RESUMO
The retinas of the vast majority of vertebrate species are termed "duplex," that is, they contain both rod and cone photoreceptor neurons in different ratios. The retina of little skate (Leucoraja erinacea) is a rarity among vertebrates because it contains only a single photoreceptor cell type and is thus "simplex." This unique retina provides us with an important comparative model and an exciting opportunity to study retinal circuitry within the context of a visual system with a single photoreceptor cell type. What is perhaps even more intriguing is the fact that the Leucoraja retina is able use that single photoreceptor cell type to function under both scotopic and photopic ranges of illumination. Although some ultrastructural characteristics of skate photoreceptors have been examined previously, leading to a general description of them as "rods" largely based on outer segment (OS) morphology and rhodopsin expression, a detailed study of the fine anatomy of the entire cell and its synaptic connectivity is still lacking. To address this gap in knowledge, we performed serial block-face electron microscopy imaging and examined the structure of skate photoreceptors and their postsynaptic partners. We find that skate photoreceptors exhibit unusual ultrastructural characteristics that are either common to rods or cones in other vertebrates (e.g., outer segment architecture, synaptic ribbon number, terminal extensions), or are somewhere in between those of a typical vertebrate rod or cone (e.g., number of invaginating contacts, clustering of multiple ribbons over a single synaptic invagination). We suggest that some of the ultrastructural characteristics we observe may play a role in the ability of the skate retina to function across scotopic and photopic ranges of illumination. Our findings have the potential to reveal as yet undescribed principles of vertebrate retinal design.
Assuntos
Retina , Células Fotorreceptoras Retinianas Cones , Animais , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Sinapses/ultraestruturaRESUMO
The skate retina provides a native all-rod retina suited for investigating a single type of photoreceptor regarding its properties and signaling to second order cells. Using the aspartate-induced isolated A-wave of the skate eyecup electroretinogram (ERG), it has been shown that adaptation in rods remains Weber-Fechner-like over a 6-log unit increase in background light intensity. Zinc, which can block calcium channels, has been found in the rod synaptic terminal and the synaptic cleft. Histidine is a zinc chelator. Voltage signals from neurons post-synaptic to rods indicate that histidine increases the dark release of glutamate and increases the horizontal cell light response. In histidine, the A-wave response to various light intensities in the dark-adapted retina increased more than fifty percent, corresponding to the effect on horizontal cells. In the presence of background light, although histidine-treated rod light responses remained Weber-Fechner-like, their increment threshold was raised significantly. This indicates that endogenous zinc feedback serves to increase rod sensitivity in a light-adapted retina, despite a corresponding reduction of threshold sensitivity in the dark. We propose that the increase in A-wave amplitude is a result of the increased conductance at the synaptic terminal and that the A-wave can be used to monitor changes in rod transmitter release. Furthermore, endogenous zinc may also provide the benefit of reducing metabolic stress and the risk of glutamate toxicity in the dark.
Assuntos
Eletrorretinografia , Retina , Adaptação à Escuridão , Luz , Células Fotorreceptoras , Células Fotorreceptoras de VertebradosRESUMO
Loss of primary neuronal inputs inevitably strikes every neural circuit. The deafferented circuit could propagate, amplify, or mitigate input loss, thus affecting the circuit's output. How the deafferented circuit contributes to the effect on the output is poorly understood because of lack of control over loss of and access to circuit elements. Here, we control the timing and degree of rod photoreceptor ablation in mature mouse retina and uncover compensation. Following loss of half of the rods, rod bipolar cells mitigate the loss by preserving voltage output. Such mitigation allows partial recovery of ganglion cell responses. We conclude that rod death is compensated for in the circuit because ganglion cell responses to stimulation of half of the rods in an unperturbed circuit are weaker than responses after death of half of the rods. The dominant mechanism of such compensation includes homeostatic regulation of inhibition to balance the loss of excitation.
