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Photorefractive keratectomy (PRK) is a type of eye surgery that involves removal of the corneal epithelium and its associated nerves, which causes intense acute pain in most people. We used a rat model of corneal epithelium removal (corneal abrasion) to examine underlying cellular and molecular mechanisms. In this study, we used immunohistochemistry of trigeminal ganglion (TG) to assess neuronal content of CGRP and ATF3, as well as orbital tightening (OT) to assess spontaneous pain behaviors. CGRP is an important neuropeptide in pain modulation and ATF3 is often used as a nerve injury marker. We found dynamic changes in CGRP and ATF3 in TG; both increased significantly at 24 h following corneal abrasion and females had a more pronounced increase at 24 h compared to males. Interestingly, there was no sex difference in OT behaviors. Additionally, the number of cells containing either CGRP or ATF3 in each animal correlate significantly with their OT behavior at the assessed timepoint. Since CGRP increased most in females, we tested the effectiveness of Olcegepant, a CGRP antagonist, at reducing OT behaviors following corneal abrasion in female rats. Olcegepant (1 mg/kg) was given prior to and again at 24 h after abrasion but did not change OT behaviors at any time over a 1-week period. Examination of CGRP and ATF3 together in TG showed that they rarely colocalized, indicating that the cells with upregulated CGRP are distinct from those responding to epithelial nerve injury. The studies also show that underlying molecular responses may be sex specific., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous metastatic melanoma in which patients develop vision deficits that include reduced night vision, poor contrast sensitivity, and photopsia. MAR is caused by autoantibodies targeting TRPM1, an ion channel found in melanocytes and retinal ON-bipolar cells (ON-BCs). The visual symptoms arise when TRPM1 autoantibodies enter ON-BCs and block the function of TRPM1, thus detection of TRPM1 autoantibodies in patient serum is a key criterion in diagnosing MAR. Electroretinograms are used to measure the impact of TRPM1 autoantibodies on ON-BC function and represent another important diagnostic tool for MAR. To date, MAR case reports have included one or both diagnostic components, but only for a single time point in the course of a patient's disease. Here, we report a case of MAR supported by longitudinal analysis of serum autoantibody detection, visual function, ocular inflammation, vascular integrity, and response to slow-release intraocular corticosteroids. Integrating these data with the patient's oncological and ophthalmological records reveals novel insights regarding MAR pathogenesis, progression, and treatment, which may inform new research and expand our collective understanding of the disease. In brief, we find TRPM1 autoantibodies can disrupt vision even when serum levels are barely detectable by western blot and immunohistochemistry; intraocular dexamethasone treatment alleviates MAR visual symptoms despite high levels of circulating TRPM1 autoantibodies, implicating antibody access to the retina as a key factor in MAR pathogenesis. Elevated inflammatory cytokine levels in the patient's eyes may be responsible for the observed damage to the blood-retinal barrier and subsequent entry of autoantibodies into the retina., Competing Interests: E.G.B. serves on an advisory board for Bristol Myers Squibb for which she receives consulting fees. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mosavi-Hecht, Yang, Heyer, Rosenberg, White, Berry, Duvoisin and Morgans.)

Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous metastatic melanoma in which patients develop vision deficits that include reduced night vision, poor contrast sensitivity, and photopsia. MAR is caused by autoantibodies targeting TRPM1, an ion channel found in melanocytes and retinal ON-bipolar cells (ON-BCs). The visual symptoms arise when TRPM1 autoantibodies enter ON-BCs and block the function of TRPM1, thus detection of TRPM1 autoantibodies in patient serum is a key criterion in diagnosing MAR. Electroretinograms are used to measure the impact of TRPM1 autoantibodies on ON-BC function and represent another important diagnostic tool for MAR. To date, MAR case reports have included one or both diagnostic components, but only for a single time point in the course of a patient's disease. Here, we report a case of MAR supported by longitudinal analysis of serum autoantibody detection, visual function, ocular inflammation, vascular integrity, and response to slow-release intraocular corticosteroids. Integrating these data with the patient's oncological and ophthalmological records reveals novel insights regarding MAR pathogenesis, progression, and treatment, which may inform new research and expand our collective understanding of the disease. In brief, we find TRPM1 autoantibodies can disrupt vision even when serum levels are barely detectable by western blot and immunohistochemistry; intraocular dexamethasone treatment alleviates MAR visual symptoms despite high levels of circulating TRPM1 autoantibodies, implicating antibody access to the retina as a key factor in MAR pathogenesis. Elevated inflammatory cytokine levels in the patient's eyes may be responsible for the observed damage to the blood-retinal barrier and subsequent entry of autoantibodies into the retina., Competing Interests: Conflict of Interest E.G.B. serves on an advisory board for Bristol Myers Squibb for which she receives consulting fees.

Introduction: Rod bipolar cells (RBCs) faithfully transmit light-driven signals from rod photoreceptors in the outer retina to third order neurons in the inner retina. Recently, significant work has focused on the role of leucine-rich repeat (LRR) proteins in synaptic development and signal transduction at RBC synapses. We previously identified trophoblast glycoprotein (TPBG) as a novel transmembrane LRR protein localized to the dendrites and axon terminals of RBCs., Methods: We examined the effects on RBC physiology and retinal processing of TPBG genetic knockout in mice using immunofluorescence and electron microscopy, electroretinogram recording, patch-clamp electrophysiology, and time-resolved membrane capacitance measurements., Results: The scotopic electroretinogram showed a modest increase in the b-wave and a marked attenuation in oscillatory potentials in the TPBG knockout. No effect of TPBG knockout was observed on the RBC dendritic morphology, TRPM1 currents, or RBC excitability. Because scotopic oscillatory potentials primarily reflect RBC-driven rhythmic activity of the inner retina, we investigated the contribution of TPBG to downstream transmission from RBCs to third-order neurons. Using electron microscopy, we found shorter synaptic ribbons in TPBG knockout axon terminals in RBCs. Time-resolved capacitance measurements indicated that TPBG knockout reduces synaptic vesicle exocytosis and subsequent GABAergic reciprocal feedback without altering voltage-gated Ca2 + currents., Discussion: TPBG is required for normal synaptic ribbon development and efficient neurotransmitter release from RBCs to downstream cells. Our results highlight a novel synaptic role for TPBG at RBC ribbon synapses and support further examination into the mechanisms by which TPBG regulates RBC physiology and circuit function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wakeham, Shi, Ren, Haley, Duvoisin, von Gersdorff and Morgans.)

The ability of the visual system to relay meaningful information over a wide range of lighting conditions is critical to functional vision, and relies on mechanisms of adaptation within the retina that adjust sensitivity and gain as ambient light changes. Photoreceptor synapses represent the first stage of image processing in the visual system, thus activity-driven changes at this site are a potentially powerful, yet under-studied means of adaptation. To gain insight into these mechanisms, the abundance and distribution of key synaptic proteins involved in photoreceptor to ON-bipolar cell transmission were compared between light-adapted mice and mice subjected to prolonged dark exposure (72 hours), by immunofluorescence confocal microscopy and immunoblotting. We also tested the effects on protein abundance and distribution of 0.5-4 hours of light exposure following prolonged darkness. Proteins examined included the synaptic ribbon protein, ribeye, and components of the ON-bipolar cell signal transduction pathway (mGluR6, TRPM1, RGS11, GPR179, Goα). The results indicate a reduction in immunoreactivity for ribeye, TRPM1, mGluR6, and RGS11 following prolonged dark exposure compared to the light-adapted state, but a rapid restoration of the light-adapted pattern upon light exposure. Electron microscopy revealed similar ultrastructure of light-adapted and dark-adapted photoreceptor terminals, with the exception of electron dense vesicles in dark-adapted but not light-adapted ON-bipolar cell dendrites. To assess synaptic transmission from photoreceptors to ON-bipolar cells, we recorded electroretinograms after different dark exposure times (2, 16, 24, 48, 72 hours) and measured the b-wave to a-wave ratios. Consistent with the reduction in synaptic proteins, the b/a ratios were smaller following prolonged dark exposure (48-72 hours) compared to 16 hours dark exposure (13-21%, depending on flash intensity). Overall, the results provide evidence of light/dark-dependent plasticity in photoreceptor synapses at the biochemical, morphological, and physiological levels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Haley, Hecht, Ren, Carroll, Aicher, Duvoisin and Morgans.)

