Amacrine Cells Research Articles

Sstr2 Defines the Cone Differentiation-Competent Late-Stage Retinal Progenitor Cells in the Developing Mouse Retina.

Cone cell death is a characteristic shared by various retinal degenerative disorders, such as cone-rod dystrophy, Stargardt disease, achromatopsia, and retinitis pigmentosa. This leads to conditions like color blindness and permanently impaired visual acuity. Stem cell therapy focused on photoreceptor replacement holds promise for addressing these conditions. However, identifying surface markers that aid in enriching retinal progenitor cells (RPCs) capable of differentiating into cones remains a complex task. In this study, we employed single-cell RNA sequencing to scrutinize the transcriptome of developing retinas in C57BL/6J mice. This revealed the distinctive expression of somatostatin receptor 2 (Sstr2), a surface protein, in late-stage RPCs exhibiting the potential for photoreceptor differentiation. In vivo lineage tracing experiments verified that Sstr2+ cells within the late embryonic retina gave rise to cones, amacrine and horizontal cells during the developmental process. Furthermore, Sstr2+ cells that were isolated from the late embryonic mouse retina displayed RPC markers and exhibited the capability to differentiate into cones in vitro. Upon subretinal transplantation into both wild-type and retinal degeneration 10 (rd10) mice, Sstr2+ cells survived and expressed cone-specific markers. This study underscores the ability of Sstr2 to enrich late-stage RPCs primed for cone differentiation to a large extent. It proposes the utility of Sstr2 as a biomarker for RPCs capable of generating cones for transplantation purposes.

The Atoh1-Cre Knock-In Allele Ectopically Labels a Subpopulation of Amacrine Cells and Bipolar Cells in Mouse Retina.

The retina has diverse neuronal cell types derived from a common pool of retinal progenitors. Many molecular drivers, mostly transcription factors, have been identified to promote different cell fates. In Drosophila, atonal is required for specifying photoreceptors. In mice, there are two closely related atonal homologs, Atoh1 and Atoh7 While Atoh7 is known to promote the genesis of retinal ganglion cells, there is no study on the function of Atoh1 in retinal development. Here, we crossed Atoh1Cre/+ mice to mice carrying a Cre-dependent TdTomato reporter to track potential Atoh1-lineage neurons in retinas. We characterized a heterogeneous group of TdTomato+ retinal neurons that were detected at the postnatal stage, including glutamatergic amacrine cells, AII amacrine cells, and BC3b bipolar cells. Unexpectedly, we did not observe TdTomato+ retinal neurons in the mice with an Atoh1-FlpO knock-in allele and a Flp-dependent TdTomato reporter, suggesting Atoh1 is not expressed in the mouse retina. Consistent with these data, conditional removal of Atoh1 in the retina did not cause any observable phenotypes. Importantly, we did not detect Atoh1 expression in the retina at multiple ages using mice with Atoh1-GFP knock-in allele. Therefore, we conclude that Atoh1Cre/+ mice have ectopic Cre expression in the retina and that Atoh1 is not required for retinal development.

Layers of inhibitory networks shape receptive field properties of AII amacrine cells

In the retina, rod and cone pathways mediate visual signals over a billion-fold range in luminance. AII ("A-two") amacrine cells (ACs) receive signals from both pathways via different bipolar cells, enabling AIIs to operate at night and during the day. Previous work has examined luminance-dependent changes in AII gap junction connectivity, but less is known about how surrounding circuitry shapes AII receptive fields across light levels. Here, we report that moderate contrast stimuli elicit surround inhibition in AIIs under all but the dimmest visual conditions, due to actions of horizontal cells and at least two ACs that inhibit presynaptic bipolar cells. Under photopic (daylight) conditions, surround inhibition transforms AII response kinetics, which are inherited by downstream ganglion cells. Ablating neuronal nitric oxide synthase type-1 (nNOS-1) ACs removes AII surround inhibition under mesopic (dusk/dawn), but not photopic, conditions. Our findings demonstrate how multiple layers of neural circuitry interact to encode signals across a wide physiological range.

The rod synapse in aging wildtype and Dscaml1 mutant mice.

