RESUMO
Rod-specific photoreceptor dystrophies are complicated by the delayed death of genetically normal neighboring cones. In transgenic (Tg) swine with a rod-specific (rhodopsin) gene mutation, cone photoreceptor physiology was normal for months but later declined, consistent with delayed cone cell death. Surprisingly, cone postreceptoral function was markedly abnormal when cone photoreceptor physiology was still normal. The defect was localized to hyperpolarizing cells postsynaptic to the middle wavelength-sensitive cones. Recordings throughout postnatal development indicated a failure of cone circuitry maturation, a novel mechanism of secondary cone abnormality in rod dystrophy. The results have implications for therapy for human retinal dystrophies and raise the possibility that rod afferent activity plays a role in the postnatal maturation of cone retinal circuitry.
Assuntos
Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Rodopsina/genética , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados , Adaptação à Escuridão/fisiologia , Eletrorretinografia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Potenciais da Membrana/fisiologia , Vias Neurais , Retinose Pigmentar/genética , Retinose Pigmentar/fisiopatologia , Suínos , Sinapses/fisiologiaRESUMO
During their complex life history, anguilliform eels go through a major metamorphosis when developing from a fresh water yellow eel into a deep-sea silver eel. In addition to major changes in body morphology, the visual system also adapts from a fresh water teleost duplex retina with rods and cones, to a specialized deep-sea retina containing only rods. The history of the rods is well documented with an initial switch from a porphyropsin to a rhodopsin (P523(2) to P501(1)) and then a total change in gene expression with the down regulation of a "freshwater" opsin and its concomitant replacement by the expression of a typical "deep-sea" opsin (P501(1) to P482(1)). Yellow eels possess only two spectral classes of single cones, one sensitive in the green presumably expressing an RH2 opsin gene and the second sensitive in the blue expressing an SWS2 opsin gene. In immature glass eels, entering into rivers from the sea, the cones contain mixtures of rhodopsins and porphyropsins, whereas the fully freshwater yellow eels have cone pigments that are almost pure porphyropsins with peak sensitivities at about 540-545 nm and 435-440 nm, respectively. However, during the early stages of metamorphosis, the pigments switch to rhodopsins with the maximum sensitivity of the "green"-sensitive cone shifting to about 525 nm, somewhat paralleling, but preceding the change in rods. During metamorphosis, the cones are almost completely lost.
Assuntos
Percepção de Cores/fisiologia , Enguias/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Pigmentos da Retina/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Animais , Metamorfose Biológica , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/crescimento & desenvolvimento , EspectrofotometriaRESUMO
The tiger salamander lives in shallow water with bright light in the aquatic phase, and in dim tunnels or caves in the terrestrial phase. In the aquatic phase, there are five types of photoreceptors--two types of rods and three types of cones. Our previous studies showed that the green rods and blue-sensitive cones contain the same visual pigment and have the same absorbance spectra; however, the green rods have a larger photon-catch area and thus have higher light sensitivity than the blue-sensitive cones. Here we show that after metamorphosis, the terrestrial salamander looses the blue-sensitive cones, while the density of the green rods increases. Moreover, the size of the green rod outer segments is increased in the terrestrial phase, compared to that in the aquatic phase. This switch from the blue-sensitive cones to the green rods may represent an adaptation to the dim light environment of the terrestrial phase.
Assuntos
Células Fotorreceptoras de Vertebrados/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Animais , Metamorfose Biológica , Camundongos , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Coelhos , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Pigmentos da Retina , Raios Ultravioleta , ÁguaRESUMO
EGF receptor and Notch signaling are involved in a wide variety of developmental processes. A new study has revealed a serial linkage between them, via Ebi and Strawberry Notch, which is important in determining the cone cell fate in the Drosophila eye.