Myopia is the most common eye disorder, caused by heterogeneous genetic and environmental factors. Rare progressive and stationary inherited retinal disorders are often associated with high myopia. Genes implicated in myopia encode proteins involved in a variety of biological processes including eye morphogenesis, extracellular matrix organization, visual perception, circadian rhythms, and retinal signaling. Differentially expressed genes (DEGs) identified in animal models mimicking myopia are helpful in suggesting candidate genes implicated in human myopia. Complete congenital stationary night blindness (cCSNB) in humans and animal models represents an ON-bipolar cell signal transmission defect and is also associated with high myopia. Thus, it represents also an interesting model to identify myopia-related genes, as well as disease mechanisms. While the origin of night blindness is molecularly well established, further research is needed to elucidate the mechanisms of myopia development in subjects with cCSNB. Using whole transcriptome analysis on three different mouse models of cCSNB (in Gpr179 -/- , Lrit3 -/- and Grm6 -/- ), we identified novel actors of the retinal signaling cascade, which are also novel candidate genes for myopia. Meta-analysis of our transcriptomic data with published transcriptomic databases and genome-wide association studies from myopia cases led us to propose new biological/cellular processes/mechanisms potentially at the origin of myopia in cCSNB subjects. The results provide a foundation to guide the development of pharmacological myopia therapies., Competing Interests: Declaration of competing interest None., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Electrical coupling, mediated by gap junctions, contributes to signal averaging, synchronization, and noise reduction in neuronal circuits. In addition, gap junctions may also provide alternative neuronal pathways. However, because they are small and especially difficult to image, gap junctions are often ignored in large-scale 3D reconstructions. Here, we reconstruct gap junctions between photoreceptors in the mouse retina using serial blockface-scanning electron microscopy, focused ion beam-scanning electron microscopy, and confocal microscopy for the gap junction protein Cx36. An exuberant spray of fine telodendria extends from each cone pedicle (including blue cones) to contact 40-50 nearby rod spherules at sites of Cx36 labeling, with approximately 50 Cx36 clusters per cone pedicle and 2-3 per rod spherule. We were unable to detect rod/rod or cone/cone coupling. Thus, rod/cone coupling accounts for nearly all gap junctions between photoreceptors. We estimate a mean of 86 Cx36 channels per rod/cone pair, which may provide a maximum conductance of ~1200 pS, if all gap junction channels were open. This is comparable to the maximum conductance previously measured between rod/cone pairs in the presence of a dopamine antagonist to activate Cx36, suggesting that the open probability of gap junction channels can approach 100% under certain conditions., Competing Interests: MI, JK, CM, SA, JC, JS, LJ, WL, IF, CR, SM No competing interests declared

Purpose: To determine the effect of mycophenolate mofetil (MMF) on retinal degeneration on two mouse models of retinitis pigmentosa., Methods: Intraperitoneal injections of MMF were administered daily in rd10 and c57 mice starting at postoperative day 12 (P12) and rd1 mice starting at P8. The effect of MMF was assessed with optical coherence tomography, immunohistochemistry, electroretinography, and OptoMotry. Whole retinal cyclic guanosine monophosphate (cGMP) and mycophenolic acid levels were quantified with mass spectrometry. Photoreceptor cGMP cytotoxicity was evaluated with cell counts of cGMP immunostaining., Results: MMF treatment significantly delays the onset of retinal degeneration and cGMP-dependent photoreceptor cytotoxicity in rd10 and rd1 mice, albeit a more modest effect in the latter. In rd10 mice, treatment with MMF showed robust preservation of the photoreceptors up to P22 with associated suppression of cGMP immunostaining and microglial activation; The neuroprotective effect diminished after P22, but outer retinal thickness was still significantly thicker by P35 and OptoMotry response was significantly better up to P60. Whereas cGMP immunostaining of the photoreceptors were present in rd10 and rd1 mice, hyperphysiological whole retinal cGMP levels were observed only in rd1 mice., Conclusions: Early treatment with MMF confers potent neuroprotection in two animal models of RP by suppressing the cGMP-dependent common pathway for photoreceptor cell death. The neuroprotective effect of MMF on cGMP-dependent cytotoxicity occurs independently of the presence of hyperphysiological whole retinal cGMP levels. Thus our data suggest that MMF may be an important new class of neuroprotective agent that could be useful in the treatment of patients with RP.

We recently identified the leucine-rich repeat (LRR) adhesion protein, trophoblast glycoprotein (TPBG), as a novel PKCα-dependent phosphoprotein in retinal rod bipolar cells (RBCs). Since TPBG has not been thoroughly examined in the retina, this study characterizes the localization and expression patterns of TPBG in the developing and adult mouse retina using two antibodies, one against the N-terminal LRR domain and the other against the C-terminal PDZ-interacting motif. Both antibodies labeled RBC dendrites in the outer plexiform layer and axon terminals in the IPL, as well as a putative amacrine cell with their cell bodies in the inner nuclear layer (INL) and a dense layer in the middle of the inner plexiform layer (IPL). In live transfected HEK293 cells, TPBG was localized to the plasma membrane with the N-terminal LRR domain facing the extracellular space. TPBG immunofluorescence in RBCs was strongly altered by the loss of TRPM1 in the adult retina, with significantly less dendritic and axon terminal labeling in TRPM1 knockout compared to wild type, despite no change in total TPBG detected by immunoblotting. During retinal development, TPBG expression increases dramatically just prior to eye opening with a time course closely correlated with that of TRPM1 expression. In the retina, LRR proteins have been implicated in the development and maintenance of functional bipolar cell synapses, and TPBG may play a similar role in RBCs., (© 2019 Wiley Periodicals, Inc.)

Melanoma-associated retinopathy (MAR) is a rare paraneoplastic retinal disorder usually occurring in the context of metastatic melanoma. Patients present with night blindness, photopsias and a constriction of the visual field. MAR is an auto-immune disorder characterized by the production of autoantibodies targeting retinal proteins, especially autoantibodies reacting to the cation channel TRPM1 produced in melanocytes and ON-bipolar cells. TRPM1 has at least three different isoforms which vary in the N-terminal region of the protein. In this study, we report the case of three new MAR patients presenting different anti-TRPM1 autoantibodies reacting to the three isoforms of TRPM1 with variable binding affinity. Two sera recognized all isoforms of TRPM1, while one recognized only the two longest isoforms upon immunolocalization studies on overexpressing cells. Similarly, the former two sera reacted with all TRPM1 isoforms on western blot, but an immunoprecipitation enrichment step was necessary to detect all isoforms with the latter serum. In contrast, all sera labelled ON-bipolar cells on Tprm1+/+ but not on Trpm1-/- mouse retina as shown by co-immunolocalization. This confirms that the MAR sera specifically detect TRPM1. Most likely, the anti-TRPM1 autoantibodies of different patients vary in affinity and concentration. In addition, the binding of autoantibodies to TRPM1 may be conformation-dependent, with epitopes being inaccessible in some constructs (truncated polypeptides versus full-length TRPM1) or applications (western blotting versus immunohistochemistry). Therefore, we propose that a combination of different methods should be used to test for the presence of anti-TRPM1 autoantibodies in the sera of MAR patients., Competing Interests: The authors have declared that no competing interests exist.

In the central nervous system, melastatin transient receptor potential (TRPM) channels function as receptors for the neurosteroid pregnenolone sulfate (PregS). The expression and function of TRPM3 has been explored in adult retina, although its role during development is unknown. We found, during the second postnatal week in mice, TRPM3 immunofluorescence labeled distinct subsets of inner retinal neurons, including a subset of retinal ganglion cells (RGCs), similar to what has been reported in the adult. Labeling for a TRPM3 promoter-driven reporter confirmed expression of the TRPM3 gene in RGCs and revealed additional expression in nearly all Müller glial cells. Using two-photon calcium imaging, we show that PregS and the synthetic TRPM3 agonist CIM0216 (CIM) induced prolonged calcium transients in RGCs, which were mostly absent in TRPM3 knock-out (KO) mice. These prolonged calcium transients were not associated with strong membrane depolarizations but induced c-Fos expression. To elucidate the impact of PregS-activation of TRPM3 on retinal circuits we took two sets of physiological measurements. First, PregS induced a robust increase in the frequency but not amplitude of spontaneous postsynaptic currents (PSCs). This increase was absent in the TRPM3 KO mice. Second, PregS induced a small increase in cell participation and duration of retinal waves, but this modulation persisted in TRPM3 KO mice, indicating PregS was acting on wave generating circuits independent of TRPM3 channels. Though baseline frequency of retinal waves was slightly reduced in the TRPM3 KO mice, other properties of waves were indistinguishable from wildtype. Together, these results indicate that the presence of neurosteroids impact spontaneous synaptic activity and retinal waves during development via both TRPM3-dependent and independent mechanisms., (Copyright © 2020 Webster et al.)