The retina is an intricately organized neural tissue built on cone and rod pathways for color and night vision. Genetic mutations that disrupt the proper function of the rod circuit contribute to blinding diseases including retinitis pigmentosa and congenital stationary night blindness (CSNB). Down Syndrome cell adhesion molecule like 1 (Dscaml1) is expressed by rods, rod bipolar cells (RBCs), and sub-populations of amacrine cells, and has been linked to a middle age onset of CSNB in humans. However, how Dscaml1 contributes to this visual deficit remains unexplored. Here, we probed Dscaml1's role in the maintenance of the rod-to-RBC synapse using a loss of function mouse model. We used immunohistochemistry to investigate the anatomical formation and maintenance of the rod-to-RBC synapse in the young, adult, and aging retina. We generated 3D reconstructions, using serial electron micrographs, of rod spherules and RBCs to measure the number of invaginating neurites, RBC dendritic tip number, and RBC mitochondrial morphology. We find that while rod-to-RBC synapses form and are maintained, similar to wildtype, that there is an increase in the number of invaginating neurites in rod spherules, a reduction in RBC dendritic tips, and reduced mitochondrial volume and complexity in the Dscaml1 mutant retina compared to controls. We also observed precocious sprouting of RBC dendrites into the outer nuclear layer (ONL) of the Dscaml1 mutant retina compared to controls. These results contribute to our knowledge of Dscaml1's role in rod circuit development and maintenance and give additional insight into possible genetic therapy targets for blinding diseases and disorders like CSNB.

Dysregulated CD200-CD200R signaling in early diabetes modulates microglia-mediated retinopathy.

Diabetic retinopathy (DR) is a neurovascular complication of diabetes. Recent investigations have suggested that early degeneration of the neuroretina may occur prior to the appearance of microvascular changes; however, the mechanisms underlying this neurodegeneration have been elusive. Microglia are the predominant resident immune cell in the retina and adopt dynamic roles in disease. Here, we show that ablation of retinal microglia ameliorates visual dysfunction and neurodegeneration in a type I diabetes mouse model. We also provide evidence of enhanced microglial contact and engulfment of amacrine cells, ultrastructural modifications, and transcriptome changes that drive inflammation and phagocytosis. We show that CD200-CD200R signaling between amacrine cells and microglia is dysregulated during early DR and that targeting CD200R can attenuate high glucose-induced inflammation and phagocytosis in cultured microglia. Last, we demonstrate that targeting CD200R in vivo can prevent visual dysfunction, microglia activation, and retinal inflammation in the diabetic mouse. These studies provide a molecular framework for the pivotal role that microglia play in early DR pathogenesis and identify a potential immunotherapeutic target for treating DR in patients.

Inhibition of TLR4/NF-κB pathway and endoplasmic reticulum stress by overexpressed S100A4 ameliorates retinal ischemia-reperfusion injury of mice.

Retinal ischemia exists in various ischemic retinopathies including glaucoma, contributing to the death of retinal neurons. Calcium binding protein S100A4 is important in tumors, and our previous study found that S100A4 protects retinal ganglion cells (RGCs) against retinal ischemia-reperfusion (I/R) injury. This study was aimed to further discuss the neuroprotection and mechanisms of S100A4 in retinal I/R of mice. The rAAV-EF1α-s100a4-EGFP-WPRE or rAAV-EF1α-EGFP-WPRE-Pa was injected intravitreally 4 weeks before I/R. S100A4, molecules in TLR4 signaling pathway and endoplasmic reticulum (ER) stress branches, inflammatory molecules, and surviving RGCs and cholinergic amacrine (ChAT) cells were determined by quantitative PCR, western blot, or immunofluorescent staining. The apoptosis and necrosis of retinal neurons induced by I/R were inhibited by overexpressed S100A4. RGCs, ChAT cells, and the retinal function were preserved by S100A4 overexpressing 7 days after I/R. Mechanistically, the beneficial effects of S100A4 may be mediated by inhibiting the activation of TLR4 signaling pathway and alleviating ER stress, leading to the attenuation of inflammatory response of the retina after I/R. Our findings indicated that S100A4 has neuroprotective effectagainst retinal I/R injury, and promoting S100A4 expression may be an effective strategy to inhibit retinal neurons from degeneration in ischemic retinopathy.

A common cause for nystagmus in different congenital stationary night blindness mouse models.

