Mouse Retinal Ganglion Cells Research Articles

The effect of waveform asymmetry on perception with epiretinal prostheses

ObjectiveRetinal prosthetic implants have helped improve vision in patients blinded by photoreceptor degeneration. Retinal implant users report improvements in light perception and performing visual tasks, but their ability to perceive shapes and letters is limited due to the low precision of retinal activation, which is exacerbated by axonal stimulation and high perceptual thresholds. A previous in vitro study in our lab used calcium imaging to measure the spatial activity of mouse retinal ganglion cells (RGCs) in response to electrical stimulation. Based on this study, symmetric anodic-first (SA) stimulation effectively avoided axonal activation and asymmetric anodic-first stimulation (AA) with duration ratios (ratio of the anodic to cathodic phase) greater than 10 reduced RGC activation thresholds significantly. Applying these novel stimulation strategies in clinic may increase perception precision and improve the overall patient outcomes.ApproachWe combined human subject testing and computational modeling to further examine the effect of SA and AA stimuli on perception shapes and thresholds for epiretinal stimulation of RGCs.Main resultsThreshold measurement in three Argus II participants indicated that AA stimulation could increase perception probabilities compared to a standard symmetric cathodic-first (SC) pulse, and this effect can be intensified by addition of an interphae gap (IPG). Our in silico RGC model predicts lower thresholds with AA and asymmetric cathodic-first (AC) stimuli compared to a SC pulse. This effect was more pronounced at shorter pulse widths. The most effective pulse for threshold reduction with short pulse durations (≤0.12 ms) was AA stimulation with small duration ratios (≤5) and long IPGs (≥2 ms). For the 0.5 ms pulse duration, SC stimulation with IPGs longer than 0.5 ms, or asymmetric stimuli with large duration ratios (≥20) were most effective in threshold reduction. Phosphene shape analysis did not reveal a significant change in percept elongation with SA stimulation. However, there was a significant increase in percept size (P < 0.01) with AA stimulation compared to the standard pulse in one participant.SignificaneIncluding asymmetric waveform capability will provide more flexible options for optimization and personalized fitting of retinal implants.

Type-specific dendritic integration in mouse retinal ganglion cells

Neural computation relies on the integration of synaptic inputs across a neuron’s dendritic arbour. However, it is far from understood how different cell types tune this process to establish cell-type specific computations. Here, using two-photon imaging of dendritic Ca2+ signals, electrical recordings of somatic voltage and biophysical modelling, we demonstrate that four morphologically distinct types of mouse retinal ganglion cells with overlapping excitatory synaptic input (transient Off alpha, transient Off mini, sustained Off, and F-mini Off) exhibit type-specific dendritic integration profiles: in contrast to the other types, dendrites of transient Off alpha cells were spatially independent, with little receptive field overlap. The temporal correlation of dendritic signals varied also extensively, with the highest and lowest correlation in transient Off mini and transient Off alpha cells, respectively. We show that differences between cell types can likely be explained by differences in backpropagation efficiency, arising from the specific combinations of dendritic morphology and ion channel densities.

Mouse γ-Synuclein Promoter-Mediated Gene Expression and Editing in Mammalian Retinal Ganglion Cells.

Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma, and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno-associated virus (AAV)-mediated gene transfer to perform a low-throughput in vivo screening in both male and female mouse eyes and identified the mouse γ-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can knock down pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.SIGNIFICANCE STATEMENT Here, we present an RGC-specific promoter, mouse γ-synuclein (mSncg) promoter, and perform extensive characterization and proof-of-concept studies of mSncg promoter-mediated gene expression and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing in RGCs in vivo To our knowledge, this is the first report demonstrating in vivo neuroprotection of injured RGCs and optic nerve (ON) by AAV-mediated CRISPR/Cas9 inhibition of genes that are critical for neurodegeneration. It represents a powerful tool to achieve RGC-specific gene modulation, and also opens up a promising gene therapy strategy for optic neuropathies, the most common form of eye diseases that cause irreversible blindness.

Utility of LysM-cre and Cdh5-cre Driver Mice in Retinal and Brain Research: An Imaging Study Using tdTomato Reporter Mouse.

