Exposure to Blue Light Reduces Melanopsin Expression in Intrinsically Photoreceptive Retinal Ganglion Cells and Damages the Inner Retina in Rats.

Purpose: The purpose of this study was to investigative the effects of blue light on intrinsically photoreceptive retinal ganglion cells (ipRGCs). Methods: Brown Norway rats were used. Nine rats were continuously exposed to blue light (light emitting diodes [LEDs]: 463 nm; 1000 lx) for 2 days (acute exposure [AE]); 9 rats were exposed to 12 hours of blue light and 12 hours of darkness for 10 days (long-term exposure [LTE]); 6 control rats were exposed to 12 hours of white fluorescent light (1000 lx) and 12 hours of darkness for 10 days. Whole-mount retinas were immunolabelled with melanopsin antibodies; melanopsin-positive (MP) ipRGC somas and processes were counted and measured with Neuron J. To detect apoptosis, retinal cryo-sections were stained with terminal deoxynucleotidyl transferase dUTP nick-end labeling. Ultra-thin sections were visualized with transmission electron microscopy. Results: The number of MP ipRGC somas was significantly lower in retinas from AE and LTE rats than in those from control rats (P < 0.001 and = 0.002, respectively). The mean length of MP areas of processes was significantly lower in AE rats (P < 0.001). AE rats had severe retinal damage and massive apoptosis in the outer nuclear layer; their mitochondria were damaged in the axons and dendrites of the nerve fiber layer and the inner plexiform layer. Retinal ganglion cells (RGCs) in AE rats appeared to have reduced amounts of free ribosomes and rough endoplasmic reticulum. Conclusions: AE to blue light reduces melanopsin expression and damages RGCs, likely including ipRGCs. Changes in the axons and dendrites of RGCs suggest possible disruption of intraretinal and extraretinal signal transmission.

Disrupted eye and head development in rainbow trout with reduced ultraviolet (sws1) opsin expression.

Visual opsins are proteins expressed by retinal photoreceptors that capture light to begin the process of phototransduction. In vertebrates, the two types of photoreceptors (rods and cones) express one or multiple opsins and are distributed in variable patterns across the retina. Some cones form opsin retinal gradients, as in the mouse, whereas others form more demarcated opsin domains, as in the lattice-like mosaic retinas of teleost fishes. Reduced rod opsin (rh1) expression in mouse, zebrafish, and African clawed frog results in lack of photoreceptor outer segments (i.e., the cilium that houses the opsins) and, in the case of the mouse, to retinal degeneration. The effects of diminished cone opsin expression have only been studied in the mouse where knockout of the short-wavelength sensitive 1 (sws1) opsin leads to ventral retinal cones lacking outer segments, but no retinal degeneration. Here we show that, following CRISPR/Cas9 injections that targeted knockout of the sws1 opsin in rainbow trout, fish with diminished sws1 opsin expression exhibited a variety of developmental defects including head and eye malformations, underdeveloped outer retina, mislocalized opsin expression, cone degeneration, and mosaic irregularity. All photoreceptor types were affected even though sws1 is only expressed in the single cones of wild fish. Our results reveal unprecedented developmental defects associated with diminished cone opsin expression and suggest that visual opsin genes are involved in regulatory processes that precede photoreceptor differentiation.

Visual opsin expression and morphological characterization of retinal photoreceptors in the pouched lamprey (Geotria australis, Gray).

Lampreys are extant members of the agnathan (jawless) vertebrates that diverged ~500 million years ago, during a critical stage of vertebrate evolution when image-forming eyes first emerged. Among lamprey species assessed thus far, the retina of the southern hemisphere pouched lamprey, Geotria australis, is unique, in that it possesses morphologically distinct photoreceptors and expresses five visual photopigments. This study focused on determining the number of different photoreceptors present in the retina of G. australis and whether each cell type expresses a single opsin class. Five photoreceptor subtypes were identified based on ultrastructure and differential expression of one of each of the five different visual opsin classes (lws, sws1, sws2, rh1, and rh2) known to be expressed in the retina. This suggests, therefore, that the retina of G. australis possesses five spectrally and morphologically distinct photoreceptors, with the potential for complex color vision. Each photoreceptor subtype was shown to have a specific spatial distribution in the retina, which is potentially associated with changes in spectral radiance across different lines of sight. These results suggest that there have been strong selection pressures for G. australis to maintain broad spectral sensitivity for the brightly lit surface waters that this species inhabits during its marine phase. These findings provide important insights into the functional anatomy of the early vertebrate retina and the selection pressures that may have led to the evolution of complex color vision.