In Nyxnob mice, a model for congenital nystagmus associated with congenital stationary night blindness (CSNB), synchronous oscillating retinal ganglion cells (RGCs) lead to oscillatory eye movements, i.e. nystagmus. Given the specific expression of mGluR6 and Cav 1.4 in the photoreceptor to bipolar cell synapses, as well as their clinical association with CSNB, we hypothesize that Grm6nob3 and Cav 1.4-KO mutants show, like the Nyxnob mouse, oscillations in both their RGC activity and eye movements. Using multi-electrode array recordings of RGCs and measurements of the eye movements, we demonstrate that Grm6nob3 and Cav 1.4-KO mice also show oscillations of their RGCs as well as a nystagmus. Interestingly, the preferred frequencies of RGC activity as well as the eye movement oscillations of the Grm6nob3 , Cav 1.4-KO and Nyxnob mice differ among mutants, but the neuronal activity and eye movement behaviour within a strain remain aligned in the same frequency domain. Model simulations indicate that mutations affecting the photoreceptor-bipolar cell synapse can form a common cause of the nystagmus of CSNB by driving oscillations in RGCs via AII amacrine cells. KEY POINTS: In Nyxnob mice, a model for congenital nystagmus associated with congenital stationary night blindness (CSNB), their oscillatory eye movements (i.e. nystagmus) are caused by synchronous oscillating retinal ganglion cells. Here we show that the same mechanism applies for two other CSNB mouse models - Grm6nob3 and Cav 1.4-KO mice. We propose that the retinal ganglion cell oscillations originate in the AII amacrine cells. Model simulations show that by only changing the input to ON-bipolar cells, all phenotypical differences between the various genetic mouse models can be reproduced.

Cellular migration into a subretinal honeycomb-shaped prosthesis for high-resolution prosthetic vision

In patients blinded by geographic atrophy, a subretinal photovoltaic implant with 100 µm pixels provided visual acuity closely matching the pixel pitch. However, such flat bipolar pixels cannot be scaled below 75 µm, limiting the attainable visual acuity. This limitation can be overcome by shaping the electric field with 3-dimensional (3-D) electrodes. In particular, elevating the return electrode on top of the honeycomb-shaped vertical walls surrounding each pixel extends the electric field vertically and decouples its penetration into tissue from the pixel width. This approach relies on migration of the retinal cells into the honeycomb wells. Here, we demonstrate that majority of the inner retinal neurons migrate into the 25 µm deep wells, leaving the third-order neurons, such as amacrine and ganglion cells, outside. This enables selective stimulation of the second-order neurons inside the wells, thus preserving the intraretinal signal processing in prosthetic vision. Comparable glial response to that with flat implants suggests that migration and separation of the retinal cells by the walls does not cause additional stress. Furthermore, retinal migration into the honeycombs does not negatively affect its electrical excitability, while grating acuity matches the pixel pitch down to 40 μm and reaches the 27 μm limit of natural resolution in rats with 20 μm pixels. These findings pave the way for 3-D subretinal prostheses with pixel sizes of cellular dimensions.

Nfia Is Critical for AII Amacrine Cell Production: Selective Bipolar Cell Dependencies and Diminished ERG.

The nuclear factor one (NFI) transcription factor genes Nfia, Nfib, and Nfix are all enriched in late-stage retinal progenitor cells, and their loss has been shown to retain these progenitors at the expense of later-generated retinal cell types. Whether they play any role in the specification of those later-generated fates is unknown, but the expression of one of these, Nfia, in a specific amacrine cell type may intimate such a role. Here, Nfia conditional knockout (Nfia-CKO) mice (both sexes) were assessed, finding a massive and largely selective absence of AII amacrine cells. There was, however, a partial reduction in type 2 cone bipolar cells (CBCs), being richly interconnected to AII cells. Counts of dying cells showed a significant increase in Nfia-CKO retinas at postnatal day (P)7, after AII cell numbers were already reduced but in advance of the loss of type 2 CBCs detected by P10. Those results suggest a role for Nfia in the specification of the AII amacrine cell fate and a dependency of the type 2 CBCs on them. Delaying the conditional loss of Nfia to the first postnatal week did not alter AII cell number nor differentiation, further suggesting that its role in AII cells is solely associated with their production. The physiological consequences of their loss were assessed using the ERG, finding the oscillatory potentials to be profoundly diminished. A slight reduction in the b-wave was also detected, attributed to an altered distribution of the terminals of rod bipolar cells, implicating a role of the AII amacrine cells in constraining their stratification.SIGNIFICANCE STATEMENT The transcription factor NFIA is shown to play a critical role in the specification of a single type of retinal amacrine cell, the AII cell. Using an Nfia-conditional knockout mouse to eliminate this population of retinal neurons, we demonstrate two selective bipolar cell dependencies on the AII cells; the terminals of rod bipolar cells become mis-stratified in the inner plexiform layer, and one type of cone bipolar cell undergoes enhanced cell death. The physiological consequence of this loss of the AII cells was also assessed, finding the cells to be a major contributor to the oscillatory potentials in the electroretinogram.