Adjusting to a wide range of light intensities is an essential feature of retinal rod bipolar cell (RBC) function. While persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKCα), the targets of PKCα phosphorylation in the retina have not been identified. PKCα activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKCα antibody and antibodies to phosphorylated PKC motifs. PKCα activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKCα-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKCα knockout (PKCα-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKCα-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein). Two strongly differentially expressed phosphoproteins, BORG4 and TPBG, were localized to the synaptic layers of the retina, and may play a role in PKCα-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906. SIGNIFICANCE: Retinal rod bipolar cells (RBCs), the second-order neurons of the mammalian rod visual pathway, are able to modulate their sensitivity to remain functional across a wide range of light intensities, from starlight to daylight. Evidence suggests that this modulation requires the serine/threonine kinase, PKCα, though the specific mechanism by which PKCα modulates RBC physiology is unknown. This study examined PKCα phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKCα-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKCα-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKCα-dependent modulation of RBC physiology., (Copyright © 2019. Published by Elsevier B.V.)

Purpose: Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous malignant melanoma (CMM). Visual symptoms include night blindness, photopsia, and reduced-contrast sensitivity. An abnormal ERG b-wave and the presence of anti-bipolar cell autoantibodies, including autoantibodies reacting with the ON-bipolar cell TRPM1 channel, help to confirm the diagnosis. The goal of this study was to determine if CMM patients without visual symptoms also express anti-TRPM1 autoantibodies., Methods: Serum samples from 15 CMM patients were tested using three assays: immunofluorescent labeling of TRPM1-transfected HEK cells, immunofluorescent labeling of retinal sections from wild-type and TRPM1 knockout mice, and immunoblot detection of a bacterially produced recombinant TRPM1 peptide., Results: Serum specimens from 5 of the 15 CMM patients without declared visual symptoms were positive for anti-TRPM1 autoantibodies in at least one of the three assays. One of 50 control sera from patients not known to have cancer was also weakly reactive with the TRPM1 peptide., Conclusions: Autoantibodies against TRPM1 are present in CMM patient sera without self-reported visual symptoms. Most patients had advanced (stage III and IV) disease and were undergoing aggressive treatments, including immunotherapy. It is unknown if immunotherapy affects the expression of TRPM1 autoantibodies. The presence of TRPM1 autoantibodies may predispose patients for MAR.

In the rod pathway of the mammalian retina, axon terminals of glutamatergic rod bipolar cells are presynaptic to AII and A17 amacrine cells in the inner plexiform layer. Recent evidence suggests that both amacrines express NMDA receptors, raising questions concerning molecular composition, localization, activation, and function of these receptors. Using dual patch-clamp recording from synaptically connected rod bipolar and AII or A17 amacrine cells in retinal slices from female rats, we found no evidence that NMDA receptors contribute to postsynaptic currents evoked in either amacrine. Instead, NMDA receptors on both amacrine cells were activated by ambient glutamate, and blocking glutamate uptake increased their level of activation. NMDA receptor activation also increased the frequency of GABAergic postsynaptic currents in rod bipolar cells, suggesting that NMDA receptors can drive release of GABA from A17 amacrines. A striking dichotomy was revealed by pharmacological and immunolabeling experiments, which found GluN2B-containing NMDA receptors on AII amacrines and GluN2A-containing NMDA receptors on A17 amacrines. Immunolabeling also revealed a clustered organization of NMDA receptors on both amacrines and a close spatial association between GluN2B subunits and connexin 36 on AII amacrines, suggesting that NMDA receptor modulation of gap junction coupling between these cells involves the GluN2B subunit. Using multiphoton Ca 2+ imaging, we verified that activation of NMDA receptors evoked an increase of intracellular Ca 2+ in dendrites of both amacrines. Our results suggest that AII and A17 amacrines express clustered, extrasynaptic NMDA receptors, with different and complementary subunits that are likely to contribute differentially to signal processing and plasticity. SIGNIFICANCE STATEMENT Glutamate is the most important excitatory neurotransmitter in the CNS, but not all glutamate receptors transmit fast excitatory signals at synapses. NMDA-type glutamate receptors act as voltage- and ligand-gated ion channels, with functional properties determined by their specific subunit composition. These receptors can be found at both synaptic and extrasynaptic sites on neurons, but the role of extrasynaptic NMDA receptors is unclear. Here, we demonstrate that retinal AII and A17 amacrine cells, postsynaptic partners at rod bipolar dyad synapses, express extrasynaptic (but not synaptic) NMDA receptors, with different and complementary GluN2 subunits. The localization of GluN2A-containing receptors to A17s and GluN2B-containing receptors to AIIs suggests a mechanism for differential modulation of excitability and signaling in this retinal microcircuit., (Copyright © 2019 the authors 0270-6474/19/390627-24$15.00/0.)

Purpose: Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with malignant melanoma and the presence of anti-retinal autoantibodies, including autoantibodies against transient receptor potential melanopsin 1 (TRPM1), a cation channel expressed by both melanocytes and retinal bipolar cells. The goal of this study was to further map the antigenic epitope., Methods: Patient sera were tested by immunofluorescence and Western blotting on HEK293 cells transfected with enhanced green fluorescent protein (EGFP)-TRPM1 fusion constructs and mouse retina sections., Results: The epitope recognized by MAR patient sera was mapped to a region encoded by exons 9 and 10 of the human TRPM1 gene. This region of TRPM1 is highly conserved with TRPM3, and indeed MAR sera were found to cross-react with TRPM3, a closely related channel expressed in the retinal pigment epithelium (RPE)., Conclusions: These results indicate that TRPM1 autoantibodies in MAR patient sera recognize a short, intracellular segment of TRPM1. Cross-reactivity with TRPM3 in the RPE may account for other visual symptoms that are experienced by some MAR patients such as retinal and RPE detachments. We propose that TRPM1 autoantibodies are generated in response to abnormal TRPM1 polypeptides encoded by an alternate mRNA splice variant expressed by malignant melanocytes.

Purpose: To develop diagnostic criteria for nonparaneoplastic autoimmune retinopathy (AIR) through expert panel consensus and to examine treatment patterns among clinical experts., Design: Modified Delphi process., Methods: A survey of uveitis specialists in the American Uveitis Society, a face-to-face meeting (AIR Workshop) held at the National Eye Institute, and 2 iterations of expert panel surveys were used in a modified Delphi process. The expert panel consisted of 17 experts, including uveitis specialists and researchers with expertise in antiretinal antibody detection. Supermajority consensus was used and defined as 75% of experts in agreement., Results: There was unanimous agreement among experts regarding the categorization of autoimmune retinopathies as nonparaneoplastic and paraneoplastic, including cancer-associated retinopathy and melanoma-associated retinopathy. Diagnostic criteria and tests essential to the diagnosis of nonparaneoplastic AIR and multiple supportive criteria reached consensus. For treatment, experts agreed that corticosteroids and conventional immunosuppressives should be used (prescribed) as first- or second-line treatments, though a consensus agreed that biologics and intravenous immunoglobulin were considered appropriate in the treatment of nonparaneoplastic AIR patients regardless of the stage of disease. Experts agreed that more evidence is needed to treat nonparaneoplastic AIR patients with long-term immunomodulatory therapy and that there is enough equipoise to justify randomized, placebo-controlled trials to determine if nonparaneoplastic AIR patients should be treated with long-term immunomodulatory therapy. Regarding antiretinal antibody detection, consensus agreed that a standardized assay system is needed to detect serum antiretinal antibodies. Consensus agreed that an ideal assay should have a 2-tier design and that Western blot and immunohistochemistry should be the methods used to identify antiretinal antibodies., Conclusions: Consensus was achieved using a modified Delphi process to develop diagnostic criteria for nonparaneoplastic AIR. There is enough equipoise to justify randomized, placebo-controlled trials to determine whether patients with nonparaneoplastic AIR should be treated with long-term immunomodulatory therapy. Efforts to develop a standardized 2-tier assay system for the detection of antiretinal antibodies have been initiated as a result of this study., (Published by Elsevier Inc.)