Mechanistic insights into the evolution of the differential expression of tandemly arrayed cone opsin genes in zebrafish.

The genome of many organisms contains several loci consisting of duplicated genes that are arrayed in tandem. The daughter genes produced by duplication typically exhibit differential expression patterns with each other or otherwise experience pseudogenization. Remarkably, opsin genes in fish are preserved after many duplications in different lineages. This fact indicates that fish opsin genes are characterized by a regulatory mechanism that could intrinsically facilitate the differentiation of the expression patterns. However, little is known about the mechanisms that underlie the differential expression patterns or how they were established during evolution. The loci of green (RH2)- and red (LWS)-sensitive cone opsin genes in zebrafish have been used as model systems to study the differential regulation of tandemly arrayed opsin genes. Over a decade of studies have uncovered several mechanistic features that might have assisted the differentiation and preservation of duplicated genes. Furthermore, recent progress in the understanding of the transcriptional process in general has added essential insights. In this article, the current understanding of the transcriptional regulation of differentially expressed tandemly arrayed cone opsin genes in zebrafish is summarized and a possible evolutionary scenario that could achieve this differentiation is discussed.

Illuminating insights into opsin 3 function in the skin.

Because sunlight is essential for human survival, we have developed complex mechanisms for detecting and responding to light stimuli. The eyes and skin are major organs for sensing light and express several light-sensitive opsin receptors. These opsins mediate cellular responses to spectrally-distinct wavelengths of visible and ultraviolet light. How the eyes mediate visual phototransduction is well understood, but less is known about how the skin detects light. Both human and murine skin express a wide array of opsins, with one of the most highly expressed being the functionally elusive opsin 3 (OPN3). In this review we explore light reception, opsin expression and signaling in skin cells; we compile data elucidating potential functions for human OPN3 in skin, with emphasis on recent studies investigating OPN3 regulation of melanin within epidermal melanocytes.

LWS visual pigment in owls: Spectral tuning inferred by genetics.

Owls constitute a diverse group of raptors, active at different times of the day with distinct light conditions that might be associated with multiple visual adaptations. We investigated whether shifts in the spectral sensitivity of the L cone visual pigment, as inferred by analysis of gene structure, could be one such adaptive mechanism. Using Sanger sequencing approach, we characterized the long wavelength-sensitive (LWS) opsin gene expressed in the retina of five owl species, specifically chosen to represent distinct patterns of activity. Nocturnality was epitomized by the American barn owl (Tyto furcata), the striped owl (Asio clamator), and the tropical screech owl (Megascops choliba); diurnality, by the ferruginous pygmy owl (Glaucudium brasilianum); and cathemerality, by the burrowing owl (Athene cunicularia). We also analyzed the presence of the L cone in the retinas of four species of owl (T. furcata, A. cunicularia, G. brasilianum and M. choliba) using immnunohistochemistry. Five critical sites for the spectral tuning of the LWS opsin (164, 181, 261, 269, and 292) were analyzed and compared to the sequence of other birds. The sequence of A. cunicularia showed a substitution on residue 269, with the presence of an alanine instead threonine, which generates an estimated maximum absorption (λmax) around 537 nm. No other variation was found in the spectral tuning sites of the LWS opsin, among the other species, and the λmax was estimated at around 555 nm. The presence of L cones in the retinas of the four species of owls was revealed using immunohistochemistry and we observed a reduced number of L cones in T. furcata compared to A. cunicularia, G. brasilianum and M. choliba.

Endocrine regulation of multichromatic color vision.

