptor matrix, even in control animals. Vardenafil treatment decreased total retinal thickness in the control and diabetic groups and reduced the number of nuclei in the outer nuclear layer. Müller cell activation was detectable even in the vardenafil-treated control animals, and vardenafil did not improve gliosis in the diabetic group. Vardenafil-treated animals showed complex retinal alterations with improvements in some parameters while deterioration in others. Our results point towards the retinotoxicity of vardenafil, even without diabetes, which raises doubts about the retinal safety of long-term continuous vardenafil administration. This effect needs to be considered when approving PDE inhibitors for alternative indications.

Detailed Evaluation of Possible Ganglion Cell Loss in the Retina of Zucker Diabetic Fatty (ZDF) Rats.

A thinning of the inner retina is one of the earliest potential markers of neuroretinal damage in diabetic subjects. The histological background is uncertain; retinal ganglion cell (RGC) loss and changes in the structure or thickness of the inner plexiform layer (IPL) have been suspected. Studies conducted on animal models on RGC pathology gave contradictory results. Hereby we present RGC numbers, distribution patterns and IPL thickness from Zucker Diabetic Fatty (ZDF) rats. After labelling RGCs on retinal whole mounts, isodensity maps were constructed, RGC numbers and distribution patterns analysed using a custom-built algorithm, enabling point-by-point comparison. There was no change in staining characteristics of the antibodies and no significant difference in average RGC densities was found compared to controls. The distribution patterns were also comparable and no significant difference was found in IPL thickness and stratification or in the number of apoptotic cells in the ganglion cell layer (GCL). Our results provide a detailed evaluation of the inner retina and exclude major RGC loss in ZDF rats and suggest that other factors could serve as a potential explanation for inner retinal thinning in clinical studies. Our custom-built method could be adopted for the assessment of other animal or human retinas.

Histological Evaluation of Diabetic Neurodegeneration in the Retina of Zucker Diabetic Fatty (ZDF) Rats.

In diabetes, retinal dysfunctions exist prior to clinically detectable vasculopathy, however the pathology behind these functional deficits is still not fully established. Previously, our group published a detailed study on the retinal histopathology of type 1 diabetic (T1D) rat model, where specific alterations were detected. Although the majority of human diabetic patients have type 2 diabetes (T2D), similar studies on T2D models are practically absent. To fill this gap, we examined Zucker Diabetic Fatty (ZDF) rats - a model for T2D - by immunohistochemistry at the age of 32 weeks. Glial reactivity was observed in all diabetic specimens, accompanied by an increase in the number of microglia cells. Prominent outer segment degeneration was detectable with changes in cone opsin expression pattern, without a decrease in the number of labelled elements. The immunoreactivity of AII amacrine cells was markedly decreased and changes were detectable in the number and staining of some other amacrine cell subtypes, while most other cells examined did not show any major alterations. Overall, the retinal histology of ZDF rats shows a surprising similarity to T1D rats indicating that despite the different evolution of the disease, the neuroretinal cells affected are the same in both subtypes of diabetes.

Characterization of connexin36 gap junctions in the human outer retina.

Retinal connexins (Cx) form gap junctions (GJ) in key circuits that transmit average or synchronize signals. Expression of Cx36, -45, -50 and -57 have been described in many species but there is still a disconcerting paucity of information regarding the Cx makeup of human retinal GJs. We used well-preserved human postmortem samples to characterize Cx36 GJ constituent circuits of the outer plexiform layer (OPL). Based on their location, morphometric characteristics and co-localizations with outer retinal neuronal markers, we distinguished four populations of Cx36 plaques in the human OPL. Three of these were comprised of loosely scattered Cx36 plaques; the distalmost population 1 formed cone-to-rod GJs, population 2 in the mid-OPL formed cone-to-cone GJs, whereas the proximalmost population 4 likely connected bipolar cell dendrites. The fourth population (population 3) of Cx36 plaques conglomerated beneath cone pedicles and connected dendritic tips of bipolar cells that shared a common presynaptic cone. Overall, we show that the human outer retina displays a diverse cohort of Cx36 GJ that follows the general mammalian scheme and display a great functional diversity.