Fragile X Mental Retardation Protein (FMRP) is a RNA-binding protein that modulates protein synthesis at the synapse and its function is regulated by glutamate. The retina is the first structure that participates in vision, and uses glutamate to transduce electromagnetic signals from light to electrochemical signals to neurons. FMRP has been previously detected in the retina, but its localization has not been studied yet. In this work, our objectives were to describe the localization of FMRP in the retina, to determine whether different exposure to dark or light stimulus alters FMRP expression in the retina, and to compare the pattern in two different species, the mouse and chick. We found that both FMRP mRNA and protein are expressed in the retina. By immunohistochemistry analysis we found that both mouse and chick present similar FMRP expression localized mainly in both plexiform layers and the inner retina. It was also observed that FMRP is down-regulated by 24 h dark adaptation compared to its expression in the retina of animals that were exposed to light for 1 h after 24 h in the dark. We conclude that FMRP is likely to participate in retinal physiology, since its expression changes with light exposure. In addition, the expression pattern and regulation by light of FMRP seems well conserved since it was similar in both mouse and chick., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Mutations in LRIT3 lead to complete congenital stationary night blindness (cCSNB). The exact role of LRIT3 in ON-bipolar cell signaling cascade remains to be elucidated. Recently, we have characterized a novel mouse model lacking Lrit3 [no b-wave 6, (Lrit3(nob6/nob6) )], which displays similar abnormalities to patients with cCSNB with LRIT3 mutations. Here we compare the localization of components of the ON-bipolar cell signaling cascade in wild-type and Lrit3(nob6/nob6) retinal sections by immunofluorescence confocal microscopy. An anti-LRIT3 antibody was generated. Immunofluorescent staining of LRIT3 in wild-type mice revealed a specific punctate labeling in the outer plexiform layer (OPL), which was absent in Lrit3(nob6/nob6) mice. LRIT3 did not co-localize with ribeye or calbindin but co-localized with mGluR6. TRPM1 staining was severely decreased at the dendritic tips of all depolarizing bipolar cells in Lrit3(nob6/nob6) mice. mGluR6, GPR179, RGS7, RGS11 and Gβ5 immunofluorescence was absent at the dendritic tips of cone ON-bipolar cells in Lrit3(nob6/nob6) mice, while it was present at the dendritic tips of rod bipolar cells. Furthermore, peanut agglutinin (PNA) labeling was severely reduced in the OPL in Lrit3(nob6/nob6) mice. This study confirmed the localization of LRIT3 at the dendritic tips of depolarizing bipolar cells in mouse retina and demonstrated the dependence of TRPM1 localization on the presence of LRIT3. As tested components of the ON-bipolar cell signaling cascade and PNA revealed disrupted localization, an additional function of LRIT3 in cone synapse formation is suggested. These results point to a possibly different regulation of the mGluR6 signaling cascade between rod and cone ON-bipolar cells., (© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Purpose: Protein kinase C α (PKCα) is abundantly expressed in rod bipolar cells (RBCs) in the retina, yet the physiological function of PKCα in these cells is not well understood. To elucidate the role of PKCα in visual processing in the eye, we examined the effect of genetic deletion of PKCα on the ERG and on RBC light responses in the mouse., Methods: Immunofluorescent labeling was performed on wild-type (WT), TRPM1 knockout, and PKCα knockout (PKC-KO) retina. Scotopic and photopic ERGs were recorded from WT and PKC-KO mice. Light responses of RBCs were measured using whole-cell recordings in retinal slices from WT and PKC-KO mice., Results: Protein kinase C alpha expression in RBCs is correlated with the activity state of the cell. Rod bipolar cells dendrites are a major site of PKCα phosphorylation. Electroretinogram recordings indicated that loss of PKCα affects the scotopic b-wave, including a larger peak amplitude, longer implicit time, and broader width of the b-wave. There were no differences in the ERG a- or c-wave between PKCα KO and WT mice, indicating no measurable effect of PKCα in photoreceptors or the RPE. The photopic ERG was unaffected consistent with the lack of detectable PKCα in cone bipolar cells. Whole-cell recordings from RBCs in PKC-KO retinal slices revealed that, compared with WT, RBC light responses in the PKC-KO retina are delayed and of longer duration., Conclusions: Protein kinase C alpha plays an important modulatory role in RBCs, regulating both the peak amplitude and temporal properties of the RBC light response in the rod visual pathway.

Transient receptor potential (TRP) channels constitute a large family of cation permeable ion channels that serve crucial functions in sensory systems by transducing environmental changes into cellular voltage and calcium signals. Within the retina, two closely related members of the melastatin TRP family, TRPM1 and TRPM3, are highly expressed. TRPM1 has been shown to be required for the depolarizing response to light of ON-bipolar cells, but the role of TRPM3 in the retina is unknown. Immunohistochemical staining of mouse retina with an antibody directed against the C-terminus of TRPM3 labeled the inner plexiform layer (IPL) and a subset of cells in the ganglion cell layer. Within the IPL, TRPM3 immunofluorescence was markedly stronger in the OFF sublamina than in the ON sublamina. Electroretinogram recordings showed that the scotopic and photopic a- and b-waves of TRPM3(-/-) mice are normal indicating that TRPM3 does not play a major role in visual processing in the outer retina. TRPM3 activity was measured by calcium imaging and patch-clamp recording of immunopurified retinal ganglion cells. Application of the TRPM3 agonist, pregnenolone sulfate (PS), stimulated increases in intracellular calcium in ~40% of cells from wild type and TRPM1(‑/‑) mice, and the PS-stimulated increases in calcium were blocked by co-application of mefenamic acid, a TRPM3 antagonist. No PS-stimulated changes in fluorescence were observed in ganglion cells from TRPM3(-/-) mice. Similarly, PS-stimulated currents that could be blocked by mefenamic acid were recorded from wild type retinal ganglion cells but were absent in ganglion cells from TRPM3-/- mice.

Purpose: Administration of voriconazole, an antifungal triazole, causes transient visual disturbances in patients and attenuates the b-wave of the ERG. We sought to identify the retinal target of voriconazole underlying the effect on the ERG b-wave., Methods: Electroretinograms were recorded from mice before and after intraperitoneal injection of voriconazole. The effect of voriconazole on ON-bipolar cells was tested by patch-clamp recordings of ON-bipolar cells in mouse retinal slices. Effects of voriconazole on mGluR6 and TRPM3 were assessed by patch-clamp recordings of Chinese hamster ovary (CHO) and HEK293 cells transfected with either TRPM3 or mGluR6 plus Kir3.1/Kir3.4., Results: Voriconazole attenuated the ERG b-wave in mice, and inhibited ON-bipolar cell responses evoked by application of CPPG, an mGluR6 antagonist, onto the ON-bipolar cell dendrites, indicating that voriconazole blocks a step in the mGluR6-TRPM1 signal transduction pathway. Voriconazole almost completely blocked capsaicin-activated currents in ON-bipolar cells, which have been attributed to direct activation of the TRPM1 cation channel. Furthermore, application of voriconazole to CHO cells expressing TRPM3, a closely related channel to TRPM1, showed that voriconazole reversibly blocked pregnenolone sulfate-stimulated TRPM3 currents in transfected cells. In contrast, voriconazole only slightly inhibited mGluR6-mediated activation of G-protein activated inward rectifier potassium (GIRK) currents in cotransfected cells, suggesting that mGluR6 is not the primary target of voriconazole in ON-bipolar cells., Conclusions: The visual disturbances associated with voriconazole are likely due to block of TRPM1 channels in retinal ON-bipolar cells. Other neurological effects of voriconazole may be due to block of TRPM3 channels expressed in the brain., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Simultaneous dual labeling to visualize specific RNA and protein content within the same formalin-fixed paraffin embedded (FFPE) section can be technically challenging and usually impossible, because of variables such as tissue fixation time and pretreatment methods to access the target RNA or protein. Within a specific experiment, ocular tissue sections can be a precious commodity. Thus, the ability to easily and consistently detect and localize cell-specific expression of RNA and protein within a single slide would be advantageous. In this study, we describe a simplified and reliable method for combined in situ hybridization (ISH) and immunohistochemistry (IHC) for detection of mRNA and protein, respectively, within the same FFPE ocular tissue., Methods: Whole mouse eyes were prepared for 5 micron FFPE sections after fixation for 3, 24, 48 or 72 h. Customized probes from Advanced Cell Diagnostics to detect mRNA for vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1-alpha (HIF-1α), and hypoxia-inducible factor 2-alpha (HIF-2α) were used for ISH. Various parameters were tested using the novel RNAscope method for ISH and optimized for compatibility with subsequent IHC for glial fibrillary acidic protein (GFAP) or GS-lectin within the same tissue section. Dual fluorescent visualization of Fast Red ISH and Alexa Fluor 488 IHC signal was observed with confocal microscopy., Results: A fixation time of 72 h was found to be optimal for ISH and subsequent IHC. The RNAscope probes for VEGF, HIF-1α, and HIF-2α mRNA all gave a strong Fast Red signal with both 48 h and 72 h fixed tissue, but the optimal IHC signal for either GFAP or GS-lectin within a retinal tissue section after ISH processing was observed with 72 h fixation. A pretreatment boiling time of 15 min and a dilution factor of 1:15 for the pretreatment protease solution were found to be optimal and necessary for successful ISH visualization with 72 h FFPE ocular tissue., Conclusions: The protocol presented here provides a simple and reliable method to simultaneously detect mRNA and protein within the same paraffin-embedded ocular tissue section. The procedure, after preparation of FFPE sections, can be performed over a 2-day or 4-day period. We provide an optimization strategy that may be adapted for any RNAscope probe set and antibody for determining retinal or ocular cell-specific patterns of expression.

