Nystagmus in patients with congenital stationary night blindness (CSNB) originates from synchronously firing retinal ganglion cells.

Congenital nystagmus, involuntary oscillating small eye movements, is commonly thought to originate from aberrant interactions between brainstem nuclei and foveal cortical pathways. Here, we investigated whether nystagmus associated with congenital stationary night blindness (CSNB) results from primary deficits in the retina. We found that CSNB patients as well as an animal model (nob mice), both of which lacked functional nyctalopin protein (NYX, nyx) in ON bipolar cells (BCs) at their synapse with photoreceptors, showed oscillating eye movements at a frequency of 4-7 Hz. nob ON direction-selective ganglion cells (DSGCs), which detect global motion and project to the accessory optic system (AOS), oscillated with the same frequency as their eyes. In the dark, individual ganglion cells (GCs) oscillated asynchronously, but their oscillations became synchronized by light stimulation. Likewise, both patient and nob mice oscillating eye movements were only present in the light when contrast was present. Retinal pharmacological and genetic manipulations that blocked nob GC oscillations also eliminated their oscillating eye movements, and retinal pharmacological manipulations that reduced the oscillation frequency of nob GCs also reduced the oscillation frequency of their eye movements. We conclude that, in nob mice, synchronized oscillations of retinal GCs, most likely the ON-DCGCs, cause nystagmus with properties similar to those associated with CSNB in humans. These results show that the nob mouse is the first animal model for a form of congenital nystagmus, paving the way for development of therapeutic strategies.

Intermolecular Interaction between Anchoring Subunits Specify Subcellular Targeting and Function of RGS Proteins in Retina ON-Bipolar Neurons.

In vertebrate retina, light responses generated by the rod photoreceptors are transmitted to the second-order neurons, the ON-bipolar cells (ON-BC), and this communication is indispensible for vision in dim light. In ON-BCs, synaptic transmission is initiated by the metabotropic glutamate receptor, mGluR6, that signals via the G-protein Go to control opening of the effector ion channel, TRPM1. A key role in this process belongs to the GTPase Activating Protein (GAP) complex that catalyzes Go inactivation upon light-induced suppression of glutamate release in rod photoreceptors, thereby driving ON-BC depolarization to changes in synaptic input. The GAP complex has a striking molecular complexity. It contains two Regulator of G-protein Signaling (RGS) proteins RGS7 and RGS11 that directly act on Go and two adaptor subunits: RGS Anchor Protein (R9AP) and the orphan receptor, GPR179. Here we examined the organizational principles of the GAP complex in ON-BCs. Biochemical experiments revealed that RGS7 binds to a conserved site in GPR179 and that RGS11 in vivo forms a complex only with R9AP. R9AP and GPR179 are further integrated via direct protein-protein interactions involving their cytoplasmic domains. Elimination of GPR179 prevents postsynaptic accumulation of R9AP. Furthermore, concurrent knock-out of both R9AP and RGS7 does not reconfigure the GAP complex and completely abolishes synaptic transmission, resulting in a novel mouse model of night blindness. Based on these results, we propose a model of hierarchical assembly and function of the GAP complex that supports ON-BCs visual signaling.

Mouse retinal ganglion cell signalling is dynamically modulated through parallel anterograde activation of cannabinoid and vanilloid pathways.