Assuntos
Drosophila/citologia , Proteínas de Membrana/metabolismo , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Transdução de Sinais , Animais , Linhagem da Célula , Proteínas de Drosophila , Receptores NotchRESUMO
Color vision requires the expression of opsin photopigments with different wavelength sensitivities in retinal cone photoreceptors. The basic color visual system of mammals is dichromatic, involving differential expression in the cone population of two opsins with sensitivity to short (S, blue) or medium (M, green) wavelengths. However, little is known of the factors that directly activate these opsin genes and thereby contribute to the S or M opsin identity of the cone. We report that the orphan nuclear receptor RORbeta (retinoid-related orphan receptor beta) activates the S opsin gene (Opn1sw) through binding sites upstream of the gene. RORbeta lacks a known physiological ligand and activates the Opn1sw promoter modestly alone but strongly in synergy with the retinal cone-rod homeobox factor (CRX), suggesting a cooperative means of enhancing RORbeta activity. Comparison of wild-type and mutant lacZ reporter transgenes showed that the RORbeta-binding sites in Opn1sw are required for expression in mouse retina. RORbeta-deficient mice fail to induce S opsin appropriately during postnatal cone development. Photoreceptors in these mice also lack outer segments, indicating additional functions for RORbeta in photoreceptor morphological maturation. The results identify Opn1sw as a target gene for RORbeta and suggest a key role for RORbeta in regulating opsin expression in the color visual system.
Assuntos
Regulação da Expressão Gênica , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Células Fotorreceptoras Retinianas Cones/embriologia , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Opsinas de Bastonetes/genética , Animais , Células Cultivadas , Proteínas de Ligação a DNA , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Transgênicos , Membro 2 do Grupo F da Subfamília 1 de Receptores Nucleares , Estrutura Terciária de Proteína , Elementos de Resposta , Retina/embriologia , Retina/metabolismo , Opsinas de Bastonetes/metabolismo , Homologia de Sequência do Ácido Nucleico , Transativadores/metabolismo , TransgenesRESUMO
Although abundant structural plasticity in the form of axonal retraction, neurite extension, and formation of presynaptic varicosities is displayed by photoreceptors after retinal detachment and during genetic and age-related retinal degeneration, the mechanisms involved are mostly unknown. We demonstrated recently that Ca(2+) influx through cGMP-gated channels in cones and voltage-gated L-type channels in rods is required for neurite extension in vitro (Zhang and Townes-Anderson, 2002). Here, we report that the nitric oxide (NO)-cGMP signaling pathway is active in photoreceptors and that its manipulation differentially regulates the structural plasticity of cone and rod cells. The NO receptor soluble guanylyl cyclase (sGC) was detected immunocytochemically in both cone and rod cells. Stimulation of sGC increased cGMP production in retinal cultures. In cone cells, quantitative analysis showed that NO or cGMP stimulated neuritic sprouting; this stimulatory effect was dependent on both Ca2+ influx through cGMP-gated channels and phosphorylation by protein kinase G (PKG). At the highest levels of cGMP, however, cone outgrowth was no longer increased. In rod photoreceptors, NO or cGMP consistently inhibited neuritic growth in a dose-dependent manner; this inhibitory effect required PKG. When NO-cGMP signaling was inhibited, changes in the neuritic development of cone and rod cells were also observed but in the opposite direction. These results expand the role of cGMP in axonal activity to adult neuritogenesis and suggest an explanation for the neurite sprouting observed in an autosomal recessive form of retinitis pigmentosa that is characterized by high cGMP levels in photoreceptor layers.
