RESUMO
Organogenesis requires precise interactions between a developing tissue and its environment. In vertebrates, the developing eye is surrounded by a complex extracellular matrix as well as multiple mesenchymal cell populations. Disruptions to either the matrix or periocular mesenchyme can cause defects in early eye development, yet in many cases the underlying mechanism is unknown. Here, using multidimensional imaging and computational analyses in zebrafish, we establish that cell movements in the developing optic cup require neural crest. Ultrastructural analysis reveals that basement membrane formation around the developing eye is also dependent on neural crest, but only specifically around the retinal pigment epithelium. Neural crest cells produce the extracellular matrix protein nidogen: impairing nidogen function disrupts eye development, and, strikingly, expression of nidogen in the absence of neural crest partially restores optic cup morphogenesis. These results demonstrate that eye formation is regulated in part by extrinsic control of extracellular matrix assembly.This article has an associated 'The people behind the papers' interview.
Assuntos
Membrana Basal/embriologia , Olho/embriologia , Crista Neural/embriologia , Alelos , Animais , Sistemas CRISPR-Cas , Proteínas de Ligação ao Cálcio/fisiologia , Movimento Celular , Eletroforese Capilar , Matriz Extracelular/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Mesoderma/embriologia , Microscopia Eletrônica de Transmissão , Morfogênese , Mutação , Crista Neural/citologia , Organogênese , Retina/embriologia , Epitélio Pigmentado da Retina/embriologia , Transdução de Sinais , Fator de Transcrição AP-2/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Over the past decade, the field of retinal connectomics has made huge strides in describing the precise topologies underlying retinal visual processing. The same techniques that allowed these advancements are also applicable to understanding the progression of rewiring in retinal remodeling: retinal pathoconnectomics. Pathoconnectomics is unique in its unbiased approach to understanding the impacts of deafferentation on the remaining network components and identifying aberrant connectivities leading to visual processing defects. Pathoconnectomics also paves the way for identifying underlying rules of rewiring that may be recapitulated throughout the nervous system in other neurodegenerative diseases.
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Retina , Degeneração Retiniana , Humanos , Retina/fisiologiaRESUMO
Gap junctions are ubiquitous throughout the nervous system, mediating critical signal transmission and integration, as well as emergent network properties. In mammalian retina, gap junctions within the Aii amacrine cell-ON cone bipolar cell (CBC) network are essential for night vision, modulation of day vision, and contribute to visual impairment in retinal degenerations, yet neither the extended network topology nor its conservation is well established. Here, we map the network contribution of gap junctions using a high-resolution connectomics dataset of an adult female rabbit retina. Gap junctions are prominent synaptic components of ON CBC classes, constituting 5%-25% of all axonal synaptic contacts. Many of these mediate canonical transfer of rod signals from Aii cells to ON CBCs for night vision, and we find that the uneven distribution of Aii signals to ON CBCs is conserved in rabbit, including one class entirely lacking direct Aii coupling. However, the majority of gap junctions formed by ON CBCs unexpectedly occur between ON CBCs, rather than with Aii cells. Such coupling is extensive, creating an interconnected network with numerous lateral paths both within, and particularly across, these parallel processing streams. Coupling patterns are precise with ON CBCs accepting and rejecting unique combinations of partnerships according to robust rulesets. Coupling specificity extends to both size and spatial topologies, thereby rivaling the synaptic specificity of chemical synapses. These ON CBC coupling motifs dramatically extend the coupled Aii-ON CBC network, with implications for signal flow in both scotopic and photopic retinal networks during visual processing and disease.SIGNIFICANCE STATEMENT Electrical synapses mediated by gap junctions are fundamental components of neural networks. In retina, coupling within the Aii-ON CBC network shapes visual processing in both the scotopic and photopic networks. In retinal degenerations, these same gap junctions mediate oscillatory activity that contributes to visual impairment. Here, we use high-resolution connectomics strategies to identify gap junctions and cellular partnerships. We describe novel, pervasive motifs both within and across classes of ON CBCs that dramatically extend the Aii-ON CBC network. These motifs are highly specific with implications for both signal processing within the retina and therapeutic interventions for blinding conditions. These findings highlight the underappreciated contribution of coupling motifs in retinal circuitry and the necessity of their detection in connectomics studies.