KEY POINTS: Retinal cells use vanilloid transient receptor potential (TRP) channels to integrate light-evoked signals with ambient mechanical, chemical and temperature information. Localization and function of the polymodal non-selective cation channel TRPV1 (transient receptor potential vanilloid isoform 1) remains elusive. TRPV1 is expressed in a subset of mouse retinal ganglion cells (RGCs) with peak expression in the mid-peripheral retina. Endocannabinoids directly activate TRPV1 and inhibit it through cannabinoid type 1 receptors (CB1Rs) and cAMP pathways. Activity-dependent endocannabinoid release may modulate signal gain in RGCs through simultaneous manipulation of calcium and cAMP signals mediated by TRPV1 and CB1R. ABSTRACT: How retinal ganglion cells (RGCs) process and integrate synaptic, mechanical, swelling stimuli with light inputs is an area of intense debate. The nociceptive cation channel TRPV1 (transient receptor potential vanilloid type 1) modulates RGC Ca2+ signals and excitability yet the proportion of RGCs that express it remains unclear. Furthermore, TRPV1's response to endocannabinoids (eCBs), the putative endogenous retinal activators, is unknown, as is the potential modulation by cannabinoid receptors (CBRs). The density of TRPV1-expressing RGCs in the Ai9:Trpv1 reporter mouse peaked in the mid-peripheral retina. TRPV1 agonists including capsaicin (CAP) and the eCBs anandamide and N-arachidonoyl-dopamine elevated [Ca2+ ]i in 30-40% of wild-type RGCs, with effects suppressed by TRPV1 antagonists capsazepine (CPZ) and BCTC ((4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide), and lacking in Trpv1-/- cells. The cannabinoid receptor type 1 (CB1R) colocalized with TRPV1:tdTomato expression. Its agonists 2-arachidonoylglycerol (2-AG) and WIN55,122 inhibited CAP-induced [Ca2+ ]i signals in adult, but not early postnatal, RGCs. The suppressive effect of 2-AG on TRPV1 activation was emulated by positive modulators of the protein kinase A (PKA) pathway, inhibited by the CB1R antagonist rimonabant and Gi uncoupler pertussis toxin, and absent in Cnr1-/- RGCs. We conclude that TRPV1 is a modulator of Ca2+ homeostasis in a subset of RGCs that show non-uniform distribution across the mouse retina. Non-retrograde eCB-mediated modulation of RGC signalling involves a dynamic push-pull between direct TRPV1 activation and PKA-dependent regulation of channel inactivation, with potential functions in setting the bandwidth of postsynaptic responses, sensitivity to mechanical/excitotoxic stress and neuroprotection.

A missense mutation in Grm6 reduces but does not eliminate mGluR6 expression or rod depolarizing bipolar cell function.

GRM6 encodes the metabotropic glutamate receptor 6 (mGluR6) used by retinal depolarizing bipolar cells (DBCs). Mutations in GRM6 lead to DBC dysfunction and underlie the human condition autosomal recessive complete congenital stationary night blindness. Mouse mutants for Grm6 are important models for this condition. Here we report a new Grm6 mutant, identified in an electroretinogram (ERG) screen of mice maintained at The Jackson Laboratory. The Grm6nob8 mouse has a reduced-amplitude b-wave component of the ERG, which reflects light-evoked DBC activity. Sequencing identified a missense mutation that converts a highly conserved methionine within the ligand binding domain to leucine (p.Met66Leu). Consistent with prior studies of Grm6 mutant mice, the laminar size and structure in the Grm6nob8 retina were comparable to control. The Grm6nob8 phenotype is distinguished from other Grm6 mutants that carry a null allele by a reduced but not absent ERG b-wave, decreased but present expression of mGluR6 at DBC dendritic tips, and mislocalization of mGluR6 to DBC somas. Consistent with a reduced but not absent b-wave, there were a subset of retinal ganglion cells whose responses to light onset have times to peak within the range of those in control retinas. These data indicate that the p.Met66Leu mutant mGluR6 is trafficked less than control. However, the mGluR6 that is localized to the DBC dendritic tips is able to initiate DBC signal transduction. The Grm6nob8 mouse extends the Grm6 allelic series and will be useful for elucidating the role of mGluR6 in DBC signal transduction and in human disease.NEW & NOTEWORTHY This article describes a mouse model of the human disease complete congenital stationary night blindness in which the mutation reduces but does not eliminate GRM6 expression and bipolar cell function, a distinct phenotype from that seen in other Grm6 mouse models.