Assuntos
GMP Cíclico/fisiologia , Plasticidade Neuronal/fisiologia , Óxido Nítrico/fisiologia , Terminações Pré-Sinápticas/fisiologia , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Bastonetes/crescimento & desenvolvimento , Ambystoma , Animais , Células Cultivadas , GMP Cíclico/agonistas , Inibidores Enzimáticos/farmacologia , Óxido Nítrico/agonistas , Óxido Nítrico/antagonistas & inibidores , Doadores de Óxido Nítrico/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Ultraviolet-sensitive (UVS) cones disappear from the retina of salmonid fishes during a metamorphosis that prepares them for deeper/marine waters. UVS cones subsequently reappear in the retina near sexual maturation and the return migration to natal streams. Cellular mechanisms of this UVS cone ontogeny were investigated using electroretinograms, in situ hybridization, and immunohistochemistry against opsins during and after thyroid hormone (TH) treatments of rainbow trout (Oncorhynchus mykiss). Increasing TH levels led to UVS cone degeneration. Labeling demonstrated that UVS cone degeneration occurs via programmed cell death and caspase inhibitors can inhibit this death. After the cessation of TH treatment, UVS cones regenerated in the retina. Bromodeoxyuridine (BrdU) was applied after the termination of TH treatment and was detected in the nuclei of cells expressing UVS opsin. BrdU was found in UVS cones but not other cone types. The most parsimonious explanation for the data is that UVS cones degenerated and UVS cones were regenerated from intrinsic retinal progenitor cells. Regenerating UVS cones were functionally integrated such that they were able to elicit electrical responses from second-order neurons. This is the first report of cones regenerating during natural development. Both the death and regeneration of cones in retinae represent novel mechanisms for tuning visual systems to new visual tasks or environments.
Assuntos
Metamorfose Biológica , Oncorhynchus mykiss , Regeneração , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/fisiologia , Animais , Eletrorretinografia , Imuno-Histoquímica , Hibridização In Situ , Oncorhynchus mykiss/anatomia & histologia , Oncorhynchus mykiss/crescimento & desenvolvimento , Retina/anatomia & histologia , Retina/patologia , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Opsinas de Bastonetes/metabolismo , Células-Tronco/citologia , Células-Tronco/fisiologia , Hormônios Tireóideos/farmacologia , Raios UltravioletaRESUMO
Despite the great variety in chicken photoreceptors, existing morphogenetic studies only deal with two types: rods and cones. We have therefore examined by scanning electron microscopy the first appearance and maturation of different retinal photoreceptors in 36 chicken embryos (Gallus domesticus), aged 5-19 days prehatching. On day 5 of incubation, chicken retinae were only composed of proliferating ventricular cells devoid of photoreceptors. On day 8, outer mitotic cells were separated from inner differentiating photoreceptors, by the transient layer of Chievitz. Ball-like protrusions appeared at the ventricular surface, representing the first signs of photoreceptor inner segment formation. From day 10 onward, double cones, single cones, and rods could be clearly distinguished, and occasional cilia were detected at their tip. On day 12, inner segments had increased in length and diameter, and frequently carried a cilium representing the beginning of outer segment formation. On day 14, most photoreceptors displayed a distinct outer segment. On day 19, photoreceptors had essentially assumed adult morphology. Based on the shape of their outer segments, two subtypes of cones and three subtypes of double cones could be distinguished. Throughout development, we observed microvilli close to maturing photoreceptors, either originating from their lateral sides, from their tip, or from Müller cells. Microvillus density peaked between day 12 and 14, indicating an important role in photoreceptor morphogenesis. Unilateral occlusion of the eyes of posthatching chicken reduced the proportion of double cones to single cones in the retina, indicating dependence of retinal morphogenesis upon functional activity of visual cells.
Assuntos
Morfogênese , Células Fotorreceptoras Retinianas Cones/embriologia , Células Fotorreceptoras Retinianas Bastonetes/embriologia , Ambliopia/fisiopatologia , Animais , Embrião de Galinha , Galinhas , Cílios/ultraestrutura , Microscopia Eletrônica de Varredura , Microvilosidades/ultraestrutura , Fenômenos Fisiológicos Oculares , Retina/embriologia , Retina/crescimento & desenvolvimento , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Segmento Externo da Célula Bastonete/embriologia , Segmento Externo da Célula Bastonete/crescimento & desenvolvimento , Segmento Externo da Célula Bastonete/ultraestrutura , Fatores de TempoRESUMO
We investigated the effects of photoreceptor degeneration on the anatomy and physiology of inner retinal neurons in a mouse model of retinitis pigmentosa, the retinal degeneration (rd) mutant mouse. Although there is a general assumption that the inner retinal cells do not suffer from photoreceptor death, we confirmed major changes both accompanying and after this process. Changes include sprouting of horizontal cells, lack of development of dendrites of rod bipolar cells, and progressive atrophy of dendrites in cone bipolar cells. Electrophysiological recordings demonstrate a selective impairment of second-order neurons that is not predictable on the basis of a pure photoreceptor dysfunction. Our data point out the necessity to prove integrity of the inner retina before attempting restoring visual function through photoreceptor intervention. This is even more important when considering that although intervention can be performed before the onset of any symptoms in animals carrying inherited retinopathies, this is obviously not true for human subjects.