Vertebrate color vision requires spectrally selective opsin-based pigments, expressed in distinct cone photoreceptor populations. In primates and in fish, spectrally divergent opsin genes may reside in head-to-tail tandem arrays. Mechanisms underlying differential expression from such arrays have not been fully elucidated. Regulation of human red (LWS) vs. green (MWS) opsins is considered a stochastic event, whereby upstream enhancers associate randomly with promoters of the proximal or distal gene, and one of these associations becomes permanent. We demonstrate that, distinct from this stochastic model, the endocrine signal thyroid hormone (TH) regulates differential expression of the orthologous zebrafish lws1/lws2 array, and of the tandemly quadruplicated rh2-1/rh2-2/rh2-3/rh2-4 array. TH treatment caused dramatic, dose-dependent increases in abundance of lws1, the proximal member of the lws array, and reduced lws2 Fluorescent lws reporters permitted direct visualization of individual cones switching expression from lws2 to lws1 Athyroidism increased lws2 and reduced lws1, except within a small ventral domain of lws1 that was likely sustained by retinoic acid signaling. Changes in lws abundance and distribution in athyroid zebrafish were rescued by TH, demonstrating plasticity of cone phenotype in response to this signal. TH manipulations also regulated the rh2 array, with athyroidism reducing abundance of distal members. Interestingly, the opsins encoded by the proximal lws gene and distal rh2 genes are sensitive to longer wavelengths than other members of their respective arrays; therefore, endogenous TH acts upon each opsin array to shift overall spectral sensitivity toward longer wavelengths, underlying coordinated changes in visual system function during development and growth.

Degeneration of ipRGCs in Mouse Models of Huntington's Disease Disrupts Non-Image-Forming Behaviors Before Motor Impairment.

Intrinsically photosensitive retinal ganglion cells (ipRGCs), which express the photopigment melanopsin, are photosensitive neurons in the retina and are essential for non-image-forming functions, circadian photoentrainment, and pupillary light reflexes. Five subtypes of ipRGCs (M1-M5) have been identified in mice. Although ipRGCs are spared in several forms of inherited blindness, they are affected in Alzheimer's disease and aging, which are associated with impaired circadian rhythms. Huntington's disease (HD) is an autosomal neurodegenerative disease caused by the expansion of a CAG repeat in the huntingtin gene. In addition to motor function impairment, HD mice also show impaired circadian rhythms and loss of ipRGC. Here, we found that, in HD mouse models (R6/2 and N171-82Q male mice), the expression of melanopsin was reduced before the onset of motor deficits. The expression of retinal T-box brain 2, a transcription factor essential for ipRGCs, was associated with the survival of ipRGCs. The number of M1 ipRGCs in R6/2 male mice was reduced due to apoptosis, whereas non-M1 ipRGCs were relatively resilient to HD progression. Most importantly, the reduced innervations of M1 ipRGCs, which was assessed by X-gal staining in R6/2-OPN4Lacz/+ male mice, contributed to the diminished light-induced c-fos and vasoactive intestinal peptide in the suprachiasmatic nuclei (SCN), which may explain the impaired circadian photoentrainment in HD mice. Collectively, our results show that M1 ipRGCs were susceptible to the toxicity caused by mutant Huntingtin. The resultant impairment of M1 ipRGCs contributed to the early degeneration of the ipRGC-SCN pathway and disrupted circadian regulation during HD progression.SIGNIFICANCE STATEMENT Circadian disruption is a common nonmotor symptom of Huntington's disease (HD). In addition to the molecular defects in the suprachiasmatic nuclei (SCN), the cause of circadian disruption in HD remains to be further explored. We hypothesized that ipRGCs, by integrating light input to the SCN, participate in the circadian regulation in HD mice. We report early reductions in melanopsin in two mouse models of HD, R6/2, and N171-82Q. Suppression of retinal T-box brain 2, a transcription factor essential for ipRGCs, by mutant Huntingtin might mediate the reduced number of ipRGCs. Importantly, M1 ipRGCs showed higher susceptibility than non-M1 ipRGCs in R6/2 mice. The resultant impairment of M1 ipRGCs contributed to the early degeneration of the ipRGC-SCN pathway and the circadian abnormality during HD progression.

Divergent projection patterns of M1 ipRGC subtypes.