PurposeThe lysozyme 2 (Lyz2 or LysM) cre mouse is extensively used to achieve genetic manipulation in myeloid cells and it has been widely employed in retinal research. However, LysM has been recently described to be expressed in brain neurons and there is a debate on whether it is also expressed by resident microglia in addition to infiltrating macrophages.MethodsWe examined LysM-cre recombination in retinal tissue using a LysM-cre/tdTomato reporter mouse together with immunolabeling for several retinal cell markers. We further compared LysM-cre tdTomato recombination with that of Cdh5-cre driver, which is expressed in both endothelial and hematopoietic cells.ResultsLysM-cre was strongly expressed in most microglia/resident macrophages in neonatal retinas (P8) and to a lesser extent in microglia of adult retinas. In addition, there was some neuronal recombination (8 %) of LysM-cre specifically in adult retinal ganglion cells and amacrine cells. After retinal ischemia-reperfusion injury, LysM-cre was strongly expressed in microglia/infiltrating macrophages. Cdh5-cre was expressed in endothelial and myeloid cells of P8 pups retinas. Unexpectedly, Cdh5 showed additional expression in adult mouse retinal ganglion cells and brain neurons.ConclusionsLysM-cre is expressed in macrophages and a subset of microglia together with a small but significant recombination of LysM-cre in the retinal neurons of adult mice. Cdh5 also showed some neuronal expression in both retina and brain of adult mice. These findings should be taken into consideration when interpreting results from central nervous system research using LysM-cre and Cdh5-cre mice.

Spatial Expression Pattern of the Major Ca2+-Buffer Proteins in Mouse Retinal Ganglion Cells.

The most prevalent Ca2+-buffer proteins (CaBPs: parvalbumin—PV; calbindin—CaB; calretinin—CaR) are widely expressed by various neurons throughout the brain, including the retinal ganglion cells (RGCs). Even though their retinal expression has been extensively studied, a coherent assessment of topographical variations is missing. To examine this, we performed immunohistochemistry (IHC) in mouse retinas. We found variability in the expression levels and cell numbers for CaR, with stronger and more numerous labels in the dorso-central area. CaBP+ cells contributed to RGCs with all soma sizes, indicating heterogeneity. We separated four to nine RGC clusters in each area based on expression levels and soma sizes. Besides the overall high variety in cluster number and size, the peripheral half of the temporal retina showed the greatest cluster number, indicating a better separation of RGC subtypes there. Multiple labels showed that 39% of the RGCs showed positivity for a single CaBP, 30% expressed two CaBPs, 25% showed no CaBP expression, and 6% expressed all three proteins. Finally, we observed an inverse relation between CaB and CaR expression levels in CaB/CaR dual- and CaB/CaR/PV triple-labeled RGCs, suggesting a mutual complementary function.

Endogenous Apelin Is Protective Against Age-Associated Loss of Retinal Ganglion Cells in Mice.

Age-associated loss of retinal ganglion cells (RGCs) causes visual deficits, but there is not yet any therapeutic agent to prevent the loss of these cells. Herein, we report that apelin, an endogenous peptide ligand of APJ receptor, is protective against the age-related loss of RGCs in mice. The mRNA expression of apelin was reduced in the retina of old mice compared with that in young mice, whereas retinal APJ expression increased with age. Immunofluorescence staining showed that APJ was present in RGCs and their surrounding cells expressed apelin. In addition, both functional and histological analyses demonstrated that apelin deficiency accelerated the loss of RGCs associated with age in mice. These results suggest that endogenous apelin plays a protective role against the degeneration of RGCs and that the apelinergic axis may be a new target for preventing age-related visual impairment.