Novel features of neurodegeneration in the inner retina of early diabetic rats.

The literature indicates that in diabetes retinal dysfunctions related to neural retinal alterations exist prior to clinically detectable vasculopathy. In a previous report, a detailed description about the alteration of the outer retina was given, where diabetic degeneration preceded apoptotic loss of cells (Enzsöly et al., 2014). Here, we investigated the histopathology of the inner retina in early diabetes using the same specimens. We examined rat retinas with immunohistochemistry and Western blotting, 12 weeks after streptozotocin induction of diabetes. Glial reactivity was observed in all diabetic retinal specimens; however, it was not detectable all over the retina, but appeared in randomly arranged patches, with little or no glia activation in between. Similarly, immunoreactivity of parvalbumin (staining mostly AII amacrine cells) was also decreased only in some regions. We propose that these focal changes appear prior to affecting the whole retina and overt loss of cells. In contrast to these, most other markers used (calretinin, recoverin, tyrosin hydroxylase anti-Brn-3a and also calbindin in the optic part of the retina) did not show any major alterations in the intensity of immunoreactivity or in the number of stained elements. Interestingly, under diabetic conditions, the labeling pattern of PKC-α and calbindin in the ciliary retina showed a clear resemblance to the pattern described during development. This observation is in line with our previous study, reporting an increase in the number of dual cones, coexpressing two photopigments, which is another common feature with developing retinas. These data may indicate a previously uninvestigated regenerative capacity in diabetic retina.

Stratified organization and disorganization of inner plexiform layer revealed by TNAP activity in healthy and diabetic rat retina.

Tissue non-specific alkaline phosphatase (TNAP), an abundant ectophosphatase, is present in various organs including the brain and retina of several vertebrate species. Evidence is emerging that TNAP influences neural functions in multiple ways. In rat, strong TNAP activity has been found in retinal vessels, photoreceptors, and both synaptic layers. In the present study, we identified eleven strata of the inner plexiform layer (IPL) by using TNAP histochemistry alone. The TNAP strata corresponded exactly to the strata seen after combined immunohistochemistry with four canonical IPL markers (TH-ChAT-CR-PKCα). Therefore, as described in other mammalian species, our data support the existence of multiple morphologically and functionally discernible IPL strata in rats. Remarkably, the stratification pattern of the IPL was severely disrupted in a diabetic rat model, even before changes in the canonical IPL markers were detectable. These findings indicate that TNAP histochemistry offers a more straightforward, but also more sensitive, method for investigating retinal strata and their diabetes-induced degeneration.

Is a neuronal chain between the pineal body and the retina in rats and hamsters? Transneural tracing studies.

Neuronal chains between the retina and the pineal body were investigated. Transneuronal tracers, retrograde spreading pseudorabies virus (labeled with green fluorescent protein, memGreen-RV) and virus spreading in both ante- and retrograde directions (labeled with red fluorescent protein, Ka-VHS-mCherry-A-RV) were injected into the right eye of vitreous body of intact or bilaterally sympathectomized Wistar male rats. Intact golden hamsters also received memGreen-RV into the eye and Ka-VHS-mCherry-A-RV into the pineal body. Four-five days later the animals were sacrificed. Frozen sections were prepared from the removed structures. In intact rats memGreen-RV resulted in green fluorescent labeling in the trigeminal and the superior cervical ganglia, the lateral horn of the spinal cord, the paraventricular and the suprachiasmatic nuclei, the perifornical region, the ventrolateral medulla, the locus ceruleus, and the raphe nuclei. In sympathectomized rats the labeling was missing from the brainstem but further existed in the hypothalamus. This observation indicates that the hypothalamic labeling is not mediated by the sympathetic system. One labeled neuron in the pineal body was only observed in 2/13 rats. It was independent from the sympathectomy. When the animals received Ka-VHS-mCherry-A-RV the distribution of the labeling was very similar to that of the intact group receiving retrograde virus. In golden hamsters the memGreen-RV labeled structures were seen in similar places as in rats, but virus labeled nerve cell bodies were always seen in the pineal body. Injection of Ka-VHS-mCherry-A-RV into the pineal body of hamsters resulted in labeling of the retina at both sides. It was concluded that the retinopetal neuronal chain in golden hamsters is always present but in rats it is stochastic.