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Junções Comunicantes/fisiologia , Junções Comunicantes/ultraestrutura , Rede Nervosa/fisiologia , Retina/fisiologia , Retina/ultraestrutura , Animais , Feminino , CoelhosRESUMO
Retinal degenerative diseases, such as retinitis pigmentosa, are generally thought to initiate with the loss of photoreceptors, though recent work suggests that plasticity and remodeling occurs prior to photoreceptor cell loss. This degeneration subsequently leads to death of other retinal neurons, creating functional alterations and extensive remodeling of retinal networks. Retinal prosthetic devices stimulate the surviving retinal cells by applying external current using implanted electrodes. Although these devices restore partial vision, the quality of restored vision is limited. Further knowledge about the precise changes in degenerated retina as the disease progresses is essential to understand how current flows in retinas undergoing degenerative disease and to improve the performance of retinal prostheses. We developed computational models that describe current flow from rod photoreceptors to rod bipolar cells (RodBCs) in the healthy and early-stage degenerated retina. Morphologically accurate models of retinal cells with their synapses are constructed based on retinal connectome datasets, created using serial section transmission electron microscopy (TEM) images of 70 nm-thick slices of either healthy (RC1) or early-stage degenerated (RPC1) rabbit retina. The passive membrane and active ion currents of each cell are implemented using conductance-based models in the Neuron simulation environment. In response to photocurrent input at rod photoreceptors, the simulated membrane potential at RodBCs in early degenerate tissue is approximately 10-20 mV lower than that of RodBCs of that observed in wild type retina. Results presented here suggest that although RodBCs in RPC1 show early, altered morphology compared to RC1, the lower membrane potential is primarily a consequence of reduced rod photoreceptor input to RodBCs in the degenerated retina. Frequency response and step input analyses suggest that individual cell responses of RodBCs in either healthy or early-degenerated retina, prior to substantial photoreceptor cell loss, do not differ significantly.
Assuntos
Simulação por Computador , Retina/fisiologia , Células Bipolares da Retina/fisiologia , Degeneração Retiniana/fisiopatologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Transdução de Sinais/fisiologia , Animais , Biologia Computacional , Conectoma , Plasticidade Neuronal/fisiologia , Coelhos , Sinapses/fisiologiaRESUMO
Connectomics has demonstrated that synaptic networks and their topologies are precise and directly correlate with physiology and behavior. The next extension of connectomics is pathoconnectomics: to map neural network synaptology and circuit topologies corrupted by neurological disease in order to identify robust targets for therapeutics. In this report, we characterize a pathoconnectome of early retinal degeneration. This pathoconnectome was generated using serial section transmission electron microscopy to achieve an ultrastructural connectome with 2.18nm/px resolution for accurate identification of all chemical and gap junctional synapses. We observe aberrant connectivity in the rod-network pathway and novel synaptic connections deriving from neurite sprouting. These observations reveal principles of neuron responses to the loss of network components and can be extended to other neurodegenerative diseases.
