RESUMO
Purpose: The three-dimensional configurations of rod and cone bipolar cell (BC) dendrites and horizontal cell (HC) processes outside rod and cone synaptic terminals have not been fully elucidated. We reveal how these neurites are mutually arranged to coordinate formation and maintenance of the postsynaptic complex of ribbon synapses in mouse and monkey retinas. Methods: Serial section transmission electron microscopy was utilized to reconstruct BC and HC neurites in macaque monkey and mouse, including metabotropic glutamate receptor 6 (mGluR6)-knockout mice. Results: Starting from sporadically distributed branching points, rod BC and HC neurites (B and H, respectively) took specific paths to rod spherules by gradually adjusting their mutual positions, which resulted in a closed alternating pattern of HâBâHâB neurites at the rod spherule aperture. This order corresponded to the array of elements constituting the postsynaptic complex of ribbon synapses. We identified novel helical coils of HC processes surrounding the rod BC dendrite in both mouse and macaque retinas, and these structures occurred more frequently in mGluR6-knockout than wild-type mouse retinas. Horizontal cell processes also formed hook-like protrusions that encircled cone BC and HC neurites below the cone pedicles in the macaque retina. Conclusions: Bipolar and horizontal cell neurites take specific paths to adjust their mutual positions at the rod spherule aperture. Some HC processes are helically coiled around rod BC dendrites or form hook-like protrusions around cone BC dendrites and HC processes. Loss of mGluR6 signaling may be one factor promoting unbalanced neurite growth and compensatory neurite coiling.
Assuntos
Fasciculação Axônica/fisiologia , Neuritos/ultraestrutura , Células Bipolares da Retina/ultraestrutura , Células Horizontais da Retina/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Vias Visuais/ultraestrutura , Animais , Feminino , Macaca fuscata , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Terminações Pré-Sinápticas , Receptores de Glutamato Metabotrópico/fisiologia , SinapsesRESUMO
Cajal recognized that the elaborate shape of neurons is fundamental to their function in the brain. However, there are no simple and generalizable genetic methods to study neuronal or glial cell morphology in the mammalian brain. Here, we describe four mouse lines conferring Cre-dependent sparse cell labeling based on mononucleotide repeat frameshift (MORF) as a stochastic translational switch. Notably, the optimized MORF3 mice, with a membrane-bound multivalent immunoreporter, confer Cre-dependent sparse and bright labeling of thousands of neurons, astrocytes, or microglia in each brain, revealing their intricate morphologies. MORF3 mice are compatible with imaging in tissue-cleared thick brain sections and with immuno-EM. An analysis of 151 MORF3-labeled developing retinal horizontal cells reveals novel morphological cell clusters and axonal maturation patterns. Our study demonstrates a conceptually novel, simple, generalizable, and scalable mouse genetic solution to sparsely label and illuminate the morphology of genetically defined neurons and glia in the mammalian brain.
Assuntos
Astrócitos/ultraestrutura , Encéfalo/ultraestrutura , Microglia/ultraestrutura , Neurônios/ultraestrutura , Células Horizontais da Retina/ultraestrutura , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Mutação da Fase de Leitura/genética , Proteínas de Fluorescência Verde/genética , Integrases , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Repetições de Microssatélites/genética , Neurônios/metabolismo , Neurônios/patologia , Células Horizontais da Retina/metabolismo , Células Horizontais da Retina/patologiaRESUMO
Lateral inhibition in the vertebrate retina depends on a negative feedback synapse between horizontal cells (HCs) and rod and cone photoreceptors. A change in pH is thought to be the signal for negative feedback, but its spatial profile in the synaptic cleft is unknown. Here we use three different membrane proteins, each fused to the same genetically-encoded pH-sensitive Green Fluorescent Protein (GFP) (pHluorin), to probe synaptic pH in retina from transgenic zebrafish (Danio rerio) of either sex. We used the cone transducin promoter to express SynaptopHluorin (pHluorin on vesicle-associated membrane protein (VAMP2)) or CalipHluorin (pHluorin on an L-type Ca2+ channel) and the HC-specific connexin-55.5 promoter to express AMPApHluorin (pHluorin on an AMPA receptor). Stimulus light led to increased fluorescence of all three probes, consistent with alkalinization of the synaptic cleft. The receptive field size, sensitivity to surround illumination, and response to activation of an alien receptor expressed exclusively in HCs, are consistent with lateral inhibition as the trigger for alkalinization. However, SynaptopHluorin and AMPApHluorin, which are displaced farther from cone synaptic ribbons than CalipHluorin, reported a smaller pH change. Hence, unlike feedforward glutamatergic transmission, which spills over to allow cross talk between terminals in the cone network, the pH change underlying HC feedback is compartmentalized to individual synaptic invaginations within a cone terminal, consistent with private line communication.SIGNIFICANCE STATEMENT Lateral inhibition (LI) is a fundamental feature of information processing in sensory systems, enhancing contrast sensitivity and enabling edge discrimination. Horizontal cells (HCs) are the first cellular substrate of LI in the vertebrate retina, but the synaptic mechanisms underlying LI are not completely understood, despite decades of study. This paper makes a significant contribution to our understanding of LI, by showing that each HC-cone synapse is a "private-line" that operates independently from other HC-cone connections. Using transgenic zebrafish expressing pHluorin, a pH-sensitive GFP variant spliced onto three different protein platforms expressed either in cones or HCs we show that the feedback pH signal is constrained to individual cone terminals, and more stringently, to individual synaptic contact sites within each terminal.