Intravitreal delivery of a novel AAV vector targets ON bipolar cells and restores visual function in a mouse model of complete congenital stationary night blindness.

Adeno-associated virus (AAV) effectively targets therapeutic genes to photoreceptors, pigment epithelia, Müller glia and ganglion cells of the retina. To date, no one has shown the ability to correct, with gene replacement, an inherent defect in bipolar cells (BCs), the excitatory interneurons of the retina. Targeting BCs with gene replacement has been difficult primarily due to the relative inaccessibility of BCs to standard AAV vectors. This approach would be useful for restoration of vision in patients with complete congenital stationary night blindness (CSNB1), where signaling through the ON BCs is eliminated due to mutations in their G-protein-coupled cascade genes. For example, the majority of CSNB1 patients carry a mutation in nyctalopin (NYX), which encodes a protein essential for proper localization of the TRPM1 cation channel required for ON BC light-evoked depolarization. As a group, CSNB1 patients have a normal electroretinogram (ERG) a-wave, indicative of photoreceptor function, but lack a b-wave due to defects in ON BC signaling. Despite retinal dysfunction, the retinas of CSNB1 patients do not degenerate. The Nyx(nob) mouse model of CSNB1 faithfully mimics this phenotype. Here, we show that intravitreally injected, rationally designed AAV2(quadY-F+T-V) containing a novel 'Ple155' promoter drives either GFP or YFP_Nyx in postnatal Nyx(nob) mice. In treated Nyx(nob) retina, robust and targeted Nyx transgene expression in ON BCs partially restored the ERG b-wave and, at the cellular level, signaling in ON BCs. Our results support the potential for gene delivery to BCs and gene replacement therapy in human CSNB1.

GPR179 is required for high sensitivity of the mGluR6 signaling cascade in depolarizing bipolar cells.

Parallel visual pathways are initiated at the first retinal synapse by signaling between the rod and cone photoreceptors and two general classes of bipolar cells. For normal function, ON or depolarizing bipolar cells (DBCs) require the G-protein-coupled receptor, mGluR6, an intact G-protein-coupled cascade and the transient receptor potential melastatin 1 (TRPM1) cation channel. In addition, another seven transmembrane protein, GPR179, is required for DBC function and recruits the regulators of G-protein signaling (RGS) proteins, RGS7 and RGS11, to the dendritic tips of the DBCs. Here we use the Gpr179(nob5) mouse, which lacks GPR179 and has a no b-wave electroretinogram (ERG) phenotype, to demonstrate that despite the absence of both GPR179 and RGS7/RGS11, a small dark-adapted ERG b-wave remains and can be enhanced with long duration flashes. Consistent with the ERG, the mGluR6-mediated gating of TRPM1 can be evoked pharmacologically in Gpr179(nob5) and RGS7(-/-)/RGS11(-/-) rod BCs if strong stimulation conditions are used. In contrast, direct gating of TRPM1 by capsaicin in RGS7(-/-)/RGS11(-/-) and WT rod BCs is similar, but severely compromised in Gpr179(nob5) rod BCs. Noise and standing current analyses indicate that the remaining channels in Gpr179(nob5) and RGS7(-/-)/RGS11(-/-) rod BCs have a very low open probability. We propose that GPR179 along with RGS7 and RGS11 controls the ability of the mGluR6 cascade to gate TRPM1. In addition to its role in localizing RGS7 and RGS11 to the dendritic tips, GPR179 via a direct interaction with the TRPM1 channel alters its ability to be gated directly by capsaicin.

Functional changes in Tg P23H-1 rat retinal responses: differences between ON and OFF pathway transmission to the superior colliculus.