D-Serine, an N-methyl D-aspartate receptor coagonist, and its regulatory enzymes, D-amino acid oxidase (DAO; degradation) and serine racemase (SR; synthesis), have been implicated in crucial roles of the developing central nervous system, yet the functional position that they play in regulating the availability of d-serine throughout development of the mammalian retina is not well-known. Using capillary electrophoresis and a sensitive method of enantiomeric amino acid separation, we were able to determine total levels of d-serine at specific ages during postnatal development of the mouse retina in two different strains of mice, one of which contained a loss-of-function point mutation for DAO while the other was a SR knockout line. Each mouse line was tested against conspecific wild type (WT) mice for each genetic strain. The universal trend in all WT and transgenic mice was a large amount of total retinal d-serine at postnatal age 2 (P2), followed by a dramatic decrease as the mice matured into adulthood (P70-80). SR knockout mice retinas had 41% less D-serine than WT retinas at P2, and 10 times less as an adult. DAO mutant mice retinas had significantly elevated levels of d-serine when compared to WT retinas at P2 (217%), P4 (223%), P8 (194%), and adulthood (227%).

Purpose: To report the first case of melanoma-associated retinopathy (MAR) and underlying occult melanoma diagnosed based on the presence of serum transient receptor potential melastatin 1 (TRPM1) autoantibodies., Design: Interventional case report with basic science correlation., Participants: One patient with MAR., Intervention: Testing for the presence of serum TRPM1 autoantibodies., Main Outcome Measures: Diagnosis of an occult melanoma involving the axillary lymph nodes (unknown primary site) and MAR based on the presence of TRPM1 autoantibodies in the patient's serum., Results: The patient's clinical exam was remarkable for mild intraocular inflammation in both eyes and retinal hemorrhages with an apparent choroidal neovascularization in the left eye, which was confirmed by fluorescein angiography and indocyanine green angiography testing. Humphrey visual field 30-2 SITA-fast (Humphrey Visual Field Analyzer, Carl Zeiss Meditec, Inc, Dublin, CA) demonstrated diffuse depression in both eyes out of proportion to the clinical exams, prompting electroretinography testing that revealed an electronegative response. Dark-adapted thresholds were markedly elevated and mediated by cones. Due to concern for MAR, a systemic work-up for melanoma was performed by the primary care physician that was unrevealing. Given our continued clinical suspicion for MAR, the patient's serum was sent for evaluation for TRPM1 autoantibodies. The patient's serum applied to normal human retina exhibited positivity in the inner nuclear layer. Application of the patient's serum to wild-type and TRPM1 knockout mouse retina revealed strongly labeled bipolar cells in the wild-type retina, but not in the TRPM1 knockout retina, indicating TRPM1-dependent immunoreactivity. The antigen was confirmed as TRPM1 by labeling of TRPM1-transfected human embryonic kidney 293 cells. Additional systemic work-up prompted by this finding resulted in identification of an occult metastatic melanoma involving the axillary lymph nodes with an unknown primary site. The patient underwent surgical excision of the occult melanoma without evidence of other sites of metastases. He also received intravenous immunoglobulin therapy and his vision has stabilized., Conclusions: This is the first reported case of a melanoma-associated retinopathy diagnosed utilizing the innovative approach of testing for serum TRPM1 autoantibodies., (Copyright © 2013 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

D-serine is present in the vertebrate retina and serves as a coagonist for the N-methyl-D-aspartate (NMDA) receptors of ganglion cells. Although the enzyme D-amino acid oxidase (DAO) has been implicated as a pathway for d-serine degradation, its role in the retina has not been established. In this study, we investigated the role of DAO in regulating D-serine levels using a mutant mouse line deficient in DAO (ddY/DAO(-)) and compared these results with their wild-type counterparts (ddY/DAO(+)). Our results show that DAO is functionally present in the mouse retina and normally serves to reduce the background levels of D-serine. The enzymatic activity of DAO was restricted to the inner plexiform layer as determined by histochemical analysis. Using capillary electrophoresis, we showed that mutant mice had much higher levels of D-serine. Whole cell recordings from identified retinal ganglion cells demonstrated that DAO-deficient animals had light-evoked synaptic activity strongly biased toward a high NMDA-to-AMPA receptor ratio. In contrast, recordings from wild-type ganglion cells showed a more balanced ratio between the two receptor subclasses. Immunostaining for AMPA and NMDA receptors was carried out to compare the two receptor ratios by quantitative immunofluorescence. These studies revealed that the mutant mouse had a significantly higher representation of NMDA receptors compared with the wild-type controls. We conclude that 1) DAO is an important regulatory enzyme and normally functions to reduce D-serine levels in the retina, and 2) D-serine levels play a role in the expression of NMDA receptors and the NMDA-to-AMPA receptor ratio.

Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous malignant melanoma and the presence of autoantibodies that label neurons in the inner retina. The visual symptoms and electroretinogram (ERG) phenotype characteristic of MAR resemble the congenital visual disease caused by mutations in TRPM1, a cation channel expressed by both melanocytes and retinal bipolar cells. Four serum samples from MAR patients were identified as TRPM1 immunoreactive by 1. Labeling of ON-bipolar cells in TRPM1+/+ but not TRPM1-/- mouse retina, 2. Labeling of TRPM1-transfected CHO cells; and 3. Attenuation of the ERG b-wave following intravitreal injection of TRPM1-positive MAR IgG into wild-type mouse eyes, and the appearance of the IgG in the retinal bipolar cells at the conclusion of the experiment. Furthermore, the epitope targeted by the MAR autoantibodies was localized within the amino-terminal cytoplasmic domain of TRPM1. Incubation of live retinal neurons with TRPM1-positive MAR serum resulted in the selective accumulation of IgG in ON-bipolar cells from TRPM1+/+ mice, but not TRPM1-/- mice, suggesting that the visual deficits in MAR are caused by the uptake of TRPM1 autoantibodies into ON-bipolar cells, where they bind to an intracellular epitope of the channel and reduce the ON-bipolar cell response to light.

The P23H-1 transgenic rat carries a mutated mouse opsin gene, in addition to endogenous opsin genes, and undergoes progressive photoreceptor loss that is generally characteristic of human autosomal dominant retinitis pigmentosa (RP). Here, we examined morphological changes correlated with visual function that is comparable to clinical application in the pigmented P23H-1 rat retina as photoreceptor degeneration progressed. We found that rod function was compromised as early as postnatal day 28 and was a good indicator for tracking retinal degeneration. Cone function was normal and did not change until the thickness of the photoreceptor layer was reduced by 75%. Similar to the threshold versus intensity curves used to evaluate vision of RP patients, light-adaptation curves showed that cone thresholds depended on the number of remaining functioning cones, but not on its length of outer segments (OS). By 1 year of age, both rod and cone functions were significantly compromised. Correlating with early abnormal rod function, rods and related secondary neurons also underwent progressive degeneration, including shortening of inner and OS of photoreceptors, loss of rod bipolar and horizontal cell dendrites, thickening of the outer Müller cell processes, and reduced density of pre- and postsynaptic markers. Similar early morphological modifications were also observed in cones and their related secondary neurons. However, cone function was maintained at nearly normal level for a long period. The dramatic loss of rods at late stage of degeneration may contribute to the dysfunction of cones. Attention has to be focused on preserving cone function and identifying factors that damage cones when therapeutic regimes are applied to treat retinal degeneration. As such, these findings provide a foundation for future studies involving treatments to counter photoreceptor loss.