Assuntos
Conectoma/métodos , Degeneração Retiniana/diagnóstico , Células Fotorreceptoras Retinianas Bastonetes/patologia , Células Amácrinas/metabolismo , Células Amácrinas/patologia , Animais , Modelos Animais de Doenças , Junções Comunicantes , Coelhos , Degeneração Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sinapses/metabolismoRESUMO
Glia play important roles in neural function, including but not limited to amino acid recycling, ion homeostasis, glucose metabolism, and waste removal. During retinal degeneration and subsequent retinal remodeling, Müller cells (MCs) are the first cells to show metabolic and morphological alterations in response to stress. Metabolic alterations in MCs chaotically progress in retina undergoing photoreceptor degeneration; however, what relationship these alterations have with neuronal stress, synapse maintenance, or glia-glia interactions is currently unknown. The work described here reconstructs a MC from a pathoconnectome of early retinal remodeling retinal pathoconnectome 1 (RPC1) and explores relationships between MC structural and metabolic phenotypes in the context of neighboring neurons and glia. Here we find variations in intensity of osmication inter- and intracellularly, variation in small molecule metabolic content of MCs, as well as morphological alterations of glial endfeet. RPC1 provides a framework to analyze these relationships in early retinal remodeling through ultrastructural reconstructions of both neurons and glia. These reconstructions, informed by quantitative metabolite labeling via computational molecular phenotyping (CMP), allow us to evaluate neural-glial interactions in early retinal degeneration with unprecedented resolution and sensitivity.
Assuntos
Conectoma , Células Ependimogliais/patologia , Neurônios/citologia , Degeneração Retiniana/fisiopatologia , Humanos , Retina/citologia , Retina/patologiaRESUMO
Müller cells play a critical role in retinal metabolism and are among the first cells to demonstrate metabolic changes in retinal stress or disease. The timing, extent, regulation, and impacts of these changes are not yet known. We evaluated metabolic phenotypes of Müller cells in the degenerating retina. Retinas harvested from wild-type (WT) and rhodopsin Tg P347L rabbits were fixed in mixed aldehydes and resin embedded for computational molecular phenotyping (CMP). CMP facilitates small molecule fingerprinting of every cell in the retina, allowing evaluation of metabolite levels in single cells. CMP revealed signature variations in metabolite levels across Müller cells from TgP347L retina. In brief, neighboring Müller cells demonstrated variability in taurine, glutamate, glutamine, glutathione, glutamine synthetase (GS), and CRALBP. This variability showed no correlation across metabolites, implying the changes are functionally chaotic rather than simply heterogeneous. The inability of any clustering algorithm to classify Müller cell as a single class in the TgP347L retina is a formal proof of metabolic variability in the present in degenerating retina. Although retinal degeneration is certainly the trigger, Müller cell metabolic alterations are not a coherent response to the microenvironment. And while GS is believed to be the primary enzyme responsible for the conversion of glutamate to glutamine in the retina, alternative pathways appear to be unmasked in degenerating retina. Somehow, long term remodeling involves loss of Müller cell coordination and identity, which has negative implications for therapeutic interventions that target neurons alone.
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Células Ependimogliais/metabolismo , Neuroglia/metabolismo , Degeneração Retiniana/patologia , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Células Ependimogliais/patologia , Neuroglia/patologia , Fenótipo , Coelhos , Degeneração Retiniana/metabolismoRESUMO
Functional deficiency of the X-linked methyl-CPG binding protein 2 (MeCP2) leads to the neurodevelopmental disorder Rett syndrome (RTT). Due to random X-chromosome inactivation (XCI), most RTT patients are females who are heterozygous for the MECP2 mutation and therefore mosaic in MeCP2 deficiency. Some MECP2 heterozygote females are found to have unbalanced XCI, which may affect the severity of neurological symptoms seen in these patients; however, whether MeCP2 deficiency affects XCI in the postnatal and adult brain is unclear. Here we developed a novel MeCP2 mosaic mouse model in which the X chromosome containing the wild-type Mecp2 expresses a green fluorescent protein (GFP) transgene, while the X chromosome harboring the mutant Mecp2 does not. Due to random XCI, the neurons in the female MeCP2 mosaic mice express either wild-type MeCP2 (GFP+) or mutant MeCP2 (GFP-), and the two can be distinguished by GFP fluorescence. Using this mouse model, we evaluated XCI in female heterozygote mice from 3 to 9 months after birth. We found that MeCP2 deficiency does not affect XCI at 3 months of age, but does alter the proportion of wild-type MeCP2-expressing neurons at later ages, suggesting that MeCP2 impacts XCI patterns in an age-dependent manner. Given the important function of MeCP2 in neuronal development, our data could shed light on how MeCP2 deficiency affects postnatal brain functions and the dynamic changes in the neurological symptoms of RTT.