The morphological consequences of retinal photoreceptor degeneration are well documented. Much less is known about changes in visual function during degeneration and whether central visual structures directly reflect changes in retinal ganglion cell (RGC) function. To address this, we compared changes in visual function of RGCs and cells in the superior colliculus (SC) in transgenic (Tg) P23H-1 rats, a model of retinitis pigmentosa (RP), and wild-type (WT) rats at postnatal days 35-50 (P35-50) and P300. RGCs were classified on the basis of their responses to light: onset (ON), offset (OFF), or both (ON-OFF). The distribution of ON, OFF, and ON-OFF RGCs is similar between WT and P35 Tg P23H-1 rats. By P300, many Tg P23H-1 RGCs are nonresponsive (NR). At this age, there is a sharp decline in ON and ON-OFF RGCs, and the majority that remain are OFF RGCs. Spontaneous rhythmic activity was observed in many RGCs at P300, but only in OFF or NR RGCs. In the SC, WT and P50 Tg P23H-1 responses are similar. At P300, Tg P23H-1 ON SC responses declined but OFF responses increased. We examined postsynaptic glutamate receptor expression located on the bipolar cells (BC), where the ON and OFF pathways arise. At P150, metabotropic glutamate receptor 6 (mGluR6) expression is lower than in WT, consistent with a decrease in ON RGC responses. GluR4 expression, an ionotropic glutamate receptor associated with OFF BCs, appears similar to that in WT. The loss of ON responses in Tg P23H-1 RGCs and in the SC is conserved and related to reduced mGluR6 signaling.

GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness.

Complete congenital stationary night blindness (cCSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impairment of night vision, absence of the electroretinogram (ERG) b-wave, and variable degrees of involvement of other visual functions. We report here that mutations in GPR179, encoding an orphan G protein receptor, underlie a form of autosomal-recessive cCSNB. The Gpr179(nob5/nob5) mouse model was initially discovered by the absence of the ERG b-wave, a component that reflects depolarizing bipolar cell (DBC) function. We performed genetic mapping, followed by next-generation sequencing of the critical region and detected a large transposon-like DNA insertion in Gpr179. The involvement of GPR179 in DBC function was confirmed in zebrafish and humans. Functional knockdown of gpr179 in zebrafish led to a marked reduction in the amplitude of the ERG b-wave. Candidate gene analysis of GPR179 in DNA extracted from patients with cCSNB identified GPR179-inactivating mutations in two patients. We developed an antibody against mouse GPR179, which robustly labeled DBC dendritic terminals in wild-type mice. This labeling colocalized with the expression of GRM6 and was absent in Gpr179(nob5/nob5) mutant mice. Our results demonstrate that GPR179 plays a critical role in DBC signal transduction and expands our understanding of the mechanisms that mediate normal rod vision.

GlyRα2, not GlyRα3, modulates the receptive field surround of OFF retinal ganglion cells.

Receptive fields (RFs) of most retinal ganglion cells (RGCs) consist of an excitatory center and suppressive surround. The RF center arises from the summation of excitatory bipolar cell glutamatergic inputs, whereas the surround arises from lateral inhibitory inputs. In the retina, both gamma amino butyric acid (GABA) and glycine are inhibitory neurotransmitters. A clear role for GABAergic inhibition modulating the RGC RF surround has been demonstrated across species. Glycinergic inhibition is more commonly associated with RF center modulation, although there is some evidence that it may contribute to the RF surround. The synaptic glycinergic chloride channels are formed by three homomeric ß and two homomeric α subunits that can be glycine receptor (GlyR) α1, α2, α3, or α4. GlyRα composition is responsible for currents with distinct decay kinetics. Their expression within the inner plexiform laminae and neuronal subtypes also differ. We studied the role of GlyR subunit selective modulation of RGC RF surrounds, using mice lacking GlyRα2 (Glra2 -/-), GlyRα3 (Glra3 -/-), or both (Glra2/3 -/-). We chose this molecular genetic approach instead of pharmacological manipulation because there are no subunit selective antagonists and strychnine blocks all GlyRs. Comparisons of annulus-evoked responses among wild type (WT) and GlyRα knockouts (Glra2 -/-, Glra3 -/- and Glra2/3 -/-) show that GlyRα2 inhibition enhances RF surround suppression and post-stimulus excitation in only WT OFF RGCs. Similarities in the responses in Glra2 -/- and Glra2/3 -/- RGCs verify these conclusions. Based on previous and current data, we propose that GlyRα2-mediated input uses a crossover inhibitory circuit. Further, we suggest that GlyRα2 modulates the OFF RGC RF center and surround independently. In summary, our results define a selective GlyR subunit-specific control of RF surround suppression in OFF RGCs.