Assuntos
Retina/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Vias Visuais/fisiopatologia , Animais , CamundongosRESUMO
Retinal responses to photons originate in rod photoreceptors and are transmitted to the ganglion cell output of the retina through the primary rod bipolar pathway. At the first synapse of this pathway, input from multiple rods is pooled into individual rod bipolar cells. This architecture is called convergence. Convergence serves to improve sensitivity of rod vision when photons are sparse. Establishment of convergence depends on the development of a proper complement of dendritic tips and transduction proteins in rod bipolar cells. How the dendrites of rod bipolar cells develop and contact the appropriate number of rods is unknown. To answer this question we visualized individual rod bipolar cells in mouse retina during postnatal development and quantified the number of dendritic tips, as well as the expression of transduction proteins within dendrites. Our findings show that the number of dendritic tips in rod bipolar cells increases monotonically during development. The number of tips at P21, P30, and P82 exceeds the previously reported rod convergence ratios, and the majority of these tips are proximal to a presynaptic rod release site, suggesting more rods provide input to a rod bipolar cell. We also show that dendritic transduction cascade members mGluR6 and TRPM1 appear in tips with different timelines. These finding suggest that (a) rod bipolar cell dendrites elaborate without pruning during development, (b) the convergence ratio between rods and rod bipolar cells may be higher than previously reported, and (c) mGluR6 and TRPM1 are trafficked independently during development.
Assuntos
Dendritos/ultraestrutura , Neurogênese/fisiologia , Retina/crescimento & desenvolvimento , Células Bipolares da Retina/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Sinapses/ultraestrutura , Animais , Dendritos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transporte Proteico/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Retina/metabolismo , Células Bipolares da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Sinapses/metabolismo , Canais de Cátion TRPM/metabolismoRESUMO
The chemical signal of light onset, a decrease in glutamate release from rod and cone photoreceptors, is processed by a postsynaptic G protein signaling cascade in ON-bipolar cells (BPCs). The metabotropic glutamate receptor mGluR6, along with other cascade elements, is localized synaptically at the BPC dendritic tips. The effector ion channel protein transient receptor potential melastatin-1 (TRPM1), in contrast, is located not only at the dendritic tips but also in BPC bodies and axons. Little is known about the intracellular localization of TRPM1, or its trafficking route to the dendritic tip plasma membrane. Recombinant TRPM1 expressed in mammalian cells colocalized with endoplasmic reticulum (ER) markers, with little or none detected at the plasma membrane. In mouse retina, somatic TRPM1 was similarly intracellular, and not at the plasma membrane. Labeling of ER membranes by expression of a fluorescent marker showed that in BPCs the ER extends into axons and dendrites, but not dendritic tips. In cell bodies, TRPM1 colocalized with the ER, and not with the Golgi apparatus. Fluorescence protease protection (FPP) assays with TRPM1-GFP fusions in heterologous cells revealed that the N and C termini are both accessible to the cytoplasm, consistent with the transmembrane domain topology of related TRP channels. These results indicate that the majority of TRPM1 is present in the ER, from which it can potentially be transported to the dendritic tips as needed for ON light responses. The excess of ER-resident TRPM1 relative to the amount needed at the dendritic tips suggests a potential new function for TRPM1 in the ER.
Assuntos
Retículo Endoplasmático/metabolismo , Células Bipolares da Retina/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Axônios/metabolismo , Dendritos/metabolismo , Feminino , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Transporte ProteicoRESUMO
The transient receptor potential channel TRPM1 is required for synaptic transmission between photoreceptors and the ON subtype of bipolar cells (ON-BPC), mediating depolarization in response to light. TRPM1 is present in the somas and postsynaptic dendritic tips of ON-BPCs. Monoclonal antibodies generated against full-length TRPM1 were found to have differential labeling patterns when used to immunostain the mouse retina, with some yielding reduced labeling of dendritic tips relative to the labeling of cell bodies. Epitope mapping revealed that those antibodies that poorly label the dendritic tips share a binding site (N2d) in the N-terminal arm near the transmembrane domain. A major splice variant of TRPM1 lacking exon 19 does not contain the N2d binding site, but quantitative immunoblotting revealed no enrichment of this variant in synaptsomes. One explanation of the differential labeling is masking of the N2d epitope by formation of a synapse-specific multiprotein complex. Identifying the binding partners that are specific for the fraction of TRPM1 present at the synapses is an ongoing challenge for understanding TRPM1 function.