In addition to its well-known role in pattern vision, light influences a wide range of non-image forming, subconscious visual behaviors including circadian photoentrainment, sleep, mood, learning, and the pupillary light reflex. Each of these behaviors is thought to require input from the M1 subtype of melanopsin-expressing, intrinsically photosensitive retinal ganglion cell (ipRGC). Recent work has demonstrated that the M1 subtype of ipRGC can be further subdivided based on expression of the transcription factor Brn3b. Brn3b-positive M1 ipRGCs project to the olivary pretectal nucleus and are necessary for the pupillary light reflex, while Brn3b-negative M1 ipRGCs project to the suprachiasmatic nucleus (SCN) and are sufficient for circadian photoentrainment. However, beyond the circadian and pupil systems, little is known about the projection patterns of M1 ipRGC subtypes. Here we show that Brn3b-positive M1 ipRGCs comprise the majority of sparse M1 ipRGC inputs to the thalamus, midbrain, and hypothalamus. Our data demonstrate that very few brain targets receive convergent input from both M1 ipRGC subpopulations, suggesting that each subpopulation drives a specific subset of light-driven behaviors.

Seeing the light to change colour: An evolutionary perspective on the role of melanopsin in neuroendocrine circuits regulating light-mediated skin pigmentation.

Melanopsin photopigments, Opn4x and Opn4m, were evolutionary selected to "see the light" in systems that regulate skin colour change. In this review, we analyse the roles of melanopsins, and how critical evolutionary developments, including the requirement for thermoregulation and ultraviolet protection, the emergence of a background adaptation mechanism in land-dwelling amphibian ancestors and the loss of a photosensitive pineal gland in mammals, may have helped sculpt the mechanisms that regulate light-controlled skin pigmentation. These mechanisms include melanopsin in skin pigment cells directly inducing skin darkening for thermoregulation/ultraviolet protection; melanopsin-expressing eye cells controlling neuroendocrine circuits to mediate background adaptation in amphibians in response to surface-reflected light; and pineal gland secretion of melatonin phased to environmental illuminance to regulate circadian and seasonal variation in skin colour, a process initiated by melanopsin-expressing eye cells in mammals, and by as yet unknown non-visual opsins in the pineal gland of non-mammals.

Melanopsin-expressing retinal ganglion cells are relatively resistant to excitotoxicity induced by N-methyl-d-aspartate.

Excitotoxicity plays an important role in neuronal loss in glaucoma. Previous studies indicate melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are more injury-resistant. However, whether melanopsin-expressing ipRGCs are resistant to N-methyl-d-aspartate (NMDA) induced excitotoxicity is not well understood. In the present study, we investigated the effects of NMDA-induced excitotoxicity in conventional retinal ganglion cells (RGCs) and melanopsin-expressing ipRGCs in adult mice. The loss of RGCs and the reduction of the thickness of inner plexiform layer (IPL) were studied by histology, immunofluorescence, TUNEL assay and optical coherence tomography (OCT). The remaining conventional RGCs and ipRGCs were quantified on the 1st, 3rd, 7th, and 21st day after NMDA injection using immunofluorescence. NMDA mediated acute and severe damage of conventional RGCs damage in a time-dependent manner, and approximately 85% of the conventional RGCs were lost on the 21st days. Furthermore, a significant reduction of the IPL thickness was observed. Moreover, compared to the PBS-injected eyes, the density of total melanopsin-positive RGCs decreased by 25% on the 1st day after NMDA injection, and then the density was constant at other time points. Our results suggest that melanopsin-expressing ipRGCs are relatively resistant to excitotoxicity induced by NMDA.

Loss of Melanopsin-Expressing Retinal Ganglion Cells in Patients With Diabetic Retinopathy.

Purpose: Photo-entrainment of the circadian clock is mediated by melanopsin-expressing retinal ganglion cells (mRGCs) located in the retina. Patients suffering from diabetic retinopathy (DR) show impairment of light regulated circadian activity such as sleep disorders, altered blood pressure, and abnormal melatonin secretion. The aim of this study was to assess the impact of DR on the expression of mRGCs in the human retina. Methods: The expression of mRGCs and RGCs was determined in eye sections containing retinal tissue from patients with DR (n = 6) and respective age-matched controls (n = 8) using immunohistochemistry by costaining with antibodies against RNA binding protein with multiple splicing (RBPMS), which identified RGCs and melanopsin which identified the mRGCs. Results: The expression of RGCs in the retina from patients with severe DR was significantly reduced to a density of 146 ± 76 cells/mm2 compared with controls at 1280 ± 249 cells/mm2. The density of mRGCs was also significantly reduced from 3.12 ± 0.54 cells/mm2 in controls to 0.72 ± 0.18 cells/mm2 in patients with DR, with significant loss of 73.5% and 81.9% in mRGC density in the ganglion cell layer (GCL) and inner nuclear layer (INL), respectively. Conclusions: Our findings show that DR affects the expression of mRGCs in the human retina and could explain the abnormal circadian activity observed in patients with DR.