TNAP activity is localized at critical sites of retinal neurotransmission across various vertebrate species.

Evidence is emerging with regard to the role of tissue non-specific alkaline phosphatase (TNAP) in neural functions. As an ectophosphatase, this enzyme might influence neural activity and synaptic transmission in diverse ways. The localization of the enzyme in known neural circuits, such as the retina, might significantly advance an understanding of its role in normal and pathological functioning. However, the presence of TNAP in the retina is scarcely investigated. Our multispecies comparative study (zebrafish, cichlid, frog, chicken, mouse, rat, golden hamster, guinea pig, rabbit, sheep, cat, dog, ferret, squirrel monkey, human) using enzyme histochemistry and Western blots has shown the presence of TNAP activity in the retina of several mammalian species, including humans. Although the TNAP activity pattern varies across species, we have observed the following trends: (1) in all investigated species (except golden hamster), retinal vessels display TNAP activity; (2) TNAP activity consistently occurs in the photoreceptor layer; (3) in majority of the investigated species, marked TNAP activity is present in the outer and inner plexiform layers. In zebrafish, frog, chicken, guinea pig, and rat, TNAP histochemistry has revealed several sublayers of the inner plexiform layer. Frog, golden hamster, guinea pig, mouse, and human retinas possess a subpopulation of amacrine cells positively staining for TNAP activity. The expression of TNAP in critical sites of retinal signal transmission across a wide range of species suggests its fundamental, evolutionally conserved role in vision.

Pathologic alterations of the outer retina in streptozotocin-induced diabetes.

PURPOSE: Neurodegeneration as an early event of diabetic retinopathy preceding clinically detectable vascular alterations is a widely proven issue today. While there is evidence for the impairment of color vision and contrast sensitivity in early diabetes, suggesting deteriorated photoreceptor function, the underlying neuropathology of these functional alterations is still unknown. The aim of the present study was to investigate the effects of early diabetes on the outer retinal cells. METHODS: The retinal pigment epithelium, photopigment expression, and density and morphology of photoreceptors were studied using immunocytochemistry in streptozotocin-induced diabetes in two rat strains. The fine structure of photoreceptors and pigment epithelium was also investigated with transmission electron microscopy. RESULTS: Here we found that retinal thickness was unchanged in diabetic animals and that no significant increase in the number of apoptotic cells was present. Although the density of cones expressing middle (M)- and shortwave (S)-sensitive opsins was similar in diabetic and control retinas, we detected remarkable morphologic signs of degeneration in the outer segments of diabetic rods, most M-cones, and some S-cones. A decrease in thickness and RPE65 protein immunoreactivity of the pigment epithelium were evident. Furthermore, an increased number of dual cones, coexpressing both M- and S-opsins, was detected at the peripheral retina of diabetic rats. CONCLUSIONS: Degenerative changes of photoreceptors and pigment epithelium shown here prior to apoptotic loss of photoreceptors may contribute to functional alterations reported in diabetic human patients and different animal models, thus may serve as a potential model for testing the efficacy of neuroprotective agents in diabetes.

Immunocytochemical analysis of misplaced rhodopsin-positive cells in the developing rodent retina.

During the first postnatal weeks of the developing rodent retina, rhodopsin can be detected in a number of neuron-like cells in the inner retina. In the present study, we aim to characterize the morphology, number and staining characteristics of this peculiar population. Misplaced rhodopsin-positive cells (MRCs) were analyzed on retinas of four rodent species, labeled with various rhodopsin-specific antibodies. To investigate their possible relation with non-photoreceptor cells, sections were double-stained against distinct retinal cell types and proteins of the phototransduction cascade. The possibility of synapse formation and apoptosis were also investigated. In all species studied, misplaced cells comprised a few percent of all rhodopsin-positive elements. This ratio declined from the end of the second week and MRCs disappeared nearly completely from the retina by P24. MRCs resembled resident neurons of the inner retina, while outer segment-like processes were seen only rarely. MRCs expressed no other photopigment types and showed no colocalization with any of the bipolar, horizontal, amacrine and ganglion cell markers used. While all MRCs colabeled for arrestin and recoverin, other proteins of the phototransduction cascade were only detectable in a minority of the population. Only a few MRCs were shown to form synaptic-like endings. Our results showed that, during development, some rhodopsin-expressing cells are displaced to the inner retinal layers. Although most MRCs lack morphological features of photoreceptors, they contain some but not all, elements of the phototransduction cascade, indicating that they are most probably misplaced rods that failed to complete differentiation and integrate into the photoreceptor mosaic.