To investigate the role of mGluR8 in modulating the synaptic responses of retinal ganglion cells, we used a recently identified positive allosteric modulator of mGluR8, AZ12216052 (AZ) and the mGluR8-specific orthosteric agonist (S)-3,4-dicarboxyphenylglycine (DCPG). These agents were applied to whole-cell voltage-clamped ganglion cells from an isolated, superfused mouse retina preparation. DCPG reduced OFF-ganglion cell excitatory currents, whereas AZ enhanced the peak excitatory currents in ON-, OFF-, and ON-OFF-ganglion cells. The effects on ganglion cell inhibitory currents were more varied. The effects of the allosteric modulator were stronger for bright stimuli than for dim stimuli, consistent with receptor stimulation by endogenous glutamate being stronger during bright light stimulation and with mGluR8 receptors mainly being localized away from glutamate release sites, immuno-labeled with VGLUT1. The differential sensitivity of ganglion cell light responses to DCPG and AZ supports multiple sites where mGluR8 modulates the light responses of ganglion cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose: The advances in stem cell biology hold a great potential to treat retinal degeneration. Importantly, specific cell types can be generated efficiently with small molecules and maintained stably over numerous passages. Here, we investigated whether neural stem cell (NSC) derived from human embryonic stem cells (hESC) by small molecules can preserve vision following grafting into the Royal College Surgeon (RCS) rats; a model for retinal degeneration., Methods: A cell suspension containing 3 × 10 4 NSCs or NSCs labeled with green fluorescent protein (GFP) was injected into the subretinal space or the vitreous cavity of RCS rats at postnatal day (P) 22; animals injected with cell-carry medium and those left untreated were used as controls. The efficacy of treatment was evaluated by testing optokinetic response, recording luminance threshold, and examining retinal histology., Results: NSCs offered significant preservation of both photoreceptors and visual function. The grafted NSCs survived for long term without evidence of tumor formation. Functionally, NSC treated eyes had significantly better visual acuity and lower luminance threshold than controls. Morphologically, photoreceptors and retinal connections were well preserved. There was an increase in expression of cillary neurotrophic factor (CNTF) in Müller cells in the graft-protected retina., Conclusions: This study reveals that NSCs derived from hESC by small molecules can survive and preserve vision for long term following subretinal transplantation in the RCS rats. These cells migrate extensively in the subretinal space and inner retina; there is no evidence of tumor formation or unwanted changes after grafting into the eyes., Translational Relevance: The NSCs derived from hESC by small molecules can be generated efficiently and provide an unlimited supply of cells for the treatment of some forms of human outer retinal degenerative diseases. The capacity of NSCs migrating into inner retina offers a potential as a vehicle to delivery drugs/factors to treat inner retinal disorders.

Horizontal cells mediate inhibitory feedforward and feedback lateral interactions in the outer retina at photoreceptor terminals and bipolar cell dendrites; however, the mechanisms that underlie synaptic transmission from mammalian horizontal cells are poorly understood. The localization of a vesicular γ-aminobutyric acid (GABA) transporter (VGAT) to horizontal cell processes in primate and rodent retinae suggested that mammalian horizontal cells release transmitter in a vesicular manner. Toward determining whether the molecular machinery for vesicular transmitter release is present in horizontal cells, we investigated the expression of SNAP25 (synaptosomal-associated protein of 25 kDa), a key SNARE protein, by immunocytochemistry with cell type-specific markers in the retinae of mouse, rat, rabbit, and monkey. Different commercial antibodies to SNAP25 were tested on vertical sections of retina. We report the robust expression of SNAP25 in both plexiform layers. Double labeling with SNAP25 and calbindin antibodies demonstrated that horizontal cell processes and their endings in photoreceptor triad synapses were strongly labeled for both proteins in mouse, rat, rabbit, and monkey retinae. Double labeling with parvalbumin antibodies in monkey retina verified SNAP25 immunoreactivity in all horizontal cells. Pre-embedding immunoelectron microscopy in rabbit retina confirmed expression of SNAP25 in lateral elements within photoreceptor triad synapses. The SNAP25 immunoreactivity in the plexiform layers and outer nuclear layer fell into at least three patterns depending on the antibody, suggesting a differential distribution of SNAP25 isoforms. The presence of SNAP25a and SNAP25b isoforms in mouse retina was established by reverse transcriptase-polymerase chain reaction. SNAP25 expression in mammalian horizontal cells along with other SNARE proteins is consistent with vesicular exocytosis., (Copyright © 2010 Wiley-Liss, Inc.)

For almost 30 years the ion channel that initiates the ON visual pathway in vertebrate vision has remained elusive. Recent findings now indicate that the pathway, which begins with unbinding of glutamate from the metabotropic glutamate receptor 6 (mGluR6), ends with the opening of the transient receptor potential (TRP)M1 cation channel. As a component of the mGluR6 signal transduction pathway, mutations in TRPM1 would be expected to cause congenital stationary night blindness (CSNB), and several such mutations have already been identified in CSNB families. Furthermore, expression of TRPM1 in both the retina and skin raises the possibility that a genetic link exists between certain types of visual and skin disorders.

The rate-limiting step in the recovery of the photoreceptor light response is the hydrolysis of GTP by transducin, a reaction that is accelerated by the RGS9-Gbeta5 complex, and its membrane anchor, R9AP. Similar complexes, including RGS7, RGS11, and Gbeta5, are found in retinal ON-bipolar cell dendrites. Here, we present evidence that R9AP is also expressed in the dendritic tips of ON-bipolar cells. Immunofluorescent staining for R9AP revealed a punctate pattern of labeling in the outer plexiform layer, where it colocalized with mGluR6. In photoreceptors, R9AP is required for proteolytic stability of the entire regulator of G protein signaling complex, and we found that genetic deletion of R9AP also results in a marked reduction in the levels of RGS11 and Gbeta5 in the bipolar cell dendrites; the level of RGS7 was unaffected, suggesting the presence of another interaction partner to stabilize RGS7. To determine the effect of R9AP deletion on the response kinetics of ON-bipolar cells, we compared the electroretinogram (ERG) between wild-type and R9AP-deficient mice. The ERG b-wave, reflecting ON-bipolar cell activity, was delayed and larger in the R9AP-deficient mice. Our data indicate that R9AP is required for stable expression of RGS11-Gbeta5 in ON-bipolar cell dendrites. Furthermore, they suggest that the RGS11-Gbeta5-R9AP complex accelerates the initial ON-bipolar cell response to light.

We tested whether calcium-induced calcium release (CICR) contributes to synaptic release from rods in mammalian retina. Electron micrographs and immunofluorescent double labeling for the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2) and synaptic ribbon protein, ribeye, showed a close association between ER and synaptic ribbons in mouse rod terminals. Stimulating CICR with 10 microM ryanodine evoked Ca(2+) increases in rod terminals from mouse retinal slices visualized using confocal microscopy with the Ca(2+)-sensitive dye, Fluo-4. Ryanodine also stimulated membrane depolarization of individual mouse rods. Inhibiting CICR with a high concentration of ryanodine (100 microM) reduced the electroretinogram (ERG) b-wave but not a-wave consistent with inhibition of synaptic transmission from rods. Ryanodine (100 microM) also inhibited light-evoked voltage responses of individual rod bipolar cells (RBCs) and presumptive horizontal cells recorded with perforated patch recording techniques. A presynaptic site of action for ryanodine's effects is further indicated by the finding that ryanodine (100 microM) did not alter currents evoked in voltage-clamped RBCs by puffing the mGluR6 antagonist, (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), onto bipolar cell dendrites in the presence of the mGluR6 agonist L-(+)-2-amino-4-phosphonobutyric acid (L-AP4). Ryanodine (100 microM) also inhibited glutamatergic outward currents in RBCs evoked by electrical stimulation of rods using electrodes placed in the outer segment layer. Together, these results indicate that, like amphibian retina, CICR contributes to synaptic release from mammalian (mouse) rods. By boosting synaptic release in darkness, CICR may improve the detection of small luminance changes by post-synaptic neurons.

Purpose: The retinal ON-bipolar cell (ON-BPC) light response is initiated upon deactivation of the metabotropic glutamate receptor mGluR6 and the G protein Go. G protein-based signaling cascades are typically accelerated by interaction of the G protein alpha subunit with a member of the regulator of G protein signaling (RGS) protein family. The goal of this study was to determine whether RGS7 and/or -11 serve this function in retinal ON-BPCs., Methods: Retinas from mice lacking RGS11 (RGS11(-/-)), or with a deletion mutation in RGS7 (RGS7(Delta/Delta)), or both, were compared to wild-type (WT) by immunofluorescence confocal microscopy. The retinal light response was measured with the electroretinogram (ERG). The kinetics of simulated light responses from individual rod bipolar cells were recorded by whole-cell patch-clamp electrophysiology., Results: Levels of the R7 RGS interaction partners, Gbeta5 and R9AP, were reduced in the outer plexiform layer of the RGS11(-/-) and RGS7(Delta/Delta)/RGS11(-/-) mice. ERG recordings demonstrated a delay in the rising phase of the ERG b-wave, larger photopic b-wave amplitudes, and increased scotopic threshold response sensitivity in the RGS11(-/-) and RGS7(Delta/Delta)/RGS11(-/-) mice. The ERG measured from the RGS7(Delta/Delta) retina was normal. Patch-clamp recordings of chemically simulated light responses of rod BPCs revealed a 25-ms delay in the onset of the ON-BPC response in the RGS7(Delta/Delta)/RGS11(-/-) mouse compared with the WT., Conclusions: RGS11 plays a role in the deactivation of Galphao, which precedes activation of the depolarizing current in ON-BPCs. RGS7 must also serve a role as changes in RGS7(Delta/Delta)/RGS11(-/-) mice were greater than those in RGS11(-/-) mice.