Protective effects of human iPS-derived retinal pigmented epithelial cells on retinal degenerative disease

BackgroundRetinitis pigmentosa (RP) is an inherited retinal disease characterized by progressive loss of photoreceptor cells. This study aim at exploring the effect of retinal pigment epithelium (RPE) derived from human-induced pluripotent stem cell (hiPSC-RPE) on the retina of retinal degeneration 10 (rd10) mice, which are characterized with progressive photoreceptor death.MethodsWe generated RPE from hiPSCs by sequential supplementation with retinal-inducing factors and RPE specification signaling factors. The three-dimensional (3D) spheroid culture method was used to obtain optimal injectable hiPSC-RPE cells. Subretinal space transplantation was conducted to deliver hiPSC-RPE cells into the retina of rd10 mice. Neurotrophic factor secretion from transplanted hiPSC-RPE cells was detected by enzyme-linked immunosorbent assay (ELISA). Immunostaining, Western blotting, electroretinography (ERG), and visual behavior testing were performed to determine the effects of hiPSC-RPE on the retinal visual function in rd10 mice.ResultsOur data demonstrated that hiPSC-RPE cells exhibited classic RPE properties and phenotype after the sequential RPE induction from hiPSCs. hiPSC-RPE cells co-cultured with mouse retinal explants or retinal ganglion cells 5 (RGC5) exhibited decreased apoptosis. The viability and functional properties of hiPSC-RPE cells were enhanced by 3D spheroid culture. Transplanted hiPSC-derived RPE cells were identified by immunostaining with human nuclear antigen staining in the retina of rd10 14 days after subretinal space injection. The pigment epithelium-derived factor level was increased significantly. The expression of CD68, microglial activation marker, reduced after transplantation. The light avoidance behavior and ERG visual function in rd10 mice improved by the transplantation of hiPSC-RPE cells.ConclusionOur findings suggest that injectable hiPSC-RPE cells after 3D spheroid culture can rescue the structure and function of photoreceptors by sub-retinal transplantation, which lay the foundation for future clinical cell therapy to treat RP and other retinal degeneration diseases.

Dynamic Expression of HDAC3 in db/db Mouse RGCs and Its Relationship with Apoptosis and Autophagy.

Background Diabetic retinopathy (DR) is a severe complication of diabetes mellitus. DR is considered as a neurovascular disease. Retinal ganglion cell (RGC) loss plays an important role in the vision function disorder of diabetic patients. Histone deacetylase3 (HDAC3) is closely related to injury repair and nerve regeneration. The correlation between HDAC3 and retinal ganglion cells in diabetic retinopathy is still unclear yet. Methods To investigate the chronological sequence of the abnormalities of retinal ganglion cells in diabetic retinopathy, we choose 15 male db/db mice (aged 8 weeks, 12 weeks, 16 weeks, 18 weeks, and 25 weeks; each group had 3 mice) as diabetic groups and 3 male db/m mice (aged 8 weeks) as the control group. In this study, we examined the morphological and immunohistochemical changes of HDAC3, Caspase3, and LC3B in a sequential manner by characterizing the process of retinal ganglion cell variation. Results Blood glucose levels and body weights of db/db mice were significantly higher than that of the control group, P < 0.01. Compared with the control group, the number of retinal ganglion cells decreased with the duration of disease increasing. HDAC3 expression gradually increased in RGCs of db/db mice. Caspase3 expression gradually accelerated in RGCs of db/db mice. LC3B expression dynamically changed in RGCs of db/db mice. HDAC3 was positively correlated with Caspase3 expression (r = 0.7424), P < 0.01. Compared with the control group, the number of retinal ganglion cells decreased with the duration of disease increasing. HDAC3 expression gradually increased in RGCs of db/db mice. Caspase3 expression gradually accelerated in RGCs of db/db mice. LC3B expression dynamically changed in RGCs of db/db mice. HDAC3 was positively correlated with Caspase3 expression (r = 0.7424), P < 0.01. Compared with the control group, the number of retinal ganglion cells decreased with the duration of disease increasing. HDAC3 expression gradually increased in RGCs of db/db mice. Caspase3 expression gradually accelerated in RGCs of db/db mice. LC3B expression dynamically changed in RGCs of db/db mice. HDAC3 was positively correlated with Caspase3 expression (Discussion. We clarified the dynamic expression changes of HDAC3, Caspase3, and LC3B in retinal ganglion cells of db/db mice. Our results suggest the HDAC3 expression has a positive correlation with apoptosis and autophagy.