Inner retinal preservation in rat models of retinal degeneration implanted with subretinal photovoltaic arrays.

Photovoltaic arrays (PVA) implanted into the subretinal space of patients with retinitis pigmentosa (RP) are designed to electrically stimulate the remaining inner retinal circuitry in response to incident light, thereby recreating a visual signal when photoreceptor function declines or is lost. Preservation of inner retinal circuitry is critical to the fidelity of this transmitted signal to ganglion cells and beyond to higher visual targets. Post-implantation loss of retinal interneurons or excessive glial scarring could diminish and/or eliminate PVA-evoked signal transmission. As such, assessing the morphology of the inner retina in RP animal models with subretinal PVAs is an important step in defining biocompatibility and predicting success of signal transmission. In this study, we used immunohistochemical methods to qualitatively and quantitatively compare inner retinal morphology after the implantation of a PVA in two RP models: the Royal College of Surgeons (RCS) or transgenic S334ter-line 3 (S334ter-3) rhodopsin mutant rat. Two PVA designs were compared. In the RCS rat, we implanted devices in the subretinal space at 4 weeks of age and histologically examined them at 8 weeks of age and found inner retinal morphology preservation with both PVA devices. In the S334ter-3 rat, we implanted devices at 6-12 weeks of age and again, inner retinal morphology was generally preserved with either PVA design 16-26 weeks post-implantation. Specifically, the length of rod bipolar cells and numbers of cholinergic amacrine cells were maintained along with their characteristic inner plexiform lamination patterns. Throughout the implanted retinas we found nonspecific glial reaction, but none showed additional glial scarring at the implant site. Our results indicate that subretinally implanted PVAs are well-tolerated in rodent RP models and that the inner retinal circuitry is preserved, consistent with our published results showing implant-evoked signal transmission.

Pig Models in Retinal Research and Retinal Disease.

The pig has been used as a large animal model in biomedical research for many years and its use continues to increase because induced mutations phenocopy several inherited human diseases. In addition, they are continuous breeders, can be propagated by artificial insemination, have large litter sizes (on the order of mice), and can be genetically manipulated using all of the techniques that are currently available in mice. The pioneering work of Petters and colleagues set the stage for the use of the pig as a model of inherited retinal disease. In the last 10 years, the pig has become a model of choice where specific disease-causing mutations that are not phenocopied in rodents need to be studied and therapeutic approaches explored. The pig is not only used for retinal eye disease but also for the study of the cornea and lens. This review attempts to show how broad the use of the pig has become and how it has contributed to the assessment of treatments for eye disease. In the last 10 years, there have been several reviews that included the use of the pig in biomedical research (see body of the review) that included information about retinal disease. None directly discuss the use of the pig as an animal model for retinal diseases, including inherited diseases, where a single genetic mutation has been identified or for multifactorial diseases such as glaucoma and diabetic retinopathy. Although the pig is used to explore diseases of the cornea and lens, this review focuses on how and why the pig, as a large animal model, is useful for research in neural retinal disease and its treatment.

Selective glycine receptor α2 subunit control of crossover inhibition between the on and off retinal pathways.