Assuntos
Modelos Animais de Doenças , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Síndrome de Rett/genética , Inativação do Cromossomo X , Fatores Etários , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Heterozigoto , Humanos , Masculino , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos Endogâmicos ICR , Camundongos Knockout , Síndrome de Rett/metabolismo , Síndrome de Rett/patologiaRESUMO
Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by the loss of functional fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that can regulate the translation of specific mRNAs. Adult neurogenesis, a process considered important for neuroplasticity and memory, is regulated at multiple molecular levels. In this study, we investigated whether Fmrp deficiency affects adult neurogenesis. We show that in a mouse model of fragile X syndrome, adult neurogenesis is indeed altered. The loss of Fmrp increases the proliferation and alters the fate specification of adult neural progenitor/stem cells (aNPCs). We demonstrate that Fmrp regulates the protein expression of several components critical for aNPC function, including CDK4 and GSK3beta. Dysregulation of GSK3beta led to reduced Wnt signaling pathway activity, which altered the expression of neurogenin1 and the fate specification of aNPCs. These data unveil a novel regulatory role for Fmrp and translational regulation in adult neurogenesis.
Assuntos
Diferenciação Celular/genética , Proliferação de Células , Proteína do X Frágil da Deficiência Intelectual/genética , Regulação da Expressão Gênica , Neurogênese , Células-Tronco/citologia , Animais , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Células-Tronco/metabolismoRESUMO
The lens proteome undergoes dramatic composition changes during development and maturation. A defective developmental process leads to congenital cataracts that account for about 30% of cases of childhood blindness. Gene mutations are associated with approximately 50% of early-onset forms of lens opacity, with the remainder being of unknown etiology. To gain a better understanding of cataractogenesis, we utilized a transgenic mouse model expressing a mutant ubiquitin protein in the lens (K6W-Ub) that recapitulates most of the early pathological changes seen in human congenital cataracts. We performed mass spectrometry-based tandem-mass-tag quantitative proteomics in E15, P1, and P30 control or K6W-Ub lenses. Our analysis identified targets that are required for early normal differentiation steps and altered in cataractous lenses, particularly metabolic pathways involving glutathione and amino acids. Computational molecular phenotyping revealed that glutathione and taurine were spatially altered in the K6W-Ub cataractous lens. High-performance liquid chromatography revealed that both taurine and the ratio of reduced glutathione to oxidized glutathione, two indicators of redox status, were differentially compromised in lens biology. In sum, our research documents that dynamic proteome changes in a mouse model of congenital cataracts impact redox biology in lens. Our findings shed light on the molecular mechanisms associated with congenital cataracts and point out that unbalanced redox status due to reduced levels of taurine and glutathione, metabolites already linked to age-related cataract, could be a major underlying mechanism behind lens opacities that appear early in life.
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Catarata , Proteoma , Humanos , Animais , Camundongos , Glutationa , Modelos Animais de Doenças , Camundongos Transgênicos , Proteínas Mutantes , Oxirredução , Taurina , Catarata/genéticaRESUMO
In retinal degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), the photoreceptors become stressed and start to degenerate in the early stages of the disease. Retinal prosthetic devices have been developed to restore vision in patients by applying electrical stimulation to the surviving retinal cells. However, these devices provide limited visual perception as the therapeutic interventions are generally considered in the later stages of the disease when only inner retinal layer cells are left. A potential treatment option for retinal degenerative diseases in the early stages can be stimulating bipolar cells, which receive presynaptic signals from photoreceptors. In this work, we constructed computational models of healthy and degenerated (both ON and OFF-type) cone bipolar cells (CBCs) with realistic morphologies extracted from connectomes of the healthy and early-stage degenerated rabbit retina. We examined these cells' membrane potential and axon terminal calcium current differences when subjected to electrical stimulation. In addition, we investigated how differently healthy and degenerated cells behave with respect to various stimulation parameters, including pulse duration and cells' distance from the stimulating electrode. The results suggested that regardless of the position of the OFF CBCs in the retina model, there is not a significant difference between the membrane potential of healthy and degenerate cells when electrically stimulated. However, the healthy ON CBC axon terminal membrane potential rising time-constant is shorter (0.29 ± 0.03 ms) than the degenerated cells (0.8 ± 0.07 ms). Moreover, the ionic calcium channels at the axon terminals of the cells have a higher concentration and higher current in degenerated cells (32.24 ± 6.12 pA) than the healthy cells (13.64 ± 2.88 pA) independently of the cell's position.