New Features of Receptive Fields in Mouse Retina through Spike-triggered Covariance.

Retinal ganglion cells (RGCs) encode various spatiotemporal features of visual information into spiking patterns. The receptive field (RF) of each RGC is usually calculated by spike-triggered average (STA), which is fast and easy to understand, but limited to simple and unimodal RFs. As an alternative, spike-triggered covariance (STC) has been proposed to characterize more complex patterns in RFs. This study compares STA and STC for the characterization of RFs and demonstrates that STC has an advantage over STA for identifying novel spatiotemporal features of RFs in mouse RGCs. We first classified mouse RGCs into ON, OFF, and ON/OFF cells according to their response to full-field light stimulus, and then investigated the spatiotemporal patterns of RFs with random checkerboard stimulation, using both STA and STC analysis. We propose five sub-types (T1–T5) in the STC of mouse RGCs together with their physiological implications. In particular, the relatively slow biphasic pattern (T1) could be related to excitatory inputs from bipolar cells. The transient biphasic pattern (T2) allows one to characterize complex patterns in RFs of ON/OFF cells. The other patterns (T3–T5), which are contrasting, alternating, and monophasic patterns, could be related to inhibitory inputs from amacrine cells. Thus, combining STA and STC and considering the proposed sub-types unveil novel characteristics of RFs in the mouse retina and offer a more holistic understanding of the neural coding mechanisms of mouse RGCs.

Cell types and cell circuits in human and non-human primate retina.

This review summarizes our current knowledge of primate including human retina focusing on bipolar, amacrine and ganglion cells and their connectivity. We have two main motivations in writing. Firstly, recent progress in non-invasive imaging methods to study retinal diseases mean that better understanding of the primate retina is becoming an important goal both for basic and for clinical sciences. Secondly, genetically modified mice are increasingly used as animal models for human retinal diseases. Thus, it is important to understand to which extent the retinas of primates and rodents are comparable. We first compare cell populations in primate and rodent retinas, with emphasis on how the fovea (despite its small size) dominates the neural landscape of primate retina. We next summarise what is known, and what is not known, about the postreceptoral neurone populations in primate retina. The inventories of bipolar and ganglion cells in primates are now nearing completion, comprising ~12 types of bipolar cell and at least 17 types of ganglion cell. Primate ganglion cells show clear differences in dendritic field size across the retina, and their morphology differs clearly from that of mouse retinal ganglion cells. Compared to bipolar and ganglion cells, amacrine cells show even higher morphological diversity: they could comprise over 40 types. Many amacrine types appear conserved between primates and mice, but functions of only a few types are understood in any primate or non-primate retina. Amacrine cells appear as the final frontier for retinal research in monkeys and mice alike.

On Laplacian spectrum of dendrite trees

For dendrite graphs from biological experiments on mouse's retinal ganglion cells, a paper by Nakatsukasa, Saito and Woei reveals a mysterious phase transition phenomenon in the spectra of the corresponding graph Laplacian matrices. While the bulk of the spectrum can be well understood by structures resembling starlike trees, mysteries about the spikes, that is, isolated eigenvalues outside the bulk spectrum, remain unexplained. In this paper, we bring new insights on these mysteries by considering a class of uniform trees. Exact relationships between the number of such spikes and the number of T-junctions are analyzed in function of the number of vertices separating the T-junctions. Using these theoretical results, predictions are proposed for the number of spikes observed in real-life dendrite graphs. Interestingly enough, these predictions match well the observed numbers of spikes, thus confirm the practical meaningness of our theoretical results.