Melanopsin-expressing retinal ganglion cells are resistant to cell injury, but not always.

Melanopsin retinal ganglion cells (mRGCs) are intrinsically photosensitive RGCs deputed to non-image forming functions of the eye such as synchronization of circadian rhythms to light-dark cycle. These cells are characterized by unique electrophysiological, anatomical and biochemical properties and are usually more resistant than conventional RGCs to different insults, such as axotomy and different paradigms of stress. We also demonstrated that these cells are relatively spared compared to conventional RGCs in mitochondrial optic neuropathies (Leber's hereditary optic neuropathy and Dominant Optic Atrophy). However, these cells are affected in other neurodegenerative conditions, such as glaucoma and Alzheimer's disease. We here review the current evidences that may underlie this dichotomy. We also present our unpublished data on cell experiments demonstrating that melanopsin itself does not explain the robustness of these cells and some preliminary data on immunohistochemical assessment of mitochondria in mRGCs.

Melanopsin expressing human retinal ganglion cells: Subtypes, distribution, and intraretinal connectivity.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin belong to a heterogenic population of RGCs which regulate the circadian clock, masking behavior, melatonin suppression, the pupillary light reflex, and sleep/wake cycles. The different functions seem to be associated to different subtypes of melanopsin cells. In rodents, subtype classification has associated subtypes to function. In primate and human retina such classification has so far, not been applied. In the present study using antibodies against N- and C-terminal parts of human melanopsin, confocal microscopy and 3D reconstruction of melanopsin immunoreactive (-ir) RGCs, we applied the criteria used in mouse on human melanopsin-ir RGCs. We identified M1, displaced M1, M2, and M4 cells. We found two other subtypes of melanopsin-ir RGCs, which were named "gigantic M1 (GM1)" and "gigantic displaced M1 (GDM1)." Few M3 cells and no M5 subtypes were labeled. Total cell counts from one male and one female retina revealed that the human retina contains 7283 ± 237 melanopsin-ir (0.63-0.75% of the total number of RGCs). The melanopsin subtypes were unevenly distributed. Most significant was the highest density of M4 cells in the nasal retina. We identified input to the melanopsin-ir RGCs from AII amacrine cells and directly from rod bipolar cells via ribbon synapses in the innermost ON layer of the inner plexiform layer (IPL) and from dopaminergic amacrine cells and GABAergic processes in the outermost OFF layer of the IPL. The study characterizes a heterogenic population of human melanopsin-ir RGCs, which most likely are involved in different functions.

Assessment of Rod, Cone, and Intrinsically Photosensitive Retinal Ganglion Cell Contributions to the Canine Chromatic Pupillary Response.

Purpose: The purpose of this study was to evaluate a chromatic pupillometry protocol for specific functional assessment of rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) in dogs. Methods: Chromatic pupillometry was tested and compared in 37 dogs in different stages of primary loss of rod, cone, and combined rod/cone and optic nerve function, and in 5 wild-type (WT) dogs. Eyes were stimulated with 1-s flashes of dim (1 cd/m2) and bright (400 cd/m2) blue light (for scotopic conditions) or bright red (400 cd/m2) light with 25-cd/m2 blue background (for photopic conditions). Canine retinal melanopsin/Opn4 was cloned, and its expression was evaluated using real-time quantitative reverse transcription-PCR and immunohistochemistry. Results: Mean ± SD percentage of pupil constriction amplitudes induced by scotopic dim blue (scDB), scotopic bright blue (scBB), and photopic bright red (phBR) lights in WT dogs were 21.3% ± 10.6%, 50.0% ± 17.5%, and 19.4% ± 7.4%, respectively. Melanopsin-mediated responses to scBB persisted for several minutes (7.7 ± 4.6 min) after stimulus offset. In dogs with inherited retinal degeneration, loss of rod function resulted in absent scDB responses, followed by decreased phBR responses with disease progression and loss of cone function. Primary loss of cone function abolished phBR responses but preserved those responses to blue light (scDB and scBB). Although melanopsin/Opn4 expression was diminished with retinal degeneration, melanopsin-expressing ipRGCs were identified for the first time in both WT and degenerated canine retinas. Conclusions: Pupil responses elicited by light stimuli of different colors and intensities allowed differential functional assessment of canine rods, cones, and ipRGCs. Chromatic pupillometry offers an effective tool for diagnosing retinal and optic nerve diseases.