Assuntos
Degeneração Retiniana , Retinose Pigmentar , Animais , Coelhos , Degeneração Retiniana/terapia , Retina/fisiologia , Retinose Pigmentar/terapia , Axônios/fisiologia , Estimulação Elétrica/métodosRESUMO
The retinal degenerative diseases retinitis pigmentosa and age-related macular degeneration are a leading cause of irreversible vision loss. Both present with progressive photoreceptor degeneration that is further complicated by processes of retinal remodeling. In this perspective, we discuss the current state of the field of retinal remodeling and its implications for vision-restoring therapeutics currently in development. Here, we discuss the challenges and pitfalls retinal remodeling poses for each therapeutic strategy under the premise that understanding the features of retinal remodeling in totality will provide a basic framework with which therapeutics can interface. Additionally, we discuss the potential for approaching therapeutics using a combined strategy of using diffusible molecules in tandem with other vision-restoring therapeutics. We end by discussing the potential of the retina and retinal remodeling as a model system for more broadly understanding the progression of neurodegeneration across the central nervous system.
RESUMO
The maturation of young neurons is regulated by complex mechanisms and dysregulation of this process is frequently found in neurodevepmental disorders. MicroRNAs have been implicated in several steps of neuronal maturation including dendritic and axonal growth, spine development, and synaptogenesis. We demonstrate that one brain-enriched microRNA, miR-137, has a significant role in regulating neuronal maturation. Overexpression of miR-137 inhibits dendritic morphogenesis, phenotypic maturation, and spine development both in brain and cultured primary neurons. On the other hand, a reduction in miR-137 had opposite effects. We further show that miR-137 targets the Mind bomb one (Mib1) protein through the conserved target site located in the 3' untranslated region of Mib1 messenger RNA. Mib1 is an ubiquitin ligase known to be important for neurodevelopment. We show that exogenously expressed Mib1 could partially rescue the phenotypes associated with miR-137 overexpression. These results demonstrate a novel miRNA-mediated mechanism involving miR-137 and Mib1 that function to regulate neuronal maturation and dendritic morphogenesis during development.
Assuntos
Diferenciação Celular , MicroRNAs/genética , Neurônios/citologia , Neurônios/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Dendritos/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Biossíntese de Proteínas , Ubiquitina-Proteína Ligases/genéticaRESUMO
Müller cells are glia that play important regulatory roles in retinal metabolism. These roles have been evolutionarily conserved across at least 300 million years. Müller cells have a tightly locked metabolic signature in the healthy retina, which rapidly degrades in response to insult and disease. This variation in metabolic signature occurs in a chaotic fashion, involving some central metabolic pathways. The cause of this divergence of Müller cells, from a single class with a unique metabolic signature to numerous separable metabolic classes, is currently unknown and illuminates potential alternative metabolic pathways that may be revealed in disease. Understanding the impacts of this heterogeneity on degenerate retinas and the implications for the metabolic support of surrounding neurons will be critical to long-term integration of retinal therapeutics for the restoration of visual perception following photoreceptor degeneration.