The ON pathway of the visual system, which detects increases in light intensity, is established at the first retinal synapse between photoreceptors and ON-bipolar cells. Photoreceptors hyperpolarize in response to light and reduce the rate of glutamate release, which in turn causes the depolarization of ON-bipolar cells. This ON-bipolar cell response is mediated by the metabotropic glutamate receptor, mGluR6, which controls the activity of a depolarizing current. Despite intensive research over the past two decades, the molecular identity of the channel that generates this depolarizing current has remained elusive. Here, we present evidence indicating that TRPM1 is necessary for the depolarizing light response of ON-bipolar cells, and further that TRPM1 is a component of the channel that generates this light response. Gene expression profiling revealed that TRPM1 is highly enriched in ON-bipolar cells. In situ hybridization experiments confirmed that TRPM1 mRNA is found in cells of the retinal inner nuclear layer, and immunofluorescent confocal microscopy showed that TRPM1 is localized in the dendrites of ON-bipolar cells in both mouse and macaque retina. The electroretinogram (ERG) of TRPM1-deficient (TRPM1(-/-)) mice had a normal a-wave, but no b-wave, indicating a loss of bipolar cell response. Finally, whole-cell patch-clamp recording from ON-bipolar cells in mouse retinal slices demonstrated that genetic deletion of TRPM1 abolished chemically simulated light responses from rod bipolar cells and dramatically altered the responses of cone ON-bipolar cells. Identification of TRPM1 as a mGluR6-coupled cation channel reveals a key step in vision, expands the role of the TRP channel family in sensory perception, and presents insights into the evolution of vertebrate vision.

The Synaptic Vesicle Protein 2 (SV2) family of transporter-like proteins is expressed exclusively in vesicles that undergo calcium-regulated exocytosis. Of the three isoforms expressed in mammals, SV2B is the most divergent. Here we report studies of SV2B location and function in the retina. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. In mice lacking SV2B, synaptic transmission at the synapse between photoreceptors and bipolar neurons was decreased, as evidenced by a significant reduction in the amplitude of the b-wave in electroretinogram recordings. Quantitative immunoblot analyses of whole eyes revealed that loss of SV2B was associated with reduced levels of synaptic vesicle proteins including synaptotagmin, VAMP, synaptophysin and the vesicular glutamate transporter V-GLUT1. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. Thus, SV2B contributes to the modulation of synaptic vesicle exocytosis and plays a significant role in regulating synaptic protein content.

The time course of G-protein-coupled responses is largely determined by the kinetics of GTP hydrolysis by the G protein alpha subunit, which is accelerated by interaction with regulator of G-protein signaling (RGS) proteins. Light responses of ON-bipolar cells of the vertebrate retina require rapid inactivation of the G protein Galphao, which is activated in the dark by metabotropic glutamate receptor, mGluR6, in their dendritic tips. It is not yet known, however, which RGS protein(s) might be responsible for rapid inactivation kinetics. By immunofluorescence and co-immunoprecipitation, we have identified complexes of the Galphao-selective RGS proteins RGS7 and RGS11, with their obligate binding partner, Gbeta5, that are localized to the dendritic tips of murine rod and cone ON-bipolar cells, along with mGluR6. Experiments using pre- and post-synaptic markers, and a dissociated bipolar cell preparation, clearly identified the location of these complexes as the ON-bipolar cell dendritic tips and not the adjacent photoreceptor terminals or horizontal cell dendrites. In mice lacking mGluR6, the distribution of RGS11, RGS7 and Gbeta5 shifts away from the dendritic tips, implying a functional relationship with mGluR6. The precise co-localization of Gbeta5-RGS7 and Gbeta5-RGS11 with mGluR6, and the dependence of localization on the presence of mGluR6, suggests that Gbeta5-RGS7 and Gbeta5-RGS11 function specifically in the mGluR6 signal transduction pathway, where they may stimulate the GTPase activity of Galphao, thus accelerating the ON-bipolar cell light response, in a manner analogous to the acceleration of photoreceptor light responses by the Gbeta5-RGS9-1 complex.

In the brain and the retina metabotropic glutamate receptors (mGluRs) modulate synaptic transmission; in particular, L-2-amino-4-phosphonobutyrate-sensitive group-III mGluRs are generally presynaptic and provide negative feedback of neurotransmitter release. We performed a comparative immunohistochemical analysis of the distribution of all group-III mGluRs in the mouse retina. mGluR6 expression was limited to the outer plexiform layer. Discrete, punctate immunolabeling, exclusively in the inner plexiform layer (IPL), was observed for each of the remaining group-III mGluRs. mGluR4 immunostaining was most abundant in IPL sublamina 1; mGluR7 immunoreactivity was organized in four bands, corresponding to sublaminae 1-4; and mGluR8 was localized in two broad bands, one each in the OFF and ON layers of the IPL. mGluR8 immunoreactivity was evident in the OFF plexus of cholinergic amacrine cell processes. Surprisingly, we found little overlap between group-III mGluR immunolabeling and that for the vesicular glutamate transporter VGLUT1. Instead, we found that mGluR4 and mGluR7 were located close to bipolar cell ribbons. No compensatory changes in the distribution of group-III mGluRs, or of several other markers also showing a stratified localization in the IPL, were observed in genetically engineered mice lacking either mGluR4, mGluR8, or both mGluR4 and mGluR8. The unique pattern of expression of each receptor suggests that they have distinct functions in the retina, and their asymmetric distribution in the ON and OFF layers of the IPL suggests distinct roles in the processing of light-ON and light-OFF stimuli., ((c) 2007 Wiley-Liss, Inc.)

The nob2 mouse carries a null mutation in the Cacna1f gene, which encodes the pore-forming subunit of the L-type calcium channel, Ca(v)1.4. The loss of the electroretinogram b-wave in these mice suggests a severe reduction in transmission between photoreceptors and second-order neurons in the retina and supports a central role for the Ca(v)1.4 calcium channel at photoreceptor ribbon synapses, to which it has been localized. Here we show that the loss of Ca(v)1.4 leads to the aberrant outgrowth of rod bipolar cell dendrites and horizontal cell processes into the outer nuclear layer (ONL) of the nob2 retina and to the formation of ectopic synaptic contacts with rod photoreceptors in the ONL. Ectopic contacts are predominantly between rods and rod bipolar cells, with horizontal cell processes also present at some sites. Ectopic contacts contain apposed pre- and postsynaptic specializations, albeit with malformed synaptic ribbons. Cone photoreceptor terminals do not participate in ectopic contacts in the ONL. During retinal development, ectopic contacts appear in the days after eye opening, appearing progressively farther into the ONL at later postnatal stages. Ectopic contacts develop at the tips of rod bipolar cell dendrites and are less frequently associated with the tips of horizontal cell processes, consistent with the adult phenotype. The relative occurrence of pre- and postsynaptic markers in the ONL during development suggests a mechanism for the formation of ectopic synaptic contacts that is driven by the retraction of rod photoreceptor terminals and neurite outgrowth by rod bipolar cell dendrites., ((c) 2006 Wiley-Liss, Inc.)

We performed genome-wide chemical mutagenesis of C57BL/6J mice using N-ethyl-N-nitrosourea (ENU). Electroretinographic screening of the third generation offspring revealed two G3 individuals from one G1 family with a normal a-wave but lacking the b-wave that we named nob4. The mutation was transmitted with a recessive mode of inheritance and mapped to chromosome 11 in a region containing the Grm6 gene, which encodes a metabotropic glutamate receptor protein, mGluR6. Sequencing confirmed a single nucleotide substitution from T to C in the Grm6 gene. The mutation is predicted to result in substitution of Pro for Ser at position 185 within the extracellular, ligand-binding domain and oocytes expressing the homologous mutation in mGluR6 did not display robust glutamate-induced currents. Retinal mRNA levels for Grm6 were not significantly reduced, but no immunoreactivity for mGluR6 protein was found. Histological and fundus evaluations of nob4 showed normal retinal morphology. In contrast, the mutation has severe consequences for visual function. In nob4 mice, fewer retinal ganglion cells (RGCs) responded to the onset (ON) of a bright full field stimulus. When ON responses could be evoked, their onset was significantly delayed. Visual acuity and contrast sensitivity, measured with optomotor responses, were reduced under both photopic and scotopic conditions. This mutant will be useful because its phenotype is similar to that of human patients with congenital stationary night blindness and will provide a tool for understanding retinal circuitry and the role of ganglion cell encoding of visual information.