Assuntos
Retroalimentação Fisiológica/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Horizontais da Retina/fisiologia , Sinapses/fisiologia , Animais , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/fisiologia , Conexinas/metabolismo , Feminino , Glutamatos/fisiologia , Concentração de Íons de Hidrogênio , Masculino , Prótons , Receptores de AMPA/metabolismo , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Células Horizontais da Retina/ultraestrutura , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologia , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/fisiologia , Peixe-ZebraRESUMO
Ganglion cells (GCs) are fundamental to retinal neural circuitry, processing photoreceptor signals for transmission to the brain via their axons. However, much remains unknown about their role in vision and their vulnerability to disease leading to blindness. A major bottleneck has been our inability to observe GCs and their degeneration in the living human eye. Despite two decades of development of optical technologies to image cells in the living human retina, GCs remain elusive due to their high optical translucency. Failure of conventional imaging-using predominately singly scattered light-to reveal GCs has led to a focus on multiply-scattered, fluorescence, two-photon, and phase imaging techniques to enhance GC contrast. Here, we show that singly scattered light actually carries substantial information that reveals GC somas, axons, and other retinal neurons and permits their quantitative analysis. We perform morphometry on GC layer somas, including projection of GCs onto photoreceptors and identification of the primary GC subtypes, even beneath nerve fibers. We obtained singly scattered images by: (i) marrying adaptive optics to optical coherence tomography to avoid optical blurring of the eye; (ii) performing 3D subcellular image registration to avoid motion blur; and (iii) using organelle motility inside somas as an intrinsic contrast agent. Moreover, through-focus imaging offers the potential to spatially map individual GCs to underlying amacrine, bipolar, horizontal, photoreceptor, and retinal pigment epithelium cells, thus exposing the anatomical substrate for neural processing of visual information. This imaging modality is also a tool for improving clinical diagnosis and assessing treatment of retinal disease.
Assuntos
Células Amácrinas/ultraestrutura , Óptica e Fotônica/métodos , Células Bipolares da Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Células Ganglionares da Retina/ultraestrutura , Células Horizontais da Retina/ultraestrutura , Tomografia de Coerência Óptica/métodos , Adulto , Células Amácrinas/fisiologia , Contagem de Células , Voluntários Saudáveis , Humanos , Pessoa de Meia-Idade , Fibras Nervosas/fisiologia , Fibras Nervosas/ultraestrutura , Óptica e Fotônica/instrumentação , Células Bipolares da Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Ganglionares da Retina/fisiologia , Células Horizontais da Retina/fisiologia , Tomografia de Coerência Óptica/instrumentação , Visão Ocular/fisiologiaRESUMO
The functional and morphological connectivity between various horizontal cell (HC) types (H1, H2, H3, and H4) and photoreceptors was studied in zebrafish retina. Since HCs are strongly coupled by gap junctions and feedback from HCs to photoreceptors depends strongly on connexin (Cx) hemichannels, we characterized the various HC Cxs (Cx52.6, Cx52.7, Cx52.9, and Cx55.5) in Xenopus oocytes. All Cxs formed hemichannels that were conducting at physiological membrane potentials. The Cx hemichannels differed in kinetic properties and voltage dependence, allowing for specific tuning of the coupling of HCs and the feedback signal from HCs to cones. The morphological connectivity between HC layers and cones was determined next. We used zebrafish expressing green fluorescent protein under the control of Cx promoters. We found that all HCs showed Cx55.5 promoter activity. Cx52.7 promoter activity was exclusively present in H4 cells, while Cx52.9 promoter activity occurred only in H1 cells. Cx52.6 promoter activity was present in H4 cells and in the ventral quadrant of the retina also in H1 cells. Finally, we determined the spectral sensitivities of the HC layers. Three response types were found. Monophasic responses were generated by HCs that contacted all cones (H1 cells), biphasic responses were generated by HCs that contacted M, S, and UV cones (H2 cells), and triphasic responses were generated by HCs that contacted either S and UV cones (H3 cells) or rods and UV cones (H4 cells). Electron microscopy confirms that H4 cells innervate cones. This indicates that rod-driven HCs process spectral information during photopic and luminance information during scotopic conditions.