Assuntos
Células Fotorreceptoras Retinianas Cones/anormalidades , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/anormalidades , Retinose Pigmentar/patologia , Retinose Pigmentar/fisiopatologia , Animais , Axônios/ultraestrutura , Dendritos/ultraestrutura , Eletrorretinografia , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Retina/citologia , Retina/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Bastonetes/ultraestruturaRESUMO
An antibody against recoverin, the calcium-binding protein, labels photoreceptors, cone bipolar cells, and a subpopulation of cells in the ganglion cell layer. In the present study, we sought to establish the origin and identity of the cells expressing recoverin in the ganglion cell layer of the rat retina. By double labeling with rhodopsin, we demonstrate that early in development some of the recoverin-positive cells in the ganglion cell layer are photoreceptors. During the first postnatal week, these rhodopsin-positive cells are eliminated from the ganglion cell layer, but such neurons remain in the inner nuclear layer well into the first postnatal month. Another contingent of recoverin-positive cells, with morphological features equivalent to those of bipolar cells, is present in the postnatal retina, and approximately 50% of these neurons survive to maturity. The incidence of such cells in the ganglion cell layer was not affected by early transection of the optic nerve, a manipulation that causes rapid loss of retinal ganglion cells. These recoverin-positive cells were not double-labeled by cell-specific markers expressed by photoreceptors, rod bipolar cells, or horizontal and amacrine cells. Based on their staining with recoverin and salient morphological features, these ectopic profiles in the ganglion cell layer are most likely cone bipolar cells. Collectively, the results provide evidence for photoreceptors in the ganglion cell and inner nuclear layers of the developing retina, and a more permanent subpopulation of cone bipolar cells displaced to the ganglion cell layer.
Assuntos
Proteínas do Olho , Lipoproteínas , Proteínas do Tecido Nervoso , Células Fotorreceptoras de Vertebrados/citologia , Retina/embriologia , Retina/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/citologia , Células Ganglionares da Retina/citologia , Animais , Proteínas de Ligação ao Cálcio/análise , Hipocalcina , Imuno-Histoquímica , Microscopia Confocal , Células Fotorreceptoras de Vertebrados/química , Ratos , Ratos Long-Evans , Recoverina , Células Fotorreceptoras Retinianas Cones/embriologia , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Células Ganglionares da Retina/química , Rodopsina/análiseRESUMO
PURPOSE: To assess cone photoreceptor and cone-mediated postreceptoral retinal function in infants. METHODS: ERG responses to a 1.8-log unit range of long-wavelength flashes on a white, rod-saturating background were recorded in 4-week-old (n = 22) and 10-week-old (n = 28) infants and control adults and children, 8 to 40 years of age (n = 13). A model of the activation of cone phototransduction was fit to the a-waves. Sensitivity (S(CONE)) and saturated-response amplitude (R(CONE)) were calculated. The amplitude and implicit time of the b-wave were examined as a function of stimulus intensity. The cone photoresponse parameters were compared to the rod photoresponse parameters (S(ROD) and R(ROD)) in the same subjects. RESULTS: S(CONE) and R(CONE) in infants were significantly smaller than in the mature control subjects. The mean S(CONE) was 64% and 68%, and the mean R(CONE) was 63% and 72% in 4- and 10-week-olds, respectively. The mean rod photoresponse parameters were considerably less mature, as the mean S(ROD) was 35% and 46%, and the mean R(ROD) was 39% and 43% of mature values at 4 and 10 weeks. The b-wave stimulus-response functions in the 4- and 10-week-old infants did not show the photopic hill that was characteristic of the children's and adults' photopic b-waves. CONCLUSIONS: Peripheral cone function is relatively more mature than rod function in young infants. The lack of a photopic hill is hypothesized to result from immaturity in the relative contributions of ON and OFF bipolar cell responses.