Dynamics of morphological changes in the cellular layers of retina in the long-term experimental opioid exposure

Relevance of research. There is enough information in the foreign and domestic specialized literature on the issue of morphological reorganization of cells and tissues during experimental opioid exposure and other types of toxic effects on the body today. At present, the issue of structural reorganization in the layers of the retina and disruption of its blood supply processes during subchronic and chronic periods of experimental opioid exposure remains unclear. That is why we decided to supplement, expand and deepen information about these processes by conducting our own research in this direction. The purpose of the study is to clarify the dynamics of pathomorphological changes in the layers of the retina and the links of its hemomicrocirculatory channel in the long term of experimental opioid exposure.Material and methods. The research material was sexually mature, outbred, white male rats, 56 animals in number, weighing 160-270 g, aged 4.5-7.5 months. The initial dose of nalbuphine during the first 2 weeks was 0.212 mg/kg, the next 2 weeks (III-IV weeks) – 0.225 mg/kg, the next (V-VI weeks) – 0.252 mg/kg, the next (VI-VIII weeks ) – 0.260 mg/kg, the next (VIII – X weeks) – 0.283 mg/kg, the next (X – XII weeks) – 0.3 mg/kg, and during (XII – XIV weeks) – 0.45 mg/ kg. Before taking the material of the experimental site, the animal was removed from the experiment with the help of diethyl ether. Rat eyeballs were used as a material for ultrastructural research, considering the preservation of the topographic ratio of the eye membranes. Ultrastructural preparations were prepared according to the generally accepted method. Research results. At the end of the eighth week and the tenth week, persistent hyperemia of the choroidal vessels, degenerative and necrotic changes in the cells of the pigment layer, degeneration of the outer segments of photoreceptors, shortening and fragmentation of mitochondrial cristae of the inner segments of photoreceptors, necrotic changes in the nuclei of individual photoreceptors, degeneration of axons of the outer and inner reticular layers, as well as degenerative and necrotic changes of bipolar and amacrine cells, hypertrophy of processes of Müller cells, degeneration of ganglion cells, hyperemia and perivascular edema of vessels of the outer and inner reticular layers were revealed. After twelve and fourteen weeks, the initial stages of development with subsequent progression of sclerotic changes of Bruchʹs membrane were first detected. The phenomena of degenerative and necrotic changes in the cells of the pigment layer, destruction of the membrane of the outer segments of photoreceptors, degenerative changes in the axons of the outer and inner reticular layers and the layer of nerve fibers, degenerative and necrotic changes in bipolar and amacrine cells, hypertrophic changes in processes of Muller cells, hyperemia and stasis gradually increased, perivascular swellings in the vessels of the inner reticular layer, ganglionic layer and layer of nerve fibers, reactive changes in Muller cells and protoplasmic astrocytes. Conclusion. In the long-term course of experimental opioid exposure, irreversible pathomorphological changes occurred and subsequently progressed, which were caused by an increase in dyscirculatory manifestations in the superficial and deep vascular networks of the retina, causing the deepening of edema phenomena with further compression and hypoxia in the thickness of the retinal layers.

A sign-inverted receptive field of inhibitory interneurons provides a pathway for ON-OFF interactions in the retina

A fundamental organizing plan of the retina is that visual information is divided into ON and OFF streams that are processed in separate layers. This functional dichotomy originates in the ON and OFF bipolar cells, which then make excitatory glutamatergic synapses onto amacrine and ganglion cells in the inner plexiform layer. We have identified an amacrine cell (AC), the sign-inverting (SI) AC, that challenges this fundamental plan. The glycinergic, ON-stratifying SI-AC has OFF light responses. In opposition to the classical wiring diagrams, it receives inhibitory inputs from glutamatergic ON bipolar cells at mGluR8 synapses, and excitatory inputs from an OFF wide-field AC at electrical synapses. This “inhibitory ON center - excitatory OFF surround” receptive-field of the SI-AC allows it to use monostratified dendrites to conduct crossover inhibition and push-pull activation to enhance light detection by ACs and RGCs in the dark and feature discrimination in the light.