Assuntos
Epitopos/química , Células Bipolares da Retina/metabolismo , Sinapses/fisiologia , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Animais , Anticorpos Monoclonais , Western Blotting , Mapeamento de Epitopos , Epitopos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Reação em Cadeia da Polimerase , Isoformas de Proteínas , Transmissão Sináptica/fisiologiaRESUMO
The mammalian retina consists of multiple cell layers including photoreceptor cells, which are light sensing neurons that play essential functions in the visual process. Previously, we identified mutations in SPATA7, encoding spermatogenesis associated protein 7, in families with Leber Congenital Amaurosis (LCA) and juvenile Retinitis Pigmentosa (RP), and showed that Spata7 null mice recapitulate the human disease phenotype of retinal degeneration. SPATA7 is expressed in the connecting cilium of photoreceptor (PR) cells in the mouse retina, as well as in retinal pigment epithelium (RPE) cells, but the functional role of Spata7 in the RPE remains unknown. To investigate whether Spata7 is required in PRs, the RPE, or both, we conditionally knocked out Spata7 in photoreceptors and RPE cells using Crx-Cre and Best1-Cre transgenic mouse lines, respectively. In Spata7 photoreceptor-specific conditional (cKO) mice, both rod and cone photoreceptor dysfunction and degeneration is observed, characterized by progressive thinning of the outer nuclear layer and reduced response to light; however, RPE-specific deletion of Spata7 does not impair retinal function or cell survival. Furthermore, our findings show that both Rhodopsin and RPGRIP1 are mislocalized in the Spata7Flox/-; Crx-Cre cKO mice, suggesting that loss of Spata7 in photoreceptors alone can result in altered trafficking of these proteins in the connecting cilium. Together, our findings suggest that loss of Spata7 in photoreceptors alone is sufficient to cause photoreceptor degeneration, but its function in the RPE is not required for photoreceptor survival; therefore, loss of Spata7 in photoreceptors alters both rod and cone function and survival, consistent with the clinical phenotypes observed in LCA and RP patients with mutations in SPATA7.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinose Pigmentar/patologia , Animais , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Eletrorretinografia , Camundongos , Camundongos Knockout , Proteínas/metabolismo , Retina/metabolismo , Rodopsina/metabolismoRESUMO
Phosphoinositides play important roles in numerous intracellular membrane pathways. Little is known about the regulation or function of these lipids in rod photoreceptor cells, which have highly active membrane dynamics. Using new assays with femtomole sensitivity, we determined that whereas levels of phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate were below detection limits, phosphatidylinositol-3-phosphate (PI(3)P) levels in rod inner/outer segments increased more than 30-fold after light exposure. This increase was blocked in a rod-specific knockout of the PI-3 kinase Vps34, resulting in failure of endosomal and autophagy-related membranes to fuse with lysosomes, and accumulation of abnormal membrane structures. At early ages, rods displayed normal morphology, rhodopsin trafficking, and light responses, but underwent progressive neurodegeneration with eventual loss of both rods and cones by twelve weeks. The degeneration is considerably faster than in rod knockouts of autophagy genes, indicating defects in endosome recycling or other PI(3)P-dependent membrane trafficking pathways are also essential for rod survival.
Assuntos
Classe III de Fosfatidilinositol 3-Quinases/genética , Fosfatos de Fosfatidilinositol/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Rodopsina/genética , Animais , Autofagia/genética , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/genética , Sobrevivência Celular , Classe III de Fosfatidilinositol 3-Quinases/deficiência , Endossomos/metabolismo , Regulação da Expressão Gênica , Luz , Transdução de Sinal Luminoso , Lisossomos/metabolismo , Fusão de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Rodopsina/metabolismoRESUMO
The rod cell has an extraordinarily specialized structure that allows it to carry out its unique function of detecting individual photons of light. Both the structural features of the rod and the metabolic processes required for highly amplified light detection seem to have rendered the rod especially sensitive to structural and metabolic defects, so that a large number of gene defects are primarily associated with rod cell death and give rise to blinding retinal dystrophies. The structures of the rod, especially those of the sensory cilium known as the outer segment, have been the subject of structural, biochemical, and genetic analysis for many years, but the molecular bases for rod morphogenesis and for cell death in rod dystrophies are still poorly understood. Recent developments in imaging technology, such as cryo-electron tomography and super-resolution fluorescence microscopy, in gene sequencing technology, and in gene editing technology are rapidly leading to new breakthroughs in our understanding of these questions. A summary is presented of our current understanding of selected aspects of these questions, highlighting areas of uncertainty and contention as well as recent discoveries that provide new insights. Examples of structural data from emerging imaging technologies are presented.