Loss of Melanopsin-Expressing Retinal Ganglion Cells in Severely Staged Glaucoma Patients.

PURPOSE: Multiple studies have shown overwhelming evidence supporting the impairment of melanopsin function due to glaucoma. However, few studies have been carried out in humans analyzing the histology of melanopsin-expressing retinal ganglion cells (mRGCs) in retinas with glaucoma. The aim of this study was to analyze the pattern of expression of mRGCs relative to RGCs in the normal retina and retinas harboring varying stages of glaucoma. METHODS: Paraffin-embedded human donor eyes with glaucoma (n = 11) and age-matched controls (n = 10) were obtained from Department of Pathology at Rigshospital (Copenhagen, Denmark) for detection of RNA binding protein with multiple splicing (RBPMS) and melanopsin by immunohistochemistry. The density of RBPMS-expressing RGCs and mRGCs in each retina was estimated as the total cell count in the total retinal area analyzed (cell counts/mm2). RESULTS: No significant difference was observed in mRGC expression in the normal retinas and mild-staged retinas with glaucoma; the densities of mRGCs were 3.08 ± 0.47 and 3.00 ± 0.13 cell counts/mm2, respectively. However, the severely staged retinas with glaucoma showed a significant loss in mRGCs density, 1.09 ± 0.35 cell counts/mm2, with 75% of all retained mRGCs occurring in the inner nuclear layer. CONCLUSIONS: This is the first report illustrating histologic evidence for reduced mRGC density in the ganglion cell layer of retinas with severely staged glaucoma compared with age-matched controls. This result proposes evaluation of mRGCs integrity as a basis for assessing the pathophysiologic disease progression of glaucoma.

Exploring visual plasticity: dietary carotenoids can change color vision in guppies (Poecilia reticulata).

Differences in color vision can play a key role in an organism's ability to perceive and interact with the environment across a broad range of taxa. Recently, species have been shown to vary in color vision across populations as a result of differences in regulatory sequence and/or plasticity of opsin gene expression. For decades, biologists have been intrigued by among-population variation in color-based mate preferences of female Trinidadian guppies. We proposed that some of this variation results from variation in color vision caused by plasticity in opsin expression. Specifically, we asked about the role of dietary carotenoid availability, because carotenoids (1) are the precursors for vitamin A, which is essential for the creation of photopigments and (2) have been linked to variation in female mate choice. We raised guppies on different carotenoid-level diets and measured opsin expression. Guppies raised on high-carotenoid diets expressed higher levels of long wavelength sensitive opsin (LWS) opsins than those raised on lower levels of carotenoids. These results suggest that dietary effects on opsin expression represent a previously unaccounted for mechanism by which ecological differences across populations could lead to mate choice differences.

Melanopsin-expressing ganglion cells on macaque and human retinas form two morphologically distinct populations.

The long-term goal of this research is to understand how retinal ganglion cells that express the photopigment melanopsin, also known as OPN4, contribute to vision in humans and other primates. Here we report the results of anatomical studies using our polyclonal antibody specifically against human melanopsin that confirm and extend previous descriptions of melanopsin cells in primates. In macaque and human retina, two distinct populations of melanopsin cells were identified based on dendritic stratification in either the inner or the outer portion of the inner plexiform layer (IPL). Variation in dendritic field size and cell density with eccentricity was confirmed, and dendritic spines, a new feature of melanopsin cells, were described. The spines were the sites of input from DB6 diffuse bipolar cell axon terminals to the inner stratifying type of melanopsin cells. The outer stratifying melanopsin type received inputs from DB6 bipolar cells via a sparse outer axonal arbor. Outer stratifying melanopsin cells also received inputs from axon terminals of dopaminergic amacrine cells. On the outer stratifying melanopsin cells, ribbon synapses from bipolar cells and conventional synapses from amacrine cells were identified in electron microscopic immunolabeling experiments. Both inner and outer stratifying melanopsin cell types were retrogradely labeled following tracer injection in the lateral geniculate nucleus (LGN). In addition, a method for targeting melanopsin cells for intracellular injection using their intrinsic fluorescence was developed. This technique was used to demonstrate that melanopsin cells were tracer coupled to amacrine cells and would be applicable to electrophysiological experiments in the future. J. Comp. Neurol. 524:2845-2872, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Retinofugal Projections from Melanopsin-Expressing Retinal Ganglion Cells Revealed by Intraocular Injections of Cre-Dependent Virus.