Complete X-linked congenital stationary night blindness (CSNB1) is a hereditary visual disease characterized by abnormalities in both the dark- and light-adapted electroretinogram, consistent with a defect in synaptic transmission between photoreceptors and ON-bipolar cells. The gene responsible for CSNB1, NYX, encodes a novel, leucine-rich repeat protein, nyctalopin. Consistent with its predicted glycosylphosphatidylinositol linkage, we show that recombinant nyctalopin is targeted to the extracellular cell surface in transfected HEK293 cells. Within the retina, strong nyctalopin immunoreactivity is present in the outer plexiform layer, the site of the photoreceptor to bipolar cell synapses. Double labelling of nyctalopin and known synaptic proteins in the outer plexiform layer indicate that nyctalopin is associated with the ribbon synapses of both rod and cone terminals. In the inner plexiform layer, nyctalopin immunoreactivity is associated with rod bipolar cell terminals. Our findings support a role for nyctalopin in synaptic transmission and/or synapse formation at ribbon synapses in the retina.

Glutamate release from photoreceptor terminals is controlled by voltage-dependent calcium channels (VDCCs). In humans, mutations in the Cacna1f gene, encoding the alpha1F subunit of VDCCs, underlie the incomplete form of X-linked congenital stationary night blindness (CSNB2). These mutations impair synaptic transmission from rod and cone photoreceptors to bipolar cells. Here, we report anatomical and functional characterizations of the retina in the nob2 (no b-wave 2) mouse, a naturally occurring mutant caused by a null mutation in Cacna1f. Not surprisingly, the b-waves of both the light- and dark-adapted electroretinogram are abnormal in nob2 mice. The outer plexiform layer (OPL) is disorganized, with extension of ectopic neurites through the outer nuclear layer that originate from rod bipolar and horizontal cells, but not from hyperpolarizing bipolar cells. These ectopic neurites continue to express mGluR6, which is frequently associated with profiles that label with the presynaptic marker Ribeye, indicating potential points of ectopic synapse formation. However, the morphology of the presynaptic Ribeye-positive profiles is abnormal. While cone pedicles are present their morphology also appears compromised. Characterizations of visual responses in retinal ganglion cells in vivo, under photopic conditions, demonstrate that ON-center cells have a reduced dynamic range, although their basic center-surround organization is retained; no alteration in the responses of OFF-center cells was evident. These results indicate that nob2 mice are a valuable model in which to explore the pathophysiological mechanisms associated with Cacna1f mutations causing CSNB2, and the subsequent effects on visual information processing. Further, the nob2 mouse represents a model system in which to define the signals that guide synapse formation and/or maintenance in the OPL.

The genetic locus for incomplete congenital stationary night blindness (CSNB2) has been identified as the CACNA1f gene, encoding the alpha 1F calcium channel subunit, a member of the L-type family of calcium channels. The electroretinogram associated with CSNB2 implicates alpha 1F in synaptic transmission between retinal photoreceptors and bipolar cells. Using a recently developed monoclonal antibody to alpha 1F, we localize the channel to ribbon active zones in rod photoreceptor terminals of the mouse retina, supporting a role for alpha 1F in mediating glutamate release from rods. Detergent extraction experiments indicate that alpha 1F is part of a detergent-resistant active zone complex, which also includes the synaptic ribbons. Comparison of native mouse rod calcium currents with recombinant alpha 1F currents reveals that the current-voltage relationship for the native current is shifted approximately 30 mV to more hyperpolarized potentials than for the recombinant alpha 1F current, suggesting modulation of the native channel by intracellular factors. Lastly, we present evidence for L-type alpha 1D calcium channel subunits in cone terminals of the mouse retina. The presence of alpha 1D channels in cones may explain the residual visual abilities of individuals with CSNB2.

Particular types of amacrine cells of the vertebrate retina show oscillatory membrane potentials (OMPs) in response to light stimulation. Historically it has been thought the oscillations arose as a result of circuit properties. In a previous study we found that in some amacrine cells, the ability to oscillate was an intrinsic property of the cell. Here we characterized the ionic mechanisms responsible for the oscillations in wide-field amacrine cells (WFACs) in an effort to better understand the functional properties of the cell. The OMPs were found to be calcium (Ca2+) dependent; blocking voltage-gated Ca2+ channels eliminated the oscillations, whereas elevating extracellular Ca2+ enhanced them. Strong intracellular Ca2+ buffering (10 mM EGTA or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid) eliminated any attenuation in the OMPs as well as a Ca2+-dependent inactivation of the voltage-gated Ca2+ channels. Pharmacological and immunohistochemical characterization revealed that WFACs express L- and N-type voltage-sensitive Ca2+ channels. Block of the L-type channels eliminated the OMPs, but omega-conotoxin GVIA did not, suggesting a different function for the N-type channels. The L-type channels in WFACs are functionally coupled to a set of calcium-dependent potassium (K(Ca)) channels to mediate OMPs. The initiation of OMPs depended on penitrem-A-sensitive (BK) K(Ca) channels, whereas their duration is under apamin-sensitive (SK) K(Ca) channel control. The Ca2+ current is essential to evoke the OMPs and triggering the K(Ca) currents, which here act as resonant currents, enhances the resonance as an amplifying current, influences the filtering characteristics of the cell membrane, and attenuates the OMPs via CDI of the L-type Ca2+ channel.

Synaptic vesicle exocytosis is triggered by rises in calcium up to 100 microM at the site of vesicle fusion. The synaptic vesicle proteins synaptotagmin 1 and 2 (Syt I and Syt II) bind calcium at similarly high concentrations and have been proposed as the calcium sensors for fast neurotransmitter release. However, 1 microM calcium produces tonic transmitter release at photoreceptor and bipolar cell synapses in the goldfish retina, suggesting that these synapses use a higher affinity calcium sensor. Immunofluorescent staining with a panel of Syt I/II antibodies detected Syt I/II in both photoreceptor and bipolar cell terminals of the rodent retina. By contrast, no staining of either photoreceptor or protein kinase C (PKC)-labeled bipolar cell terminals was detected in the goldfish retina with any of the Syt I/II antibodies. The high affinity calcium sensor synaptotagmin 3 (Syt III) was localized to the synaptic layers of both goldfish and rodent retinas; however, while Syt III was associated with PKC-labeled bipolar cell terminals in the goldfish retina, it did not co-localize with PKC in the mouse retina. These results suggest that, unlike in their mammalian counterparts, synaptic vesicle exocytosis in goldfish photoreceptor and bipolar cell terminals utilizes a calcium sensor other than Syt I/II, possibly Syt III.

Bipolar cells convey information through the retina via graded changes in their membrane potential and modulate transmitter release through the influx of calcium via L-type calcium channels. However, the molecular identity of the alpha(1) subunit has not been confirmed. We report the presence of the newly cloned alpha(1F) subunit in mouse bipolar cell synaptic terminals. The alpha(1F) subunits are localized to hot spots, possibly corresponding to active zones. We also report the physiological properties of two calcium currents present in mouse bipolar cells, a low-voltage-activated L-type current and a low-voltage-activated T-type calcium current. The physiological properties of the T-type current suggest that it is completely inactivated under physiological conditions. The L-type current may be mediated by the alpha(1F) subunit, and influx of calcium through the alpha(1F) channel may control neurotransmitter release from the bipolar cell terminal., (Copyright 2002 Wiley-Liss, Inc.)

Purpose: Mutations in the alpha(1F) subunit of voltage-dependent calcium channels (VDCCs) have been shown to cause incomplete congenital stationary night blindness (CSNB2). The purpose of this study was to dentify which of the four beta subunits of VDCCs participates in the formation of this channel at the photoreceptor synapse and to determine how its absence affects visual processing., Methods: Mice without each of the four known beta subunits of VDCCs were generated by gene targeting and transgenic rescue (CNS-beta(1), -beta(2)) or by gene targeting alone (beta(3)) or were obtained from a commercial provider (beta(4)). Retinal function and visual sensitivity were examined by electroretinography and an active avoidance behavioral test, respectively. The structure of the retina and expression of the alpha(1F) subunit were examined at the light microscopic level and by immunohistochemistry., Results: Under dark-adapted conditions, CNS-beta(2)-null mice had a normal ERG a-wave, but did not have a normal b-wave. In addition, these mice showed decreased sensitivity to light. Both the a- and b-waves appear normal in the CNS-beta(1)-, beta(3)-, and beta(4)-null mice. Histologic analyses of all four mouse lines indicated that only the CNS-beta(2)-null mice had altered retinal morphology. Eyes of these mice had a thinner outer plexiform layer (OPL) than eyes of control animals. In addition, the labeling pattern of the alpha(1F) subunit in the OPL was altered in CNS-beta(2)-null mice., Conclusions: The normal distribution of the alpha(1F) subunit of the VDCCs in the OPL is dependent on the expression of the beta(2) subunit. The expression of both of these subunits is required for normal maintenance and/or formation of the OPL and synaptic transmission.