Assuntos
Eletrorretinografia , Fenômenos Fisiológicos Oculares , Células Fotorreceptoras Retinianas Cones/fisiologia , Adolescente , Adulto , Envelhecimento/fisiologia , Criança , Humanos , Lactente , Estimulação Luminosa , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Bastonetes/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Bastonetes/fisiologiaRESUMO
In mammals, each cone had been thought to contain only one single type of photopigment. It was not until the early 1990s that photopigment coexpression was reported. In the house mouse, the distribution of color cones shows a characteristic division. Whereas in the upper retinal field the ratio of short wave to middle-to-long wave cones falls in the usual range (1:10), in the ventral retinal field M/L-pigment expression is completely missing. In the transitional zone, numerous dual cones are detectable (spatial coexpression). In other species without retinal division, dual cones appear during development, suggesting that M/L-cones develop from S-cones. Dual elements represent a transitory stage in M/L-cone differentiation that disappear with maturation (transitory coexpression). These two phenomena seem to be mutually exclusive in the species studied so far. In the comparative part of this report the retinal cone distribution of eight rodent species is reported. In two species dual cones appear in adult specimens without retinal division, and dual elements either occupy the dorsal peripheral retina, or make up the entire cone population. This is the first observation proving that all cones of a retina are of dual nature. These species are good models for the study of molecular control of opsin expression and renders them suitable sources of dual cones for investigations on the role and neural connections of this peculiar cone type. In the developmental part, the retinal maturation of other species is examined to test the hypothesis of transitory coexpression. In these species S-pigment expression precedes that of the M/L-pigment, but dual cones are either identified in a small number or they are completely missing from the developing retina. These results exclude a common mechanism for M/L-cone maturation: they either transdifferentiate from S-cones or develop independently.
Assuntos
Mamíferos/crescimento & desenvolvimento , Mamíferos/metabolismo , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/metabolismo , Pigmentos da Retina/metabolismo , Animais , Humanos , Imuno-Histoquímica , Mamíferos/anatomia & histologia , Camundongos , Células Fotorreceptoras de Vertebrados/metabolismo , Primatas , Coelhos , Retina/anatomia & histologia , Retina/crescimento & desenvolvimento , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/anatomia & histologia , Roedores , Especificidade da Espécie , TupaiidaeRESUMO
During the last decade, numerous research reports have considerably improved our knowledge about the physiopathology of retinal degenerations. Three non-mutually exclusive general areas dealing with therapeutic approaches have been proposed; gene therapy, pharmacology and retinal transplantations. The first approach involving correction of the initial mutation, will need a great deal of time and further development before becoming a therapeutic tool in human clinical practice. The observation that cone photoreceptors, even those seemingly unaffected by any described anomaly, die secondarily to rod disappearance related to mutations expressed specifically in the latter, led us to study the interactions between these two photoreceptor populations to search for possible causal links between rod degeneration and cone death. These in vivo and in vitro studies suggest that paracrine interactions between both cell types exist and that rods are necessary for continued cone survival. Since the role of cones in visual perception is essential, pending the identification of the factors mediating these interactions underway, rod replacement by transplantation and/or neuroprotection by trophic factors or alternative pharmacological means appear as promising approaches for limiting secondary cone loss in currently untreatable blinding conditions.