Assuntos
Junções Comunicantes/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Potenciais da Membrana/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Retina/citologia , Células Horizontais da Retina/fisiologia , Análise de Variância , Animais , Animais Geneticamente Modificados , Biofísica , Biotina/análogos & derivados , Biotina/metabolismo , Conexinas/genética , Conexinas/metabolismo , Estimulação Elétrica , Retroalimentação Fisiológica/fisiologia , Junções Comunicantes/ultraestrutura , Proteínas de Fluorescência Verde/genética , Microinjeções , Microscopia Confocal , Microscopia Eletrônica , Oócitos , Técnicas de Patch-Clamp , Células Fotorreceptoras de Vertebrados/classificação , Células Fotorreceptoras de Vertebrados/ultraestrutura , Células Horizontais da Retina/classificação , Células Horizontais da Retina/ultraestrutura , Transdução Genética , Xenopus laevis , Peixe-ZebraRESUMO
Here we studied the ultrastructural organization of the outer retina of the European silver eel, a highly valued commercial fish species. The retina of the European eel has an organization very similar to most vertebrates. It contains both rod and cone photoreceptors. Rods are abundantly present and immunoreactive for rhodopsin. Cones are sparsely present and only show immunoreactivity for M-opsin and not for L-, S- or UV-cone opsins. As in all other vertebrate retinas, Müller cells span the width of the retina. OFF-bipolar cells express the ionotropic glutamate receptor GluR4 and ON-bipolar cells, as identified by their PKCα immunoreactivity, express the metabotropic receptor mGluR6. Both the ON- and the OFF-bipolar cell dendrites innervate the cone pedicle and rod spherule. Horizontal cells are surrounded by punctate Cx53.8 immunoreactivity indicating that the horizontal cells are strongly electrically coupled by gap-junctions. Connexin-hemichannels were found at the tips of the horizontal cell dendrites invaginating the photoreceptor synapse. Such hemichannels are implicated in the feedback pathway from horizontal cells to cones. Finally, horizontal cells are surrounded by tyrosine hydroxylase immunoreactivity, illustrating a strong dopaminergic input from interplexiform cells.
Assuntos
Anguilla/anatomia & histologia , Células Ependimogliais/ultraestrutura , Células Fotorreceptoras/ultraestrutura , Retina/ultraestrutura , Animais , Imuno-Histoquímica , Opsinas/análise , Proteína Quinase C-alfa/análise , Receptores de AMPA/análise , Células Bipolares da Retina/ultraestrutura , Células Horizontais da Retina/ultraestruturaRESUMO
Horizontal cells in the mouse retina are of the axon-bearing B-type and contribute to the gain control of photoreceptors and to the center-surround organization of bipolar cells by providing feedback and feedforward signals to photoreceptors and bipolar cells, respectively. Horizontal cells form two independent networks, coupled by dendro-dendritic and axo-axonal gap junctions composed of connexin57 (Cx57). In Cx57-deficient mice, occasionally the residual tracer coupling of horizontal cell somata was observed. Also, negative feedback from horizontal cells to photoreceptors, potentially mediated by connexin hemichannels, appeared unaffected. These results point to the expression of a second connexin in mouse horizontal cells. We investigated the expression of Cx50, which was recently identified in axonless A-type horizontal cells of the rabbit retina. In the mouse retina, Cx50-immunoreactive puncta were predominantly localized on large axon terminals of horizontal cells. Electron microscopy did not reveal any Cx50-immunolabeling at the membrane of horizontal cell tips invaginating photoreceptor terminals, ruling out the involvement of Cx50 in negative feedback. Moreover, Cx50 colocalized only rarely with Cx57 on horizontal cell processes, indicating that both connexins form homotypic rather than heterotypic or heteromeric gap junctions. To check whether the expression of Cx50 is changed when Cx57 is lacking, we compared the Cx50 expression in wildtype and Cx57-deficient mice. However, Cx50 expression was unaffected in Cx57-deficient mice. In summary, our results indicate that horizontal cell axon terminals form two independent sets of homotypic gap junctions, a feature which might be important for light adaptation in the retina.