Cell-type expression and activation by light of neuropsins in the developing and mature Xenopus retina.

Photosensitive opsins detect light and perform image- or nonimage-forming tasks. Opsins such as the "classical" visual opsins and melanopsin are well studied. However, the retinal expression and functions of a novel family of neuropsins are poorly understood. We explored the developmental time-course and cell-type specificity of neuropsin (opn5, 6a, 6b, and 8) expression in Xenopus laevis by in situ hybridization and immunohistochemistry. We compared the Xenopus results with publicly available single cell RNA sequencing (scRNA-seq) data from zebrafish, chicken, and mouse. Additionally, we analyzed light-activation of neuropsin-expressing cells through induction of c-fos mRNA. opn5 and opn8 expression begins at stage 37/38 when the retinal circuits begin to be activated. Once retinal circuits connect to the brain, opn5 mRNA is distributed across multiple retinal cell types, including bipolar (~70%-75%), amacrine (~10%), and retinal ganglion (~20%) cells, with opn8 present in amacrine (~70%) and retinal ganglion (~30%) cells. opn6a and opn6b mRNAs emerge in newborn-photoreceptors (stage 35), and are colocalized in rods and cones by stage 37/38. Interestingly, in the mature larval retina (stage 43/44), opn6a and opn6b mRNAs become preferentially localized to rods and cones, respectively, while newborn photoreceptors bordering the proliferative ciliary marginal zone express both genes. In zebrafish, opn6a and opn6b are also expressed in photoreceptors, while Müller glia and amacrine cells express opn8c. Most neuropsin-expressing retinal ganglion cells display c-fos expression in response to light, as do over half of the neuropsin-expressing interneurons. This study gave a better understanding of retinal neuropsin-expressing cells, their developmental onset, and light activation.

Single-cell RNA sequencing of retina revealed novel transcriptional landscape in high myopia and underlying cell-type-specific mechanisms.

High myopia is a leading cause of blindness worldwide with increasing prevalence. Retina percepts visual information and triggers myopia development, but the underlying etiology is not fully understood because of cellular heterogeneity. In this study, single-cell RNA sequencing analysis was performed on retinas of mouse highly myopic and control eyes to dissect the involvement of each cell type during high myopia progression. For highly myopic photoreceptors, Hk2 inhibition underlying metabolic remodeling from aerobic glycolysis toward oxidative phosphorylation and excessive oxidative stress was identified. Importantly, a novel Apoe + rod subpopulation was specifically identified in highly myopic retina. In retinal neurons of highly myopic eyes, neurodegeneration was generally discovered, and the imbalanced ON/OFF signaling driven by cone-bipolar cells and the downregulated dopamine receptors in amacrine cells were among the most predominant findings, indicating the aberrant light processing in highly myopic eyes. Besides, microglia exhibited elevated expression of cytokines and TGF-β receptors, suggesting enhanced responses to inflammation and the growth-promoting states involved in high myopia progression. Furthermore, cell-cell communication network revealed attenuated neuronal interactions and increased glial/vascular interactions in highly myopic retinas. In conclusion, this study outlines the transcriptional landscape of highly myopic retina, providing novel insights into high myopia development and prevention.

Asymmetric connections with starburst amacrine cells underlie the upward motion selectivity of J-type retinal ganglion cells.

Motion is an important aspect of visual information. The directions of visual motion are encoded in the retina by direction-selective ganglion cells (DSGCs). ON-OFF DSGCs and ON DSGCs co-stratify with starburst amacrine cells (SACs) in the inner plexiform layer and depend on SACs for their direction selectivity. J-type retinal ganglion cells (J-RGCs), a type of OFF DSGCs in the mouse retina, on the other hand, do not co-stratify with SACs, and how direction selectivity in J-RGCs emerges has not been understood. Here, we report that both the excitatory and inhibitory synaptic inputs to J-RGCs are direction-selective (DS), with the inhibitory inputs playing a more important role for direction selectivity. The DS inhibitory inputs come from SACs, and the functional connections between J-RGCs and SACs are spatially asymmetric. Thus, J-RGCs and SACs form functionally important synaptic contacts even though their dendritic arbors show little overlap. These findings underscore the need to look beyond the neurons' stratification patterns in retinal circuit studies. Our results also highlight the critical role of SACs for retinal direction selectivity.