In the retina, the receptive fields (RFs) of almost all ganglion cells (GCs) are comprised of an excitatory center and a suppressive surround. The RF center arises from local excitatory bipolar cell (BC) inputs and the surround from lateral inhibitory inputs. Selective antagonists have been used to define the roles of GABA(A) and GABA(C) receptor-mediated input in RF organization. In contrast, the role of glycine receptor (GlyR) subunit-specific inhibition is less clear because the only antagonist, strychnine, blocks all GlyR subunit combinations. We used mice lacking the GlyRα2 (Glra2(-/-)) and GlyRα3 (Glra3(-/-)) subunits, or both (Glra2/3(-/-)), to explore their roles in GC RF organization. By comparing spontaneous and visually evoked responses of WT with Glra2(-/-), Glra3(-/-) and Glra2/3(-/-) ON- and OFF-center GCs, we found that both GlyRα2 and GlyRα3 modulate local RF interactions. In the On pathway, both receptors enhance the excitatory center response; however, the underlying inhibitory mechanisms differ. GlyRα2 participates in crossover inhibition, whereas GlyRα3 mediates serial inhibition. In the Off pathway, GlyRα2 plays a similar role, again using crossover inhibition and enhancing excitatory responses within the RF center. Comparisons of single and double KOs indicate that GlyRα2 and GlyRα3 inhibition are independent and additive, consistent with the finding that they use different inhibitory circuitry. These findings are the first to define GlyR subunit-specific control of visual function and GlyRα2 subunit-specific control of crossover inhibition in the retina.

Binocular benefit following monocular subretinal AAV injection in a mouse model of autosomal dominant retinitis pigmentosa (adRP).

Autosomal dominant retinitis pigmentosa (adRP) is frequently caused by mutations in RHO, the gene for rhodopsin. In previous experiments in dogs with the T4R mutation in RHO, an AAV2/5 vector expressing an shRNA directed to human and dog RHO mRNA and an shRNA-resistant human RHO cDNA (AAV-RHO820-shRNA820) prevented retinal degeneration for more than eight months following injection. It is crucial, however, to determine if this RNA replacement vector acts in a mutation-independent and species-independent manner. We, therefore, injected mice transgenic for human P23H RHO with this vector unilaterally at postnatal day 30. We monitored their retinal structure by using spectral-domain optical coherence tomography (SD-OCT) and retinal function using electroretinography (ERG) for nine months. We compared these to P23H RHO transgenic mice injected unilaterally with a control vector. Though retinas continued to thin over time, compared to control injected eyes, treatment with AAV-RHO820-shRNA820 slowed the loss of photoreceptor cells and the decrease in ERG amplitudes during the nine-month study period. Unexpectedly, we also observed the preservation of retinal structure and function in the untreated contralateral eyes of AAV-RHO820-shRNA820 treated mice. PCR analysis and western blots showed that a low amount of vector from injected eyes was present in uninjected eyes. In addition, protective neurotrophic factors bFGF and GDNF were elevated in both eyes of treated mice. Our finding suggests that using this or similar RNA replacement vectors in human gene therapy may provide clinical benefit to both eyes of patients with adRP.

Depolarizing bipolar cell dysfunction due to a Trpm1 point mutation.

Mutations in TRPM1 are found in humans with an autosomal recessive form of complete congenital stationary night blindness (cCSNB). The Trpm1(-/-) mouse has been an important animal model for this condition. Here we report a new mouse mutant, tvrm27, identified in a chemical mutagenesis screen. Genetic mapping of the no b-wave electroretinogram (ERG) phenotype of tvrm27 localized the mutation to a chromosomal region that included Trpm1. Complementation testing with Trpm1(-/-) mice confirmed a mutation in Trpm1. Sequencing identified a nucleotide change in exon 23, converting a highly conserved alanine within the pore domain to threonine (p.A1068T). Consistent with prior studies of Trpm1(-/-) mice, no anatomical changes were noted in the Trpm1(tvrm27/tvrm27) retina. The Trpm1(tvrm27/tvrm27) phenotype is distinguished from that of Trpm1(-/-) by the retention of TRPM1 expression on the dendritic tips of depolarizing bipolar cells (DBCs). While ERG b-wave amplitudes of Trpm1(+/-) heterozygotes are comparable to wild type, those of Trpm1(+/tvrm27) mice are reduced by 32%. A similar reduction in the response of Trpm1(+/tvrm27) DBCs to LY341495 or capsaicin is evident in whole cell recordings. These data indicate that the p.A1068T mutant TRPM1 acts as a dominant negative with respect to TRPM1 channel function. Furthermore, these data indicate that the number of functional TRPM1 channels at the DBC dendritic tips is a key factor in defining DBC response amplitude. The Trpm1(tvrm27/tvrm27) mutant will be useful for elucidating the role of TRPM1 in DBC signal transduction, for determining how Trpm1 mutations impact central visual processing, and for evaluating experimental therapies for cCSNB.