To understand visual functions mediated by intrinsically photosensitive melanopsin-expressing retinal ganglion cells (mRGCs), it is important to elucidate axonal projections from these cells into the brain. Initial studies reported that melanopsin is expressed only in retinal ganglion cells within the eye. However, recent studies in Opn4-Cre mice revealed Cre-mediated marker expression in multiple brain areas. These discoveries complicate the use of melanopsin-driven genetic labeling techniques to identify retinofugal projections specifically from mRGCs. To restrict labeling to mRGCs, we developed a recombinant adeno-associated virus (AAV) carrying a Cre-dependent reporter (human placental alkaline phosphatase) that was injected into the vitreous of Opn4-Cre mouse eyes. The labeling observed in the brain of these mice was necessarily restricted specifically to retinofugal projections from mRGCs in the injected eye. We found that mRGCs innervate multiple nuclei in the basal forebrain, hypothalamus, amygdala, thalamus and midbrain. Midline structures tended to be bilaterally innervated, whereas the lateral structures received mostly contralateral innervation. As validation of our approach, we found projection patterns largely corresponded with previously published results; however, we have also identified a few novel targets. Our discovery of projections to the central amygdala suggests a possible direct neural pathway for aversive responses to light in neonates. In addition, projections to the accessory optic system suggest that mRGCs play a direct role in visual tracking, responses that were previously attributed to other classes of retinal ganglion cells. Moreover, projections to the zona incerta raise the possibility that mRGCs could regulate visceral and sensory functions. However, additional studies are needed to investigate the actual photosensitivity of mRGCs that project to the different brain areas. Also, there is a concern of "overlabeling" with very sensitive reporters that uncover low levels of expression. Light-evoked signaling from these cells must be shown to be of sufficient sensitivity to elicit physiologically relevant responses.

Transient Downregulation of Melanopsin Expression After Retrograde Tracing or Optic Nerve Injury in Adult Rats.

PURPOSE: To investigate the effect of retrograde tracing or axotomy on melanopsin mRNA expression and immunodetection in albino and pigmented rat retinas. METHODS: Groups were (1) intact-naïve retinas; (2) optic nerve crush (ONC) analyzed at 7 days (7d) or 2 months (2m); (3) Fluorogold (FG) tracing from the superior colliculi (SCi) analyzed at 7d or 2m; (4) tracing from the intact optic nerve (ON) with FG or hydroxystilbamidine methanesulfonate (OHSt), analyzed 3d later; and (5) sham tracing from the ON or sham surgery. Brn3a and melanopsin were double stained in whole mounts to quantify and assess the distribution of orthotopic and displaced Brn3a(+) retinal ganglion cells (Brn3a(+)RGCs) and melanopsin(+)RGCs (m(+)RGCs). Freshly dissected retinas were used for melanopsin mRNA quantitative PCR. RESULTS: Tracing from the SCi did not affect the number of Brn3a(+)RGCs or m(+)RGCs counted in pigmented rats. However, only 55% of m(+)RGCs were immunodetected in albinos at 7d, although by 2m the m(+)RGCs counts returned to normal. Optic nerve tracing had a more dramatic effect (38% or 77% of m(+)RGCs were immunodetected in albino or pigmented rats) that occurred irrespectively of the tracer (OHSt or FG). This effect was not observed in the sham groups. After ONC, Brn3a(+)RGCs decreased to 37% and 8% by 7d and 2m, respectively. Melanopsin (+)RGC counts diminished to 30% at 7d, but recovered to 49% of controls by 2m. Melanopsin mRNA was downregulated after ON tracing or 7d after ONC, but did not differ from intact values 2m after ONC. CONCLUSIONS: Following ON injury or retrograde tracing there is a transient melanopsin downregulation that should be taken into account when assessing m(+)RGC survival.