Robust expression of the TRPC1 channel associated with photoreceptor loss in the rat retina

Baseline intracellular calcium levels are significantly higher in neuronal and glial cells of rat retinas with retinitis pigmentosa (RP). Although this situation could initiate multiple detrimental pathways that lead to cell death, we considered the possibility of TRPC1 being involved in maintaining calcium homeostasis in the retina by acting as a component of store-operated calcium (SOC) channels with special relevance during photoreceptor degeneration. In this study, we examined by Western blot the expression of TRPC1 in healthy control rat retinas (Sprague-Dawley, SD) and retinas with RP (P23H-1 rats). We also analyzed its specific cellular distribution by immunofluorescence to recognize changes during neurodegeneration and to determine whether its presence is consistent with high basal calcium levels and cellular survival in degenerating retinas. We found that TRPC1 immunostaining was widely distributed across the retina in both rat strains, SD and P23H, and its expression levels significantly increased in the retinas with advanced degeneration compared to the age-control SD rats. In the outer retina, TRPC1 immunoreactivity was distributed in pigment epithelium cells, the photoreceptor inner segments of older animals, and the outer plexiform layer. In the inner retina, TRPC1 labeling was detected in horizontal cells, specific somata of bipolar and amacrine cells, and cellular processes in all the strata of the inner plexiform layer. Somata and processes were also highly immunoreactive in the ganglion cell layer and astrocytes in the nerve fiber layer in all animals. In the P23H rat retinas, the TRPC1 distribution pattern changed according to advancing photoreceptor degeneration and the gliosis reaction, with TRPC1 immunoreactive Müller cells mainly in advanced stages of disease. The cellular TRPC1 immunoreactivity found in this work suggests different mechanisms of activation of these channels depending on the cell type. Furthermore, the results support the idea that photoreceptor loss due to RP is associated with robust TRPC1 protein expression in the rat inner retina and raise the possibility of TRPC1 channels contributing to maintain high basal calcium levels during neurodegeneration and/or maintenance processes of the inner retina.

Direct comparison reveals algorithmic similarities in fly and mouse visual motion detection

Evolution has equipped vertebrates and invertebrates with neural circuits that selectively encode visual motion. While similarities in the computations performed by these circuits in mouse and fruit fly have been noted, direct experimental comparisons have been lacking. Because molecular mechanisms and neuronal morphology in the two species are distinct, we directly compared motion encoding in these two species at the algorithmic level, using matched stimuli and focusing on a pair of analogous neurons, the mouse ON starburst amacrine cell (ON SAC) and Drosophila T4 neurons. We find that the cells share similar spatiotemporal receptive field structures, sensitivity to spatiotemporal correlations, and tuning to sinusoidal drifting gratings, but differ in their responses to apparent motion stimuli. Both neuron types showed a response to summed sinusoids that deviates from models for motion processing in these cells, underscoring the similarities in their processing and identifying response features that remain to be explained.

Optogenetic targeting of AII amacrine cells restores retinal computations performed by the inner retina

Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of inner retinal neurons is a promising avenue to restore vision in retinas having lost their photoreceptors. Expression of optogenetic proteins in surviving ganglion cells, the retinal output, allows them to take on the lost photoreceptive function. Nonetheless, this creates an exclusively ON retina by expression of depolarizing optogenetic proteins in all classes of ganglion cells, whereas a normal retina extracts several features from the visual scene, with different ganglion cells detecting light increase (ON) and light decrease (OFF). Refinement of this therapeutic strategy should thus aim at restoring these computations. Here we used a vector that targets gene expression to a specific interneuron of the retina called the AII amacrine cell. AII amacrine cells simultaneously activate the ON pathway and inhibit the OFF pathway. We show that the optogenetic stimulation of AII amacrine cells allows restoration of both ON and OFF responses in the retina, but also mediates other types of retinal processing such as sustained and transient responses. Targeting amacrine cells with optogenetics is thus a promising avenue to restore better retinal function and visual perception in patients suffering from retinal degeneration.