Mutations in TRPM1 are a common cause of complete congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impaired night vision and variable decreased visual acuity. We report here that six out of eight female probands with autosomal-recessive complete CSNB (cCSNB) had mutations in TRPM1, a retinal transient receptor potential (TRP) cation channel gene. These data suggest that TRMP1 mutations are a major cause of autosomal-recessive CSNB in individuals of European ancestry. We localized TRPM1 in human retina to the ON bipolar cell dendrites in the outer plexifom layer. Our results suggest that in humans, TRPM1 is the channel gated by the mGluR6 (GRM6) signaling cascade, which results in the light-evoked response of ON bipolar cells. Finally, we showed that detailed electroretinography is an effective way to discriminate among patients with mutations in either TRPM1 or GRM6, another autosomal-recessive cCSNB disease gene. These results add to the growing importance of the diverse group of TRP channels in human disease and also provide new insights into retinal circuitry.

Iodoacetic acid, but not sodium iodate, creates an inducible swine model of photoreceptor damage.

Our purpose was to find a method to create a large animal model of inducible photoreceptor damage. To this end, we tested in domestic swine the efficacy of two chemical toxins, known to create photoreceptor damage in other species: Iodoacetic Acid (IAA) and Sodium Iodate (NaIO(3)). Intravenous (IV) administration of NaIO(3) up to 90 mg/kg had no effect on retinal function and 110 mg/kg was lethal. IV administration of IAA (5-20 mg/kg) produced concentration-dependent changes in visual function as measured by full-field and multi-focal electroretinograms (ffERG and mfERG), and 30 mg/kg IAA was lethal. The IAA-induced effects measured at two weeks were stable through eight weeks post-injection, the last time point investigated. IAA at 7.5, 10, and 12 mg/kg produce a concentration-dependent reduction in both ffERG b-wave and mfERG N1-P1 amplitudes compared to baseline at all post-injection times. Comparisons of dark- and light-adapted ffERG b-wave amplitudes show a more significant loss of rod relative to cone function. The fundus of swine treated with ≥10 mg/kg IAA was abnormal with thinner retinal vessels and pale optic discs, and we found no evidence of bone spicule formation. Histological evaluations show concentration-dependent outer retinal damage that correlates with functional changes. We conclude that NaIO(3,) is not an effective toxin in swine. In contrast, IAA can be used to create a rapidly inducible, selective, stable and concentration-dependent model of photoreceptor damage in swine retina. Because of these attributes this large animal model of controlled photoreceptor damage should be useful in the investigation of treatments to replace damaged photoreceptors.

Receptor targets of amacrine cells.

Amacrine cells are a morphologically and functionally diverse group of inhibitory interneurons. Morphologically, they have been divided into approximately 30 types. Although this diversity is probably important to the fine structure and function of the retinal circuit, the amacrine cells have been more generally divided into two subclasses. Glycinergic narrow-field amacrine cells have dendrites that ramify close to their somas, cross the sublaminae of the inner plexiform layer, and create cross talk between its parallel ON and OFF pathways. GABAergic wide-field amacrine cells have dendrites that stretch long distances from their soma but ramify narrowly within an inner plexiform layer sublamina. These wide-field cells are thought to mediate inhibition within a sublamina and thus within the ON or OFF pathway. The postsynaptic targets of all amacrine cell types include bipolar, ganglion, and other amacrine cells. Almost all amacrine cells use GABA or glycine as their primary neurotransmitter, and their postsynaptic receptor targets include the most common GABA(A), GABA(C), and glycine subunit receptor configurations. This review addresses the diversity of amacrine cells, the postsynaptic receptors on their target cells in the inner plexiform layer of the retina, and some of the inhibitory mechanisms that arise as a result. When possible, the effects of GABAergic and glycinergic inputs on the visually evoked responses of their postsynaptic targets are discussed.