Assuntos
Axônios/metabolismo , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Células Horizontais da Retina/metabolismo , Animais , Axônios/ultraestrutura , Western Blotting , Conexinas/genética , Retroalimentação Fisiológica/fisiologia , Junções Comunicantes/ultraestrutura , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Células Horizontais da Retina/ultraestrutura , TransfecçãoRESUMO
Horizontal cells of the human retina contain unique tubular organelles that have a diameter which is about 10 times larger than that of microtubules (~230 nm). These macrotubuli in most cases form regular aggregates. Therefore we propose to introduce them as Macrotubuli aggregati in the Terminologia histologica. Tomographic investigation of the structures revealed that the walls of the tubules most probably consist of intermediate filaments running nearly parallel to each other and show somewhat regularly attached ribosomes on their inner and also outer surface. About 2% of the organelles exhibit double- to multiple layered walls and less than 1% resemble large scrolls. The tubules may extend 10 to over 20 µm in the cytoplasm and are also encountered in soma-near processes extending into the outer plexiform layer. It remains unclear why these structures are only present in humans and few other species and why almost only in horizontal cells. Speculations on possible functions are discussed.
Assuntos
Citoplasma/ultraestrutura , Microtúbulos/ultraestrutura , Neurônios/ultraestrutura , Organelas/ultraestrutura , Retina/ultraestrutura , Células Horizontais da Retina/ultraestrutura , Idoso de 80 Anos ou mais , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Horizontal cells are interneurons that synapse with photoreceptors in the outer retina. Their genesis during development is subject to regulation by transcription factors in a hierarchical manner. Previously, we showed that Onecut 1 (Oc1), an atypical homeodomain transcription factor, is expressed in developing horizontal cells (HCs) and retinal ganglion cells (RGCs) in the mouse retina. Herein, by knocking out Oc1 specifically in the developing retina, we show that the majority (â¼80%) of HCs fail to form during early retinal development, implying that Oc1 is essential for HC genesis. However, no other retinal cell types, including RGCs, were affected in the Oc1 knock-out. Analysis of the genetic relationship between Oc1 and other transcription factor genes required for HC development revealed that Oc1 functions downstream of FoxN4, in parallel with Ptf1a, but upstream of Lim1 and Prox1. By in utero electroporation, we found that Oc1 and Ptf1a together are not only essential, but also sufficient for determination of HC fate. In addition, the synaptic connections in the outer plexiform layer are defective in Oc1-null mice, and photoreceptors undergo age-dependent degeneration, indicating that HCs are not only an integral part of the retinal circuitry, but also are essential for the survival of photoreceptors. In sum, these results demonstrate that Oc1 is a critical determinant of HC fate, and reveal that HCs are essential for photoreceptor viability, retinal integrity, and normal visual function.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Fator 6 Nuclear de Hepatócito/metabolismo , Neurogênese/genética , Retina/citologia , Células Horizontais da Retina/metabolismo , Animais , Contagem de Células , Diferenciação Celular/genética , Sobrevivência Celular , Embrião de Mamíferos , Proteínas do Olho/genética , Proteínas de Fluorescência Verde/genética , Fator 6 Nuclear de Hepatócito/genética , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais/metabolismo , Vias Neurais/ultraestrutura , Neuroglia/metabolismo , Neuroglia/fisiologia , Neurônios/classificação , Neurônios/metabolismo , Neurônios/ultraestrutura , Proteína Quinase C-alfa/metabolismo , Retina/embriologia , Células Horizontais da Retina/ultraestrutura , Sinapses/metabolismo , Sinapses/ultraestrutura , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Homeobox SIX3RESUMO
In the brain, including the retina, interneurons show an enormous structural and functional diversity. Retinal horizontal cells represent a class of interneurons that form triad synapses with photoreceptors and ON bipolar cells. At this first retinal synapse, horizontal cells modulate signal transmission from photoreceptors to bipolar cells by feedback and feedforward inhibition. To test how the fully developed retina reacts to the specific loss of horizontal cells, these interneurons were specifically ablated from adult mice using the diphtheria toxin (DT)/DT-receptor system and the connexin57 promoter. Following ablation, the retinal network responded with extensive remodeling: rods retracted their axons from the outer plexiform layer and partially degenerated, whereas cones survived. Cone pedicles remained in the outer plexiform layer and preserved synaptic contacts with OFF but not with ON bipolar cells. Consistently, the retinal ON pathway was impaired, leading to reduced amplitudes and prolonged latencies in electroretinograms. However, ganglion cell responses showed only slight changes in time course, presumably because ON bipolar cells formed multiple ectopic synapses with photoreceptors, and visual performance, assessed with an optomotor system, was only mildly affected. Thus, the loss of an entire interneuron class can be largely compensated even by the adult retinal network.