Assuntos
Proteínas de Membrana/metabolismo , Morfogênese , Doenças Retinianas/diagnóstico , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Microscopia Crioeletrônica/métodos , Humanos , Doenças Retinianas/metabolismoRESUMO
Transient receptor potential melastatin-1 (TRPM1) is essential for the light-induced depolarization of retinal ON bipolar cells. TRPM1 likely forms a multimeric channel complex, although almost nothing is known about the structure or subunit composition of channels formed by TRPM1 or any of its close relatives. Recombinant TRPM1 was robustly expressed in insect cells, but only a small fraction was localized to the plasma membrane. Similar intracellular localization was observed when TRPM1 was heterologously expressed in mammalian cells. TRPM1 was affinity-purified from Sf9 cells and complexed with amphipol, followed by detergent removal. In blue native gels and size exclusion chromatography, TRPM1 migrated with a mobility consistent with detergent- or amphipol-bound dimers. Cross-linking experiments were also consistent with a dimeric subunit stoichiometry, and cryoelectron microscopy and single particle analysis without symmetry imposition yielded a model with approximate 2-fold symmetrical features. Finally, electron microscopy of TRPM1-antibody complexes revealed a large particle that can accommodate TRPM1 and two antibody molecules. Taken together, these data indicate that purified TRPM1 is mostly dimeric. The three-dimensional structure of TRPM1 dimers is characterized by a small putative transmembrane domain and a larger domain with a hollow cavity. Blue native gels of solubilized mouse retina indicate that TRPM1 is present in two distinct complexes: one similar in size to the recombinant protein and one much larger. Because dimers are likely not functional ion channels, these results suggest that additional partner subunits participate in forming the transduction channel required for dim light vision and the ON pathway.
Assuntos
Proteínas do Olho , Multimerização Proteica/fisiologia , Canais de Cátion TRPM , Visão Ocular/fisiologia , Animais , Proteínas do Olho/química , Proteínas do Olho/genética , Proteínas do Olho/isolamento & purificação , Proteínas do Olho/metabolismo , Células HEK293 , Humanos , Camundongos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Canais de Cátion TRPM/química , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/isolamento & purificação , Canais de Cátion TRPM/metabolismoRESUMO
Our recent studies have shown that endogenous zinc, co-released with glutamate from the synaptic terminals of vertebrate retinal photoreceptors, provides a feedback mechanism that reduces calcium entry and the concomitant vesicular release of glutamate. We hypothesized that zinc feedback may serve to protect the retina from glutamate excitotoxicity, and conducted in vivo experiments on the retina of the skate (Raja erinacea) to determine the effects of removing endogenous zinc by chelation. These studies showed that removal of zinc by injecting the zinc chelator histidine results in inner retinal damage similar to that induced by the glutamate receptor agonist kainic acid. In contrast, when an equimolar quantity of zinc followed the injection of histidine, the retinal cells were unaffected. Our results are a good indication that zinc, co-released with glutamate by photoreceptors, provides an auto-feedback system that plays an important cytoprotective role in the retina.