Assuntos
Comunicação Celular/fisiologia , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Animais , Diferenciação Celular , Transplante de Células , Humanos , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Degeneração Retiniana/fisiopatologia , Degeneração Retiniana/cirurgia , Células Fotorreceptoras Retinianas Bastonetes/crescimento & desenvolvimento , Transdução de Sinais/fisiologiaRESUMO
Mouse retinal photoreceptor cell generation and morphogenesis take place in a well-characterized temporal sequence. Both rod and cone photoreceptor differentiation and synaptogenesis occur postnatally, but the relative timing of these events has been difficult to document due to the paucity of cell-specific markers. We have found that antibodies to neuron-specific enolase (NSE) preferentially label a subpopulation of photoreceptors in the outer nuclear layer (ONL) of the mouse retina in addition to labeling ganglion, amacrine, bipolar, and horizontal cells within the inner layers of the retina. The appearance of NSE immunoreactivity in the different classes of retinal neurons during development showed a close temporal relationship to the onset of expression of the synaptic vesicle-associated protein SV2 and clearly preceded the sequential development of synaptic connections in both inner and outer synaptic layers. The NSE-immunoreactive photoreceptors were identified as cones by dual labeling of their inner segments with the lectin peanut agglutinin or by colabeling with antisera to cone photopigments. Axonal extensions of NSE-labeled cone cells were shown to interact with those of differentiating horizontal cells as early as postnatal day 3 (P3). Colocalization of NSE with SV2 indicated that cone cells began to make synaptic contacts with horizontal cell processes several days prior to the development of rod synaptic terminals. Between P4 and P11, cone photoreceptor cell nuclei were observed to be scattered at various levels throughout the ONL and thus appeared to have become displaced from their previous position directly beneath the outer limiting membrane (OLM). By P12, the cone nuclei had migrated sclerad once again and were now observed to be neatly aligned adjacent to the OLM. In the rd mouse mutant, this migratory process was delayed, so that, at P12, positioning of the cone cell nuclei within the ONL was still quite irregular. Thus, we have identified a late migratory phase for cone photoreceptors during the second week after birth that correlates with the timing of maturation of the rod synaptic terminals just prior to eye opening. The types of cues used by maturing cone cells for their eventual sclerad location remain to be elucidated.
Assuntos
Células Fotorreceptoras Retinianas Cones/fisiologia , Sinapses/fisiologia , Animais , Movimento Celular , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Morfogênese , Aglutinina de Amendoim , Fosfopiruvato Hidratase/análise , Fosfopiruvato Hidratase/biossíntese , Retina/citologia , Retina/crescimento & desenvolvimento , Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Células Ganglionares da Retina/citologia , Sinapses/ultraestruturaRESUMO
In order to facilitate emerging models of retinal development, we developed electroretinogram and in situ hybridization protocols to examine the ontogeny of photoreceptors in the retina of a land-locked salmonid, the rainbow trout (Oncorhynchus mykiss). We cloned cDNA fragments corresponding to the rod opsin and each of the four cone opsin gene families, which we utilized to produce riboprobes. We established the specificity of the in situ hybridization protocol by examining subcellular signal localization and through double-labeling experiments. We confirm the assumption that the accessory corner cones in the square mosaic are the ultraviolet wavelength-sensitive (UVS) cone photoreceptor (i.e., they express an SWS1 opsin) and observed UVS cones throughout the retina of small trout. Larger fish have a decrease in sensitivity to short wavelength light stimuli and the distribution of UVS cones in the mature retina is limited to the dorsal-temporal quadrant. These larger fish also possess differentiated UVS cones in the peripheral germinal zone (PGZ), including within areas peripheral to mature retina lacking UVS cones. These data are consistent with the loss of putative UVS cones from the PGZ of a migratory salmonid of another genus, and thus the disappearance of UVS cones appears to be general to the Family Salmonidae, regardless of life history strategy. The generation, differentiation, and subsequent loss of UVS cones in the smolt PGZ is a dramatic example of the supposition that the mechanisms of PGZ development recapitulate the retinal embryogenesis of that species.