Assuntos
Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Células Horizontais da Retina/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Oxirredutases do Álcool/metabolismo , Análise de Variância , Animais , Arrestina/metabolismo , Conexinas/genética , Sensibilidades de Contraste/efeitos dos fármacos , Sensibilidades de Contraste/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Toxina Diftérica/toxicidade , Proteína 4 Homóloga a Disks-Large , Eletrorretinografia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Guanilato Quinases/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Vias Neurais/patologia , Vias Neurais/fisiopatologia , Estimulação Luminosa , Venenos/toxicidade , Proteína Quinase C-alfa/metabolismo , Receptores de AMPA/metabolismo , Retina/efeitos dos fármacos , Retina/patologia , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/induzido quimicamente , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/fisiologia , Células Horizontais da Retina/efeitos dos fármacos , Células Horizontais da Retina/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Sinapses/genética , Sinapses/patologia , Sinapses/ultraestrutura , Fatores de Tempo , Acuidade Visual/efeitos dos fármacosRESUMO
In the vertebrate retina, neuronal circuitry required for visual perception is organized within specific laminae. Photoreceptors convey external visual information to bipolar and horizontal cells at triad ribbon synapses established within the outer plexiform layer (OPL), initiating retinal visual processing. However, the molecular mechanisms that organize these three classes of neuronal processes within the OPL, thereby ensuring appropriate ribbon synapse formation, remain largely unknown. Here we show that mice with null mutations in Sema6A or PlexinA4 (PlexA4) exhibit a pronounced defect in OPL stratification of horizontal cell axons without any apparent deficits in bipolar cell dendrite or photoreceptor axon targeting. Furthermore, these mutant horizontal cells exhibit aberrant dendritic arborization and reduced dendritic self-avoidance within the OPL. Ultrastructural analysis shows that the horizontal cell contribution to rod ribbon synapse formation in PlexA4â»/â» retinas is disrupted. These findings define molecular components required for outer retina lamination and ribbon synapse formation.
Assuntos
Neurogênese/fisiologia , Receptores de Superfície Celular/fisiologia , Células Horizontais da Retina/citologia , Células Horizontais da Retina/ultraestrutura , Segmento Externo das Células Fotorreceptoras da Retina/fisiologia , Semaforinas/fisiologia , Sinapses/fisiologia , Animais , Dendritos/ultraestrutura , Feminino , Masculino , Camundongos , Mutação , Proteínas do Tecido Nervoso , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/ultraestrutura , Receptores de Superfície Celular/genética , Células Bipolares da Retina/citologia , Células Bipolares da Retina/ultraestrutura , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Semaforinas/genética , Transdução de Sinais/fisiologia , Sinapses/ultraestruturaRESUMO
Cell transplantation is a potential strategy for treating blindness caused by the loss of photoreceptors. Although transplanted rod-precursor cells are able to migrate into the adult retina and differentiate to acquire the specialized morphological features of mature photoreceptor cells, the fundamental question remains whether transplantation of photoreceptor cells can actually improve vision. Here we provide evidence of functional rod-mediated vision after photoreceptor transplantation in adult Gnat1−/− mice, which lack rod function and are a model of congenital stationary night blindness. We show that transplanted rod precursors form classic triad synaptic connections with second-order bipolar and horizontal cells in the recipient retina. The newly integrated photoreceptor cells are light-responsive with dim-flash kinetics similar to adult wild-type photoreceptors. By using intrinsic imaging under scotopic conditions we demonstrate that visual signals generated by transplanted rods are projected to higher visual areas, including V1. Moreover, these cells are capable of driving optokinetic head tracking and visually guided behaviour in the Gnat1−/− mouse under scotopic conditions. Together, these results demonstrate the feasibility of photoreceptor transplantation as a therapeutic strategy for restoring vision after retinal degeneration.