The RLR intrinsic antiviral system is expressed in neural retina and restricts lentiviral transduction of human Mueller cells

The retinoic acid-inducible gene I (RIG)-I-like receptor (RLR) family of RNA sensor proteins plays a key role in the innate immune response to viral nucleic acids, including viral gene delivery vectors, but little is known about the expression of RLR proteins in the retina. The purpose of this study was to characterize cell-specific expression patterns of RLR proteins in non-human primate (NHP) neural retina tissue and to examine if RLR pathway signaling restricts viral gene delivery transduction. Since RLR protein signaling converges at the mitochondrial antiviral signaling protein (MAVS), experiments were performed to determine if knockdown of MAVS affected FIVGFP transduction efficiency in the human Mueller cell line MIO-M1. Immunoblotting confirmed expression of RIG-I, melanoma differentiation-associated protein 5 (MDA5), laboratory of genetics and physiology 2 (LGP2), and MAVS proteins in MIO-M1 cells and NHP retina tissue. Double label immunofluorescence (IF) studies revealed RIG-I, LGP2, and MAVS were expressed in Mueller microglial cells in the NHP retina. In addition, LGP2 and MDA5 proteins were detected in cone and retinal ganglion cells (RGC). MDA5 was also present in a subset of calretinin positive amacrine cells, and in nuclei within the inner nuclear layer (INL). Knockdown of MAVS significantly increased the transduction efficiency of the lentiviral vector FIVGFP in MIO-M1 cells, compared to control cells. FIVGFP or AAVGFP challenge did not alter expression of the LGP2, MAVS, MDA5 or RIG-I genes in MIO-M1 cells or NHP retina tissue compared to media treated controls. Our data demonstrate that innate immune response proteins involved in viral RNA sensing, including MDA5, RIG-I, LGP2, and MAVS, are expressed in several cell types within the NHP neural retina. In addition, the MAVS protein restricts non-human lentiviral transduction efficiency in MIO-M1 cells.

Presynaptic depolarization differentially regulates dual neurotransmitter release from starburst amacrine cells in the mouse retina.

The retina is comprised of diverse neural networks, signaling from photoreceptors to ganglion cells to encode images. The synaptic connections between these retinal neurons are crucial points for information transfer; however, the input-output relations of many synapses are understudied. Starburst amacrine cells in the retina are known to contribute to retinal motion detection circuits, providing a unique window for understanding neural computations. We examined the dual transmitter release of GABA and acetylcholine from starburst amacrine cells by optogenetic activation of these cells, and conducted patch clamp recordings from postsynaptic ganglion cells to record excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs). As starburst amacrine cells exhibit distinct kinetics in response to objects moving in a preferred or null direction, we mimicked their depolarization kinetics using optogenetic stimuli by varying slopes of the rising phase. The amplitudes of EPSCs and IPSCs in postsynaptic ganglion cells were reduced as the stimulus rising speed was prolonged. However, the sensitivity of postsynaptic currents to the stimulus slope differed. EPSC amplitudes were consistently reduced as the steepness of the rising phase fell. By contrast, IPSCs were less sensitive to the slope of the stimulus rise phase and maintained their amplitudes until the slope became shallow. These results indicate that distinct synaptic release mechanisms contribute to acetylcholine and GABA release from starburst amacrine cells, which could contribute to the ganglion cells' direction selectivity.

Metabolic changes and retinal remodeling in Heterozygous CRX mutant cats (CRXRDY/+)

CRX is a transcription factor essential for normal photoreceptor development and survival. The CRXRdy cat has a naturally occurring truncating mutation in CRX and is a large animal model for dominant Leber congenital amaurosis. This study investigated retinal remodeling that occurs as photoreceptors degenerate. CRXRdy/+ cats from 6 weeks to 10 years of age were investigated. In vivo structural changes of retinas were analyzed by fundus examination, confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Histologic analyses included immunohistochemistry for computational molecular phenotyping with macromolecules and small molecules. Affected cats had a cone-led photoreceptor degeneration starting in the area centralis. Initially there was preservation of inner retinal cells such as bipolar, amacrine and horizontal cells but with time migration of the deafferented neurons occurred. Early in the process of degeneration glial activation occurs ultimately resulting in formation of a glial seal. With progression the macula-equivalent area centralis developed severe atrophy including loss of retinal pigmentary epithelium. Microneuroma formation occured in advanced stages as more marked retinal remodeling occurred. This study indicates that retinal degeneration in the CrxRdy/+ cat retina follows the progressive, phased revision of retina that have been previously described for retinal remodeling. These findings suggest that therapy dependent on targeting inner retinal cells may be useful in young adults with preserved inner retinas prior to advanced stages of retinal remodeling and neuronal cell loss.