Gain control by sparse, ultra-slow glycinergic synapses.

In the retina, ON starburst amacrine cells (SACs) play a crucial role in the direction-selective circuit, but the sources of inhibition that shape their response properties remain unclear. Previous studies demonstrate that ∼95% of their inhibitory synapses are GABAergic, yet we find that the light-evoked inhibitory currents measured in SACs are predominantly glycinergic. Glycinergic inhibition is extremely slow, relying on non-canonical glycine receptors containing α4 subunits, and is driven by both the ON and OFF retinal pathways. These attributes enable glycine inputs to summate and effectively control the output gain of SACs, expanding the range over which they compute direction. Serial electron microscopic reconstructions reveal three specific types of ON and OFF narrow-field amacrine cells as the presumptive sources of glycinergic inhibition. Together, these results establish an unexpected role for specific glycinergic amacrine cells in the retinal computation of stimulus direction by SACs.

Nuclear Factor I in neurons, glia and during the formation of Müller glia-derived progenitor cells in avian, porcine and primate retinas.

The regenerative potential of Müller glia (MG) is extraordinary in fish, poor in chick and terrible in mammals. In the chick model, MG readily reprogram into proliferating Müller glia-derived progenitor cells (MGPCs), but neuronal differentiation is very limited. The factors that suppress the neurogenic potential of MGPCs in the chick are slowly being revealed. Isoforms of Nuclear Factor I (NFI) are cell-intrinsic factors that limit neurogenic potential; these factors are required for the formation of MG in the developing mouse retina and deletion of these factors reprograms MG into neuron-like cells in mature mouse retina. Accordingly, we sought to characterize the patterns of expression of NFIs in the developing, mature and damaged chick retina. In addition, we characterized patterns of expression of NFIs in the retinas of large mammals, pigs and monkeys. Using a combination of single-cell RNA-sequencing (scRNA-seq) and immunolabeling, we probed for patterns of expression. In embryonic chick, levels of NFIs are very low in early E5 (embryonic day 5) retinal progenitor cells (RPCs), upregulated in E8 RPCs, further upregulated in differentiating MG at E12 and E15. NFIs are maintained in mature resting MG, microglia and neurons. Levels of NFIs are reduced in activated MG in retinas treated with NMDA and/or insulin+FGF2, and further downregulated in proliferating MGPCs. However, levels of NFIs in MGPCs were significantly higher than those seen in RPCs. Immunolabeling for NFIA and NFIB closely matched patterns of expression revealed in different types of retinal neurons and glia, consistent with findings from scRNA-seq. In addition, we find expression of NFIA and NFIB through progenitors in the circumferential marginal zone at the far periphery of the retina. We find similar patterns of expression for NFIs in scRNA-seq databases for pig and monkey retinas. Patterns of expression of NFIA and NFIB were validated with immunofluorescence in pig and monkey retinas wherein these factors were predominantly detected in MG and a few types of inner retinal neurons. In summary, NFIA and NFIB are prominently expressed in developing chick retina and by mature neurons and glia in the retinas of chicks, pigs and monkeys. Although levels of NFIs are decreased in chick, in MGPCs these levels remain higher than those seen in neurogenic RPCs. We propose that the neurogenic potential of MGPCs in the chick retina is suppressed by NFIs.

Photobleaching reduction in modulated super-resolution microscopy.

Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases in photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.