加齢に伴う網膜神経節細胞の脱落に対する内因性アペリンの保護効果

加齢に伴う網膜神経節細胞の脱落に対する内因性アペリンの保護効果

Single-Cell Profiles of Retinal Ganglion Cells Differing in Resilience to Injury Reveal Neuroprotective Genes

Neuronal types in the central nervous system differ dramatically in their resilience to injury or other insults. Here we studied the selective resilience of mouse retinal ganglion cells (RGCs) following optic nerve crush (ONC), which severs their axons and leads to death of ∼80% of RGCs within 2weeks. To identify expression programs associated with differential resilience, we first used single-cell RNA-seq (scRNA-seq) to generate a comprehensive molecular atlas of 46 RGC types in adult retina. We then tracked their survival after ONC; characterized transcriptomic, physiological, and morphological changes that preceded degeneration; and identified genes selectively expressed by each type. Finally, using loss- and gain-of-function assays invivo, we showed that manipulating some of these genes improved neuronal survival and axon regeneration following ONC. This study provides a systematic framework for parsing type-specific responses to injury and demonstrates that differential gene expression can be used to reveal molecular targets for intervention.

In vivo imaging of the light response in mouse retinal ganglion cells based on a neuronal activity-dependent promoter

Diseases of the retinal ganglion cells (RGCs) are an important cause of blindness, yet the light response of individual RGCs is difficult to assess in vivo, particularly in mammals, due to a lack of effective methods. We report a simple in vivo platform for imaging the light response of mouse RGCs based on a fluorescent reporter-tagged enhanced synaptic activity-responsive element (E-SARE) that mediates neuronal activity–dependent gene transcription. When E-SARE-driven d2Venus, packaged into an AAV vector, was injected intravitreally, light-responsive retinal neurons expressing d2Venus were visible at single-cell resolution using confocal ophthalmoscopy. Immunohistological assessment identified the majority of these cells as RGCs. In a murine model of RGC injury, the number of d2Venus-positive cells was correlated with the amplitude of light-induced responses and with visual acuity, measured electrophysiologically at the visual cortex, indicating that the vector can be used as a tool to assess visual function in RGCs. The platform described herein allows a simple in vivo assessment of RGC function, which should help basic research into the mechanisms of RGC death and the development of treatments for diseases involving the RGCs.

The neuroprotective effects of novel estrogen receptor GPER1 in mouse retinal ganglion cell degeneration

PurposeTo investigate the potential protective effect of novel G protein coupled estrogen receptor (GPER1) against the neurotoxicity induced by NMDA in the mouse retina. MethodsWe induce retinal ganglion cells (RGCs) toxic injury through intravitreal injection of NMDA or acute ocular hypertension (AOH) induced by anterior chamber infusion with saline. Endogenous ligand 17-β-estradiol (E2), GPER1 agonist (G-1), and E2 with GPER1 antagonist (G-15) or classic estrogen receptor α and β (ERα and ERβ) antagonist tamoxifen (TAM) were subcutaneous administered before NMDA to identify the possible involved receptors. Immunofluorescence staining was performed to explore the survival of RGCs and Müller cell gliosis. TUNEL staining was used to evaluate the RGC apoptosis. The involved molecular pathway was detected via antibody array expression profiling. ResultsActivation of estrogen receptor by E2 or G-1 could significantly rescue the RGCs injury in NMDA administration. The protective effect was carried exclusively by GPER1 activation. E2 application can still mimicked the protective function when estrogen receptor α and β (ERα and ERβ) blocked by tamoxifen (TAM), while the effects was blocked by GPER1 antagonist G-15. Moreover, the TUNEL positive RGCs and GFAP expression level were both attenuated in G-1 application and the effects could be reversed by G-15. In addition, application of the PI3K/Akt antagonist LY294002 counteracted the effect of G-1. And a number of apoptosis regulatory factors decreased dramatically in the G-1 group, including Bad, Caspase 3, Caspase 7, Smad2, P-53 and TAK1. Also, similar protective effect of G-1 was spotted in acute ocular hypertension (AOH) model. ConclusionEstrogen played a protective role via a novel estrogen receptor, GPER1, instead of classical receptors ERα or ERβ. Activation of GPER1 attenuated RGCs apoptosis and Müller cells gliosis, indicating GPER1 as a potential treatment target in RGCs degeneration diseases.