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Células Ependimogliais/metabolismo , Glutamato-Amônia Ligase/metabolismo , Metaboloma , Degeneração Retiniana/metabolismo , HumanosRESUMO
Retinal remodeling is a progressive series of negative plasticity revisions that arise from retinal degeneration, and are seen in retinitis pigmentosa, age-related macular degeneration and other forms of retinal disease. These processes occur regardless of the precipitating event leading to degeneration. Retinal remodeling then culminates in a late-stage neurodegeneration that is indistinguishable from progressive central nervous system (CNS) proteinopathies. Following long-term deafferentation from photoreceptor cell death in humans, and long-lived animal models of retinal degeneration, most retinal neurons reprogram, then die. Glial cells reprogram into multiple anomalous metabolic phenotypes. At the same time, survivor neurons display degenerative inclusions that appear identical to progressive CNS neurodegenerative disease, and contain aberrant α-synuclein (α-syn) and phosphorylated α-syn. In addition, ultrastructural analysis indicates a novel potential mechanism for misfolded protein transfer that may explain how proteinopathies spread. While neurodegeneration poses a barrier to prospective retinal interventions that target primary photoreceptor loss, understanding the progression and time-course of retinal remodeling will be essential for the establishment of windows of therapeutic intervention and appropriate tuning and design of interventions. Finally, the development of protein aggregates and widespread neurodegeneration in numerous retinal degenerative diseases positions the retina as a ideal platform for the study of proteinopathies, and mechanisms of neurodegeneration that drive devastating CNS diseases.
Assuntos
Doenças Neurodegenerativas/patologia , Retina/patologia , Degeneração Retiniana/patologia , Vasos Retinianos/patologia , Remodelação Vascular , Animais , Morte Celular , Progressão da Doença , Humanos , Doenças Neurodegenerativas/fisiopatologia , Retina/fisiopatologia , Degeneração Retiniana/fisiopatologia , Neurônios Retinianos/patologia , Vasos Retinianos/fisiopatologiaRESUMO
Standard automated perimetry (SAP), the most common form of perimetry used in clinical practice, is associated with high test variability, impacting clinical decision making and efficiency. Contrast sensitivity isocontours (CSIs) may reduce test variability in SAP by identifying regions of the visual field with statistically similar patterns of change that can be analysed collectively and allow a point (disease)-to-CSI (normal) comparison in disease assessment as opposed to a point (disease)-to-point (normal) comparison. CSIs in the central visual field however have limited applicability as they have only been described using visual field test patterns with low, 6° spatial sampling. In this study, CSIs were determined within the central 20° visual field using the 10-2 test grid paradigm of the Humphrey Field Analyzer which has a high 2° sampling frequency. The number of CSIs detected in the central 20° visual field was greater than previously reported with low spatial sampling and stimulus size dependent: 6 CSIs for GI, 4 CSIs for GII and GIII, and 3 CSIs for GIV and GV. CSI number and distribution were preserved with age. Use of CSIs to assess visual function in age-related macular degeneration (AMD) found CSI guided analysis detected a significantly greater deviation in sensitivity of AMD eyes from normal compared to a standard clinical pointwise comparison (-1.40 ± 0.15 dB vs -0.96 ± 0.15 dB; p < 0.05). This work suggests detection of CSIs within the central 20° is dependent on sampling strategy and stimulus size and normative distribution limits of CSIs can indicate significant functional deficits in diseases affecting the central visual field such as AMD.