Peripheral autonomic nerves of human pineal organ terminate on vessels, their supposed role in the periodic secretion of pineal melatonin.

Nonvisual pineal and retinal photoreceptors are synchronizing circadian and circannual periodicity to the environmental light periods in the function of various organs. Melatonin of the pineal organ is secreted at night and represents an important factor of this periodic regulation. Night illumination suppressing melatonin secretion may result in pathological events like breast and colorectal cancer. Experimental works demonstrated the role of autonomic nerves in the pineal melatonin secretion. It was supposed that mammalian pineals have lost their photoreceptor capacity that is present in submammalians, and sympathetic fibers would mediate light information from the retina to regulate melatonin secretion. Retinal afferentation may reach the organ by central nerve fibers via the pineal habenulae as well. In our earlier works we have found that the pineal organ developing from lobular evaginations of the epithalamus differs from peripheral endocrine glands and is composed of a retina-like central nervous tissue that is comprised of cone-like pinealocytes, secondary pineal neurons and glial cells. Their autonomic nerves in submammalians as well as in mammalian animals do not terminate on pineal cells, rather, they run in the meningeal septa among pineal lobules and form vasomotor nerve endings. Concerning the adult human pineal there are no detailed fine structural data about the termination of autonomic fibers, therefore, in the present work we investigated the ultrastructure of the human pineal peripheral autonomic nerve fibers. It was found, that similarly to other parts of the brain, autonomic nerves do not enter the human pineal nervous tissue itself but separated by glial limiting membranes take their course in the meningeal septa of the organ and terminate on vessels by vasomotor endings. We suppose that these autonomic vasomotor nerves serve the regulation of the pineal blood supply according to the circadian and circannual changes of the metabolic activity of the organ and support by this effect the secretion of pineal neurohormones including melatonin.

Photoreceptor cell death, proliferation and formation of hybrid rod/S-cone photoreceptors in the degenerating STK38L mutant retina.

A homozygous mutation in STK38L in dogs impairs the late phase of photoreceptor development, and is followed by photoreceptor cell death (TUNEL) and proliferation (PCNA, PHH3) events that occur independently in different cells between 7-14 weeks of age. During this period, the outer nuclear layer (ONL) cell number is unchanged. The dividing cells are of photoreceptor origin, have rod opsin labeling, and do not label with markers specific for macrophages/microglia (CD18) or Müller cells (glutamine synthetase, PAX6). Nestin labeling is absent from the ONL although it labels the peripheral retina and ciliary marginal zone equally in normals and mutants. Cell proliferation is associated with increased cyclin A1 and LATS1 mRNA expression, but CRX protein expression is unchanged. Coincident with photoreceptor proliferation is a change in the photoreceptor population. Prior to cell death the photoreceptor mosaic is composed of L/M- and S-cones, and rods. After proliferation, both cone types remain, but the majority of rods are now hybrid photoreceptors that express rod opsin and, to a lesser extent, cone S-opsin, and lack NR2E3 expression. The hybrid photoreceptors renew their outer segments diffusely, a characteristic of cones. The results indicate the capacity for terminally differentiated, albeit mutant, photoreceptors to divide with mutations in this novel retinal degeneration gene.

Localization of caveolin-1 and c-src in mature and differentiating photoreceptors: raft proteins co-distribute with rhodopsin during development.