Assuntos
Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/transplante , Visão Ocular/fisiologia , Animais , Subunidades alfa de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Luz , Aprendizagem em Labirinto , Camundongos , Células Bipolares da Retina/ultraestrutura , Células Horizontais da Retina/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Transducina/deficiência , Transducina/genética , Visão Ocular/efeitos da radiação , Córtex Visual/fisiologia , Córtex Visual/efeitos da radiaçãoRESUMO
In the rabbit retina, there are two types of horizontal cell (HC). The axonless A-type HCs form a coupled network via connexin 50 (Cx50) gap junctions in the outer plexiform layer (OPL). The axon-bearing B-type HCs form two independently coupled networks; the dendritic network via gap junctions consisted of unknown Cx and the axon terminal network via Cx57. The present study was conducted to examine the localization and morphological features of Cx50 and Cx57 gap junctions in rabbit HCs at cellular and subcellular levels. The results showed that each gap junction composed of Cx50 or Cx57 showed distinct features. The larger Cx50 gap junctions were located more proximally than the smaller Cx50 gap junctions. Both Cx50 plaques formed symmetrical homotypic gap junctions, but some small ones had an asymmetrical appearance, suggesting the presence of heterotypic gap junctions or hemichannels. In contrast, Cx57 gap junctions were found in the more distal part of the OPL but never on the axon terminal endings entering the rod spherules, and they were exclusively homotypic. Interestingly, about half of the Cx57 gap junctions appeared to be invaginated. These distinct features of Cx50 and Cx57 gap junctions show the variety of HC gap junctions and may provide insights into the function of different types of HCs.
Assuntos
Conexinas/análise , Proteínas do Olho/análise , Junções Comunicantes/ultraestrutura , Células Horizontais da Retina/ultraestrutura , Animais , Dendritos/ultraestrutura , Junções Comunicantes/química , Microscopia Imunoeletrônica , Terminações Pré-Sinápticas/ultraestrutura , CoelhosRESUMO
Horizontal cells (HCs) are involved in establishing the center-surround receptive field organization of photoreceptor and bipolar cells. In many species, HCs respond differentially to colors and may play a role in color vision. An earlier study from our laboratory suggested that four types of HCs exist in the zebrafish retina: three cone HCs (H1, H2 and H3) and one rod HC. In this study, we describe their photoreceptor connections. Cones are arranged in a mosaic in which rows of alternating blue (B)- and ultraviolet (UV)-sensitive single cones alternate with rows of red (R)- and green (G)-sensitive double cones; the G cones are adjacent to UV cones and B cones adjacent to R cones. Two small-field (H1 and H2) and two large-field (H3 and rod HC) cells were observed. The cone HC dendritic terminals connected to cones with single boutons, doublets, or rosettes, whereas the rod HCs connected to rods with single boutons. The single boutons/doublets/rosettes of cone HCs were arranged in double rows separated by single rows for H1 cells, in pairs and singles for H2 cells, and in a rectilinear pattern for H3 cells. These connectivity patterns suggest that H1 cells contact R, G, and B cones, H2 cells G, B, and UV cones, and H3 cells B and UV cones. These predictions were confirmed by applying the DiI method to SWS1-GFP retinas whose UV cones express green fluorescent protein. Each rod HC was adjacent to the soma or axon of a DiI-labeled cone HC and connected to 50-200 rods.
Assuntos
Vias Neurais/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Células Horizontais da Retina/fisiologia , Peixe-Zebra/fisiologia , Animais , Dendritos/fisiologia , Microscopia Confocal , Vias Neurais/ultraestrutura , Células Fotorreceptoras de Vertebrados/ultraestrutura , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Células Horizontais da Retina/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Raios UltravioletaRESUMO
Information processing in the retina starts at the first synaptic layer, where photoreceptors and second-order neurons exhibit a complex architecture of glutamatergic and electrical synapses. To investigate the composition of this highly organized synaptic network, we determined the spatial relationship of zonula occludens-1 (ZO-1) with different connexins (Cx) and glutamate receptor (GluR) subunits in the outer plexiform layer (OPL) of rabbit, mouse, and monkey retinas. ZO-1 is well known as an intracellular component of tight and adherens junctions, but also interacts with various connexins at gap junctions. We found ZO-1 closely associated with Cx50 on dendrites of A-type horizontal cells in rabbit, and with Cx57 at dendro-dendritic gap junctions of mouse horizontal cells. The spatial arrangement of ZO-1 at the giant gap-junctional plaques in rabbit was particularly striking. ZO-1 formed a clear margin around the large Cx50 plaques instead of being colocalized with the connexin staining. Our finding suggests the involvement of ZO-1 in the composition of tight or adherens junctions around gap-junctional plaques instead of interacting with connexins directly. Furthermore, gap junctions were found to be clustered in close proximity to GluRs at the level of desmosome-like junctions, where horizontal cell dendrites converge before invaginating the cone pedicle. Based on this distinct spatial organization of gap junctions and GluRs, it is tempting to speculate that glutamate released from the photoreceptors may play a role in modulating the conductance of electrical synapses in the OPL.