Assuntos
Sensibilidades de Contraste , Campos Visuais , Adulto , Idoso , Análise por Conglomerados , Feminino , Humanos , Degeneração Macular/fisiopatologia , Masculino , Pessoa de Meia-Idade , Testes de Campo Visual , Adulto JovemRESUMO
PURPOSE: To develop location-specific models of normal, age-related changes in the macular ganglion cell layer (GCL) from optical coherence tomography (OCT). Using these OCT-derived models, we predicted visual field (VF) sensitivities and compared these results to actual VF sensitivities. DESIGN: Retrospective cohort study. METHODS: Single eyes of 254 normal participants were retrospectively enrolled from the Centre for Eye Health (Sydney, Australia). Macular GCL measurements were obtained using Spectralis OCT. Cluster algorithms were performed to identify spatial patterns demonstrating similar age-related change. Quadratic and linear regression models were subsequently used to characterize age-related GCL decline. Forty participants underwent additional testing with Humphrey VFs, and 95% prediction intervals were calculated to measure the predictive ability of structure-function models incorporating cluster-based pooling, age correction, and consideration of spatial summation. RESULTS: Quadratic GCL regression models provided a superior fit (P value <.0001-.0066), establishing that GCL decline commences in the late 30s across the macula. The equivalent linear rates of GCL decline showed eccentricity-dependent variation (0.13 µm/yr centrally vs 0.06 µm/yr peripherally); however, average, normalized GCL loss per year was consistent across the 64 macular measurement locations at 0.26%. The 95% prediction intervals describing predicted VF sensitivities were significantly narrower across all cluster-based structure-function models (3.79-4.99 dB) compared with models without clustering applied (5.66-6.73 dB, P < .0001). CONCLUSIONS: Combining spatial clustering with age-correction based on regression models allowed the development of robust models describing GCL changes with age. The resultant superior predictive ability of VF sensitivity from ganglion cell measurements may be applied to future models of disease development to improve detection of early macular GCL pathology.
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Envelhecimento/fisiologia , Modelos Teóricos , Células Ganglionares da Retina/fisiologia , Campos Visuais/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Limiar Sensorial/fisiologia , Tomografia de Coerência Óptica , Acuidade Visual/fisiologia , Adulto JovemRESUMO
All superclasses of retinal neurons, including bipolar cells (BCs), amacrine cells (ACs) and ganglion cells (GCs), display gap junctional coupling. However, coupling varies extensively by class. Heterocellular AC coupling is common in many mammalian GC classes. Yet, the topology and functions of coupling networks remains largely undefined. GCs are the least frequent superclass in the inner plexiform layer and the gap junctions mediating GC-to-AC coupling (GC::AC) are sparsely arrayed amidst large cohorts of homocellular AC::AC, BC::BC, GC::GC and heterocellular AC::BC gap junctions. Here, we report quantitative coupling for identified GCs in retinal connectome 1 (RC1), a high resolution (2 nm) transmission electron microscopy-based volume of rabbit retina. These reveal that most GC gap junctions in RC1 are suboptical. GC classes lack direct cross-class homocellular coupling with other GCs, despite opportunities via direct membrane contact, while OFF alpha GCs and transient ON directionally selective (DS) GCs are strongly coupled to distinct AC cohorts. Integrated small molecule immunocytochemistry identifies these as GABAergic ACs (γ+ ACs). Multi-hop synaptic queries of RC1 connectome further profile these coupled γ+ ACs. Notably, OFF alpha GCs couple to OFF γ+ ACs and transient ON DS GCs couple to ON γ+ ACs, including a large interstitial amacrine cell, revealing matched ON/OFF photic drive polarities within coupled networks. Furthermore, BC input to these γ+ ACs is tightly matched to the GCs with which they couple. Evaluation of the coupled versus inhibitory targets of the γ+ ACs reveals that in both ON and OFF coupled GC networks these ACs are presynaptic to GC classes that are different than the classes with which they couple. These heterocellular coupling patterns provide a potential mechanism for an excited GC to indirectly inhibit nearby GCs of different classes. Similarly, coupled γ+ ACs engaged in feedback networks can leverage the additional gain of BC synapses in shaping the signaling of downstream targets based on their own selective coupling with GCs. A consequence of coupling is intercellular fluxes of small molecules. GC::AC coupling involves primarily γ+ cells, likely resulting in GABA diffusion into GCs. Surveying GABA signatures in the GC layer across diverse species suggests the majority of vertebrate retinas engage in GC::γ+ AC coupling.