Numerous biochemical and morphological studies have provided insight into the distribution pattern of caveolin-1 and the presence of membrane rafts in the vertebrate retina. To date however, studies have not addressed the localization profile of raft specific proteins during development. Therefore the purpose of our studies was to follow the localization pattern of caveolin-1, phospho-caveolin-1 and c-src in the developing retina and compare it to that observed in adults. Specific antibodies were used to visualize the distribution of caveolin-1, c-src, a kinase phosphorylating caveolin-1, and phospho-caveolin-1. The labeling pattern of this scaffolded complex was compared to those of rhodopsin and rhodopsin kinase. Samples were analyzed at various time points during postnatal development and compared to adult retinas. The immunocytochemical studies were complemented with immunoblots and immunoprecipitation studies. In the mature retina caveolin-1 and c-src localized mainly to the cell body and IS of photoreceptors, with only very weakly labeled OS. In contrast, phospho-caveolin-1 was only detectable in the OS of photoreceptors. During development we followed the expression and distribution profile of these proteins in a temporal sequence with special attention to the period when OS formation is most robust. Double labeling immunocytochemistry and immunoprecipitation showed rhodopsin to colocalize and co-immunoprecipitate with caveolin-1 and c-src. Individual punctate structures between the outer limiting membrane and the outer plexiform layer were seen at P10 to be labeled by both rhodopsin and caveolin-1 as well as by rhodopsin and c-src, respectively. These studies suggest that membrane raft specific proteins are co-distributed during development, thereby pointing to a role for such complexes in OS formation. In addition, the presence of small punctate structures containing caveolin-1, c-src and rhodopsin raise the possibility that these proteins may transport together to OS during development and that caveolin-1 exists predominantly in a phosphorylated form in the OS.

Distribution of caveolin isoforms in the lemur retina.

The distribution of caveolin isoforms was previously evaluated in the retinas of different species, but has not yet been described in the primate retina. In this study, the distribution of caveolins was assessed via immunochemistry using isoform-specific antibodies in the retina of the black-and-white ruffed lemur. Here, we report the presence of a variety of caveolin isoforms in many layers of the lemur retina. As normal human retinas were not available for research and the retinas of primates are fairly similar to those of humans, the lemur retina can be utilized as a model for caveolin distribution in normal humans.

Different caveolin isoforms in the retina of melanoma malignum affected human eye.

PURPOSE: Caveolin-1 has been identified in Müller and pigment cells of rodents, but the distribution of caveolin isoforms has not been studied in the human retina. Since there are no relevant data in humans, we aimed to study the distribution of caveolins in the human retina. METHODS: Our samples were derived from eyes affected by melanoma malignum choroideae that were enucleated. The distribution of the caveolins was examined by immunocytochemistry using isoform-specific antibodies. RESULTS: In this study we report on the presence of different caveolin isoforms in many cell types of the human retina. These isoforms were present in several regions and layers in the human retina. Centro-peripheral changes have been detected: the distribution altered following the radier direction. CONCLUSIONS: This is the first demonstration of caveolin expression in the human retina. Our data suggest that caveolins play an important role in the function of retinal cells. Our observations refute previous assumptions that there is a shortage of caveolins in the retina. Since the retina contains a number of different neuronal and glial cell types, the caveolin expression of these cells can no longer be a matter of dispute.

The expression of the Leber congenital amaurosis protein AIPL1 coincides with rod and cone photoreceptor development.

PURPOSE: The Leber congenital amaurosis (LCA) protein AIPL1 is present only in the rod photoreceptors of the adult human retina and is excluded from the cone photoreceptors. LCA, however, is characterized by an absence of both rod and cone function at birth or shortly thereafter. Therefore, this study was conducted to determine whether AIPL1 is present in the rod and cone photoreceptors of the developing human retina. In addition, the expression of NUB1, a putative AIPL1-interacting partner, was examined. METHODS: A comprehensive spatiotemporal examination of AIPL1 distribution during development was performed by immunohistochemistry, using a previously characterized AIPL1 anti-serum. Immunofluorescence confocal microscopy was used to examine the coexpression of AIPL1 with the long/medium (L/M) and short (S) wavelength-sensitive cone photoreceptors in the developing human retina. The spatiotemporal distribution of NUB1 was also examined by immunohistochemistry, using a newly developed anti-serum to the C terminus of NUB1. RESULTS: AIPL1 protein was detected by 11.8 fetal weeks in the central fetal human retina. With continued development, AIPL1 expression spread gradually toward peripheral retina. AIPL1 was expressed in the L/M and S cone photoreceptors in addition to the rods of the developing human retina. NUB1 was localized in cell nuclei throughout the human fetal and adult eye at all time points. CONCLUSIONS: The pattern of AIPL1 expression closely follows the centroperipheral gradient in photoreceptor development. The data suggest that AIPL1 is essential for the normal development of both rod and cone photoreceptor cells and that mutations in the AIPL1 gene cause the death or dysfunction of photoreceptors early in development resulting in blindness or severely impaired vision at birth.