Assuntos
Sobrevivência Celular/fisiologia , Retina/fisiologia , Rajidae/fisiologia , Zinco/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Quelantes/farmacologia , Adaptação à Escuridão/efeitos dos fármacos , Adaptação à Escuridão/fisiologia , Interpretação Estatística de Dados , Agonistas de Aminoácidos Excitatórios/farmacologia , Olho/citologia , Ácido Glutâmico/metabolismo , Histidina/toxicidade , Ácido Caínico/farmacologia , Necrose , Células Fotorreceptoras de Vertebrados/fisiologia , Retina/efeitos dos fármacos , Zinco/metabolismoRESUMO
There is abundant experimental evidence that zinc ions (Zn(2+)) are present in the synaptic vesicles of vertebrate photoreceptors, and that they are co-released with glutamate. Here we show that increasing the concentration of extracellular zinc (2 µM-2 mM) suppresses the entry of calcium into the synaptic terminals of isolated salamander double cones. The resultant dose-dependent curve was fit by an inverse Hill equation having an IC50 of 38 µM, and Hill coefficient of 1.1. Because there is currently no reliable way to measure the concentration of extracellular zinc, it is not known whether the zinc released under normal circumstances is of physiological significance. In an attempt to circumvent this problem we used zinc chelators to reduce the available pool of endogenous zinc. This enabled us to determine how the absence of zinc affected calcium entry. We found that when intra- or extra-cellular zinc was chelated by 250 µM of membrane-permeable TPEN or 500 µM of membrane-impermeable histidine, there was a significant rise in the depolarization-induced intracellular calcium level within photoreceptor terminals. This increase in internal [Ca(2+)] will undoubtedly lead to a concomitant increase in glutamate release. In addition, we found that blocking the L-type calcium channels that are expressed on the synaptic terminals of photoreceptors with 50 µM nicardipine or 100 µM verapamil abolished the effects of zinc chelation. These findings are a good indication that, when released in vivo, the zinc concentration is sufficient to suppress voltage-gated calcium channels, and reduce the rate of glutamate release from photoreceptor terminals.
Assuntos
Cálcio/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Zinco/farmacologia , Ambystoma , Compostos de Anilina/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Quelantes/farmacologia , Relação Dose-Resposta a Droga , Etilenodiaminas/farmacologia , Corantes Fluorescentes/metabolismo , Ácido Glutâmico/metabolismo , Histidina/farmacologia , Microscopia de Fluorescência , Terminações Pré-Sinápticas/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Xantenos/metabolismoRESUMO
There is mounting evidence that zinc release from glutamatergic nerve terminals serves as a neuromodulator at synaptic sites within the retina and CNS. However, it has not been possible to reliably measure the concentration of zinc co-released with glutamate in the confines of the synaptic cleft. Thus, much of the evidence supporting this view derives from electrophysiological studies showing the modulatory effects of exogenous zinc on the membrane currents of ligand- and voltage-gated channels. In the present study, we took advantage of the unique properties of the glutamatergic photoreceptor terminal to demonstrate a feedback signal mediated by endogenous zinc at the synaptic sites from which it is discharged. Through its ability to block voltage-gated calcium channels in the photoreceptor terminal, zinc suppresses the radial dark current of the visual cell, and reduces its release of glutamate. It follows that chelation of extracellular zinc, e.g., with histidine, will lead to an increase both in the dark current and in the release of glutamate, changes that result in an enhancement of the light-evoked a-wave of the ERG and can account for the b-wave enhancement observed previously after zinc chelation when inner retinal responses were not blocked by aspartate.
Assuntos
Terminações Pré-Sinápticas/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Zinco/fisiologia , Animais , Eletrorretinografia , Retroalimentação Fisiológica/fisiologia , RajidaeRESUMO
The nucleated thrombocytes of non-mammalian vertebrates are partially flattened, ovoid cells morphologically distinct from mammalian platelets, and the extent of their functional equivalence is unknown. To test whether they resemble platelets in having similar F-actin-based post-activation stages, rapid fixation/extraction/labeling methods were developed to reveal cytoskeletal organization in dogfish thrombocytes by confocal microscopy. Unactivated cells contained cortical F-actin plus denser F-actin co-localizing with outer marginal band (MB) microtubules. In the post-activation sequence, determined for the first time by continuous observation of individual thrombocytes following thrombin perfusion, cells rounded and blebbed, spread, and eventually flattened extensively. The MB twisted and then became disorganized, with microtubule bundles remaining centrally located and associated with nuclear clefts. In contrast, F-actin occupied blebs and outward-spreading cytoplasm, initially in spiky projections, then predominantly in stress fibers, and inhibitors of F-actin assembly or myosin ATPase blocked shape changes. Thus, the post-activation stages and cytoskeletal events observed in nucleated thrombocytes were found to parallel those of platelets.