Assuntos
Junções Comunicantes/ultraestrutura , Proteínas de Membrana/análise , Fosfoproteínas/análise , Retina/química , Retina/citologia , Células Horizontais da Retina/química , Células Horizontais da Retina/ultraestrutura , Junções Aderentes/ultraestrutura , Animais , Conexinas/análise , Conexinas/metabolismo , Dendritos/ultraestrutura , Desmossomos/fisiologia , Proteínas do Olho/metabolismo , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Coelhos , Receptores de Glutamato/análise , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1RESUMO
Mouse horizontal cells are coupled by gap junctions composed of connexin57. These gap junctions are regulated by ambient light via multiple neuromodulators including dopamine. In order to analyze the distribution and structure of horizontal cell gap junctions in the mouse retina, and examine the effects of light adaptation on gap junction density, we developed antibodies that detect mouse retinal connexin57. Using immunohistochemistry in retinal slices, flat-mounted retinas, and dissociated retinal cells, we showed that connexin57 is expressed in the dendrites and axon terminal processes of mouse horizontal cells. No staining was found in retinas of connexin57-deficient mice. Significantly more connexin57-positive puncta were found in the distal than in the proximal outer plexiform layer, indicating a higher level of expression in axon terminal processes than in the dendrites. We also examined the gap junctions using immunoelectron microscopy and showed that connexin57 does not form hemichannels in the horizontal cell dendritic tips. Light adaptation resulted in a significant increase in the number of connexin57-immunoreactive plaques in the outer plexiform layer, consistent with previously reported effects of light adaptation on connexin57 expression in the mouse retina. This study shows for the first time the detailed location of connexin57 expression within mouse horizontal cells, and provides the first ultrastructural data on mouse horizontal cell gap junctions.
Assuntos
Axônios/metabolismo , Conexinas/metabolismo , Dendritos/metabolismo , Junções Comunicantes/metabolismo , Luz , Células Horizontais da Retina/metabolismo , Adaptação Ocular , Animais , Anticorpos , Axônios/ultraestrutura , Western Blotting , Conexinas/genética , Conexinas/imunologia , Dendritos/ultraestrutura , Junções Comunicantes/ultraestrutura , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Imunoeletrônica , Estimulação Luminosa , Células Horizontais da Retina/ultraestruturaRESUMO
Sensory neurons with common functions are often nonrandomly arranged and form dendritic territories that show little overlap, or tiling. Repulsive homotypic interactions underlie such patterns in cell organization in invertebrate neurons. It is unclear how dendro-dendritic repulsive interactions can produce a nonrandom distribution of cells and their spatial territories in mammalian retinal horizontal cells, as mature horizontal cell dendrites overlap substantially. By imaging developing mouse horizontal cells, we found that these cells transiently elaborate vertical neurites that form nonoverlapping columnar territories on reaching their final laminar positions. Targeted cell ablation revealed that the vertical neurites engage in homotypic interactions that result in tiling of neighboring cells before the establishment of their dendritic fields. This developmental tiling of transient neurites correlates with the emergence of a nonrandom distribution of the cells and could represent a mechanism that organizes neighbor relationships and territories of neurons before circuit assembly.