Long-term effect of retinal ganglion cell axotomy on the histomorphometry of other cells in the porcine retina.

PURPOSE: To determine the effect of retinal ganglion cell axotomy on the thickness of inner plexiform, inner nuclear, and outer plexiform layers, as well as the densities of short- and middle-to-long-wavelength cones, in the porcine retina. METHODS: Unilateral retinal ganglion cell axotomy was performed in seven domestic pigs by either surgical optic nerve section or peripapillary argon laser photocoagulation. Damage to the retinal vasculature was ruled out with fluorescein angiography. Histologic examination of the retinal tissue was performed nine months later. Cone densities were determined immunohistochemically with the anti-visual pigment antibodies COS-1 and OS-2. Image analysis of semithin retinal cross sections was used to measure the thickness of the retinal layers. The effect of axotomy was quantified by optic nerve axon counts and estimations of retinal ganglion cell counts. The data were compared between the eyes with axotomy and the contralateral normal eye using the nonparametric Wilcoxon rank sum test. RESULTS: Treatment of the peripapillary retina with the argon laser resulted in a median decrease in axon counts and retinal ganglion cell density estimates of 31%. No optic nerve axons and cells resembling retinal ganglion cells were found in the eyes with transected optic nerves. There was no significant difference in either the thickness of any retinal layers or cone densities between axotomized and normal control eyes. CONCLUSION: No signs of retrograde transsynaptic degeneration were observed in porcine retinas nine months after retinal ganglion cell axotomy.

Differentiation of cones in cultured rabbit retina: effects of retinal pigment epithelial cell-conditioned medium.

This study was aimed at investigating the postnatal differentiation of cone photoreceptors in the rabbit retina in an organotypic explant culture system. Both short wavelength (S) and middle wavelength (M) cone opsins were expressed in culture but M cones appeared only in retinal explants from the dorsal half of the eye. Stimulating the explants with retinal pigment epithelial cell (RPE) conditioned medium resulted in a suppression of opsin expression despite of an increase of the number of presumptive peanut agglutinin-labeled cones. These results suggest that at birth the immature cones are largely undetermined in terms of their final cone identity although some positional information ('dorsal' vs. 'ventral' retina) is present. Furthermore, factors from RPE may inhibit as well as stimulate different steps of cone cell differentiation.

Visual pigment coexpression in all cones of two rodents, the Siberian hamster, and the pouched mouse.

PURPOSE: To decide whether the identical topography of short- and middle-wavelength cone photoreceptors in two species of rodents reflects the presence of both opsins in all cone cells. METHODS: Double-label immunocytochemistry using antibodies directed against short-wavelength (S)-and middle- to long-wavelength (M/L)-sensitive opsin were used to determine the presence of visual pigments in cones of two species of rodents, the Siberian hamster (Phodopus sungorus) and the pouched mouse (Saccostomus campestris) from South Africa. Topographical distribution was determined from retinal whole-mounts, and the colocalization of visual pigments was examined using confocal laser scanning microscopy. Opsin colocalization was also confirmed in consecutive semithin tangential sections. RESULTS: The immunocytochemical results demonstrate that in both the Siberian hamster and the pouched mouse all retinal cones contain two visual pigments. No dorsoventral gradient in the differential expression of the two opsins is observed. CONCLUSIONS: The retina of the Siberian hamster and the pouched mouse is the first example to show a uniform coexpression of M and S cone opsins in all cones, without any topographical gradient in opsin expression. This finding makes these two species good models for the study of molecular control mechanisms in opsin coexpression in rodents, and renders them suitable as sources of dual cones for future investigations on the role and neural connections of this cone type.