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Células Amácrinas/ultraestrutura , Conectoma/métodos , Ácido Glutâmico/análise , Acoplamento Neurovascular/fisiologia , Células Ganglionares da Retina/ultraestrutura , Ácido gama-Aminobutírico/análise , Células Amácrinas/química , Animais , Junções Comunicantes/química , Carpa Dourada , Imageamento Tridimensional/métodos , Coelhos , Células Ganglionares da Retina/químicaRESUMO
Purpose: To characterize macular ganglion cell layer (GCL) changes with age and provide a framework to assess changes in ocular disease. This study used data clustering to analyze macular GCL patterns from optical coherence tomography (OCT) in a large cohort of subjects without ocular disease. Methods: Single eyes of 201 patients evaluated at the Centre for Eye Health (Sydney, Australia) were retrospectively enrolled (age range, 20-85); 8 × 8 grid locations obtained from Spectralis OCT macular scans were analyzed with unsupervised classification into statistically separable classes sharing common GCL thickness and change with age. The resulting classes and gridwise data were fitted with linear and segmented linear regression curves. Additionally, normalized data were analyzed to determine regression as a percentage. Accuracy of each model was examined through comparison of predicted 50-year-old equivalent macular GCL thickness for the entire cohort to a true 50-year-old reference cohort. Results: Pattern recognition clustered GCL thickness across the macula into five to eight spatially concentric classes. F-test demonstrated segmented linear regression to be the most appropriate model for macular GCL change. The pattern recognition-derived and normalized model revealed less difference between the predicted macular GCL thickness and the reference cohort (average ± SD 0.19 ± 0.92 and -0.30 ± 0.61 µm) than a gridwise model (average ± SD 0.62 ± 1.43 µm). Conclusions: Pattern recognition successfully identified statistically separable macular areas that undergo a segmented linear reduction with age. This regression model better predicted macular GCL thickness. The various unique spatial patterns revealed by pattern recognition combined with core GCL thickness data provide a framework to analyze GCL loss in ocular disease.
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Glaucoma/fisiopatologia , Macula Lutea/patologia , Fibras Nervosas/fisiologia , Reconhecimento Visual de Modelos , Células Ganglionares da Retina/fisiologia , Tomografia de Coerência Óptica/métodos , Campos Visuais/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Glaucoma/diagnóstico , Humanos , Macula Lutea/fisiopatologia , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Estudos Retrospectivos , Acuidade Visual , Adulto JovemRESUMO
Purpose: To determine the locus of test locations that exhibit statistically similar age-related decline in sensitivity to light increments and age-corrected contrast sensitivity isocontours (CSIs) across the central visual field (VF). We compared these CSIs with test point clusters used by the Glaucoma Hemifield Test (GHT). Methods: Sixty healthy observers underwent testing on the Humphrey Field Analyzer 30-2 test grid using Goldmann (G) stimulus sizes I-V. Age-correction factors for GI-V were determined using linear regression analysis. Pattern recognition analysis was used to cluster test locations across the VF exhibiting equal age-related sensitivity decline (age-related CSIs), and points of equal age-corrected sensitivity (age-corrected CSIs) for GI-V. Results: There was a small but significant test size-dependent sensitivity decline with age, with smaller stimuli declining more rapidly. Age-related decline in sensitivity was more rapid in the periphery. A greater number of unique age-related CSIs was revealed when using smaller stimuli, particularly in the mid-periphery. Cluster analysis of age-corrected sensitivity thresholds revealed unique CSIs for GI-V, with smaller stimuli having a greater number of unique clusters. Zones examined by the GHT consisted of test locations that did not necessarily belong to the same CSI, particularly in the periphery. Conclusions: Cluster analysis reveals statistically significant groups of test locations within the 30-2 test grid exhibiting the same age-related decline. CSIs facilitate pooling of sensitivities to reduce the variability of individual test locations. These CSIs could guide future structure-function and alternate hemifield asymmetry analyses by comparing matched areas of similar sensitivity signatures.