Numbers of Retinal Ganglion Cells in Mice with Genetic Defects in Different A1 Adrenoreceptor Subtypes

Objective. To perform a comparative study of the numbers of retinal ganglion cells (RGC) in mice with genetic defects affecting different subtypes of α1 adrenoreceptors. Materials and methods. Studies were run on 36 male laboratory mice aged over 18 months with genetic defects affecting one α1 adrenoreceptor subtype (α1a, α1b, α1d). The control group consisted of intact laboratory mice (C57Bl/6NTac) of the same age and sex distribution. An immunofluorescent method was used for differential visualization of cells in retinal wholemount preparations using the Brn3a marker allowing these cells to be counted. Results. Mice with genetic defects of α1b and α1d adrenoreceptors had more and mice with defective α1a adrenoreceptors had fewer RGC than intact mice of the same age and sex. The total numbers of retinal cells in mice of all the study strains were essentially identical. Conclusions. The results obtained here provide evidence that each α1 adrenoreceptor subtype (α1a, α1b, α1d) makes a specific contribution during the process of forming retinal topography.

Abstract # 3254 Microglia regulate cognitive function in the healthy brain

Dedicator of cytokinesis 8 (DOCK8) is a guanine nucleotide exchange factor whose loss of function results in immunodeficiency, but its role in the central nervous system (CNS) has been unclear. Microglia are the resident immune cells of the CNS and are implicated in the pathogenesis of various neurodegenerative diseases, including multiple sclerosis (MS) and glaucoma, which affects the visual system. However, the exact roles of microglia in these diseases remain unknown. Herein, we report that DOCK8 is expressed in microglia but not in neurons or astrocytes and that its expression is increased during neuroinflammation. To define the role of DOCK8 in microglial activity, we focused on the retina, a tissue devoid of infiltrating T cells. The retina is divided into distinct layers, and in a disease model of MS/optic neuritis, DOCK8-deficient mice exhibited a clear reduction in microglial migration through these layers. Moreover, neuroinflammation severity, indicated by clinical scores, visual function, and retinal ganglion cell (RGC) death, was reduced in the DOCK8-deficient mice. Furthermore, using a glaucoma disease model, we observed impaired microglial phagocytosis of RGCs in DOCK8-deficient mice. Our data demonstrate that DOCK8 is expressed in microglia and regulates microglial activity in disease states. These findings contribute to a better understanding of the molecular pathways involved in microglial activation and implicate a role of DOCK8 in several neurological diseases.

The effect of retinal GABA Depletion by Allylglycine on mouse retinal ganglion cell responses to light

The effect of retinal GABA Depletion by Allylglycine on mouse retinal ganglion cell responses to light

DOCK8 is expressed in microglia, and it regulates microglial activity during neurodegeneration in murine disease models

Dedicator of cytokinesis 8 (DOCK8) is a guanine nucleotide exchange factor whose loss of function results in immunodeficiency, but its role in the central nervous system (CNS) has been unclear. Microglia are the resident immune cells of the CNS and are implicated in the pathogenesis of various neurodegenerative diseases, including multiple sclerosis (MS) and glaucoma, which affects the visual system. However, the exact roles of microglia in these diseases remain unknown. Herein, we report that DOCK8 is expressed in microglia but not in neurons or astrocytes and that its expression is increased during neuroinflammation. To define the role of DOCK8 in microglial activity, we focused on the retina, a tissue devoid of infiltrating T cells. The retina is divided into distinct layers, and in a disease model of MS/optic neuritis, DOCK8-deficient mice exhibited a clear reduction in microglial migration through these layers. Moreover, neuroinflammation severity, indicated by clinical scores, visual function, and retinal ganglion cell (RGC) death, was reduced in the DOCK8-deficient mice. Furthermore, using a glaucoma disease model, we observed impaired microglial phagocytosis of RGCs in DOCK8-deficient mice. Our data demonstrate that DOCK8 is expressed in microglia and regulates microglial activity in disease states. These findings contribute to a better understanding of the molecular pathways involved in microglial activation and implicate a role of DOCK8 in several neurological diseases.

Characterization of receptive fields of mouse retinal ganglion cells through comparative analysis of spike-triggered average and spike-triggered covariance

Characterization of receptive fields of mouse retinal ganglion cells through comparative analysis of spike-triggered average and spike-triggered covariance