Assuntos
Neuritos/fisiologia , Neuritos/ultraestrutura , Células Horizontais da Retina/fisiologia , Células Horizontais da Retina/ultraestrutura , Envelhecimento , Animais , Animais Recém-Nascidos , Comunicação Celular/fisiologia , Movimento Celular , Embrião de Mamíferos , Proteínas de Fluorescência Verde , Substâncias Luminescentes , Camundongos , Células Horizontais da Retina/citologia , Fatores de TempoRESUMO
The nob2 mouse carries a null mutation in the Cacna1f gene, which encodes the pore-forming subunit of the L-type calcium channel, Ca(v)1.4. The loss of the electroretinogram b-wave in these mice suggests a severe reduction in transmission between photoreceptors and second-order neurons in the retina and supports a central role for the Ca(v)1.4 calcium channel at photoreceptor ribbon synapses, to which it has been localized. Here we show that the loss of Ca(v)1.4 leads to the aberrant outgrowth of rod bipolar cell dendrites and horizontal cell processes into the outer nuclear layer (ONL) of the nob2 retina and to the formation of ectopic synaptic contacts with rod photoreceptors in the ONL. Ectopic contacts are predominantly between rods and rod bipolar cells, with horizontal cell processes also present at some sites. Ectopic contacts contain apposed pre- and postsynaptic specializations, albeit with malformed synaptic ribbons. Cone photoreceptor terminals do not participate in ectopic contacts in the ONL. During retinal development, ectopic contacts appear in the days after eye opening, appearing progressively farther into the ONL at later postnatal stages. Ectopic contacts develop at the tips of rod bipolar cell dendrites and are less frequently associated with the tips of horizontal cell processes, consistent with the adult phenotype. The relative occurrence of pre- and postsynaptic markers in the ONL during development suggests a mechanism for the formation of ectopic synaptic contacts that is driven by the retraction of rod photoreceptor terminals and neurite outgrowth by rod bipolar cell dendrites.
Assuntos
Canais de Cálcio/genética , Terminações Pré-Sinápticas/ultraestrutura , Células Bipolares da Retina/ultraestrutura , Células Horizontais da Retina/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/anormalidades , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Animais , Biomarcadores/metabolismo , Canais de Cálcio Tipo L , Diferenciação Celular/genética , Coristoma/genética , Coristoma/metabolismo , Coristoma/patologia , Dendritos/metabolismo , Dendritos/ultraestrutura , Distrofina/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação/genética , Terminações Pré-Sinápticas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Células Bipolares da Retina/metabolismo , Células Horizontais da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Membranas Sinápticas/genética , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestrutura , Transmissão Sináptica/genética , Visão Ocular/genéticaRESUMO
Dendrite morphology of neurons provides a structural basis for their physiological characteristics, and is precisely regulated in a cell type-dependent manner. Using a unique transposon-mediated gene transfer system that enables conditional and cell-type specific expression of exogenous genes, we investigated the role of cadherin on dendritic morphogenesis of horizontal cells in the developing chicken retina. We first visualized single horizontal cells by overexpressing membrane-targeted EGFP, and confirmed that there were three subtypes of horizontal cells, the dendritic terminals of which projected to distinct synaptic sites in the outer plexiform layer. Expression of a dominant-negative cadherin decreased the dendritic field size, and perturbed the termination of dendritic processes onto the photoreceptor cells. The cadherin blockade also impaired the accumulation of GluR4, a postsynaptic marker, at the cone pedicles. We thus provide in vivo evidence that cadherin is required for dendrite morphogenesis of horizontal cells and subsequent synapse formation with photoreceptor cells in the vertebrate retina.
Assuntos
Caderinas/fisiologia , Dendritos/fisiologia , Morfogênese , Células Horizontais da Retina/embriologia , Sinapses/ultraestrutura , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Caderinas/análise , Caderinas/genética , Embrião de Galinha , Dendritos/ultraestrutura , Expressão Gênica , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Morfogênese/genética , Receptores de AMPA/análise , Receptores de AMPA/metabolismo , Células Fotorreceptoras Retinianas Cones/química , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Horizontais da Retina/química , Células Horizontais da Retina/ultraestrutura , Sinapses/químicaRESUMO
We examined the identities of horizontal cell (HC) lateral components in cone terminals and the expression of glutamate receptors on the tips of HC dendrites. We injected A-type horizontal cells (AHCs) with neurobiotin and demonstrated that neurobiotin labeled completely all AHCs within a patch of retina. We converted neurobiotin by using diaminobenzidine and considered labeled processes to be from AHCs and unlabeled processes to be from B-type horizontal cells (BHCs). Three possible combinations of HC dendrites could exist in cone pedicles: both lateral components originating from AHCs, both from BHCs, or one from an AHC and the other from a BHC. EM observations revealed that a majority of cone terminals contained about equal numbers of lateral components originating from each of the two types of HCs and that each of the three possible combinations was present in equal numbers. Localization of different types of glutamate receptors on HC dendritic tips showed that 55% of AHC dendritic tips expressed AMPA receptors and 30% expressed kainate receptors, whereas, in the case of BHCs, 22% of dendritic tips expressed AMPA receptors and 33% expressed kainate receptors. This study suggests that cone photoreceptors feed the light signal equally into networks of AHCs and BHCs and that differential expression of AMPA/kainate receptors by different HCs could account for different functions.