Assuntos
Oncorhynchus mykiss/fisiologia , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/efeitos da radiação , Raios Ultravioleta , Envelhecimento/fisiologia , Animais , Eletrorretinografia , Imuno-Histoquímica , Hibridização In Situ , Oncorhynchus mykiss/anatomia & histologia , Oncorhynchus mykiss/crescimento & desenvolvimento , RNA Mensageiro/metabolismo , Retina/anatomia & histologia , Retina/crescimento & desenvolvimento , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/anatomia & histologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismoRESUMO
PURPOSE: The authors have established an organ culture method in which the the postnatal development and the structural integrity of the mouse retina can be maintained for at least 6 weeks. Additionally, they have examined the emergence and in vitro morphogenesis of the photoreceptors and the development of insoluble components of the interphotoreceptor matrix. METHODS: Neural retinas and retinal pigment epithelia from 48-hour-old C3H ++/++ mice were cultured. At various ages, the tissues were fixed and cryosectioned or wholemounted. Photoreceptor development was studied by immunocytochemistry with visual pigment antibodies and by lectin cytochemistry. The ultrastructure of the photoreceptors was studied by electron microscopy. RESULTS: Immunopositive rods and short-wave sensitive cones were detectable as early as 3 days after explantation. From this time on, matrix domains around cones were also identifiable and labelled with peanut agglutinin lectin. However, the antibody specific to the middle-wave sensitive cone pigment failed to recognize any cones throughout the 6-week culture period. CONCLUSIONS: Both basic photoreceptor types appeared and developed in this organ culture system according to a timetable comparable to normal in vivo development. Surprisingly, under these circumstances, one of the two cone pigments was not expressed by any photoreceptors.
Assuntos
Células Fotorreceptoras/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Histocitoquímica , Camundongos , Camundongos Endogâmicos C3H , Morfogênese , Técnicas de Cultura de Órgãos , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo , Epitélio Pigmentado Ocular/fisiologia , Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Pigmentos da Retina/metabolismoRESUMO
PURPOSE: To characterize morphologic alterations in the retina of the visual mutant zebrafish gantenbein (gnn) and to examine whether these alterations correlate with those present in human hereditary eye diseases. METHODS: The gnn mutant was isolated by behavioral and macroscopic screening. Retinas of gnn zebrafish larvae were examined at different developmental stages from 2 to 9 days postfertilization (dpf) by standard histologic staining techniques and by immunocytochemistry. Ultrastructural alterations were examined by electron microscopy. The genetic map position of the induced mutation was identified by mapping with two candidate primer pairs on single larvae. RESULTS: The gnn mutant exhibited shortened outer photoreceptor segments and altered RPE morphology. In the photoreceptor layer of the mutant, the total number of lectin-labeled cones was reduced in all developmental stages from 2 to 7 dpf, whereas the amount of rhodopsin-positive cells remained at the wild-type (WT) level. Labeling with zebrafish opsin antibodies revealed dystrophic red cones at 5 dpf, whereas the morphology of all other cone types was largely unaffected. Electron microscopy unveiled electron-dense deposits between the discs of the double cone outer segments. In addition, the onset of progressive RPE degeneration was observed at this stage of development. At later stages, all cone types and the RPE became degenerative. The morphology of distinct second-order neurons remained largely unaffected by the mutation. The gnn mutation was located approximately 4.3 cM from the simple sequence length polymorphism (SSLP) marker Z15453 on linkage group 16. CONCLUSIONS: In gnn mutant zebrafish, cones, and especially red cones, are dystrophic in early retinal development. Subsequent to this cone dystrophy, the RPE becomes dysfunctional and starts to degenerate in later stages of development. Thus, the early developmental morphology of gnn exhibits similarities to cone dystrophies most commonly seen in age-related macular degeneration (AMD) among humans, whereas the later stages of degeneration in gnn resemble RPE alterations in retinitis pigmentosa (RP) in humans. The gnn zebrafish mutant may therefore be a useful model for examining the possible interplay and connection between cone dystrophy and RPE degeneration.
Assuntos
Epitélio Pigmentado Ocular/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Peixe-Zebra/genética , Animais , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Masculino , Mutagênese , Neurônios/ultraestrutura , Células Fotorreceptoras de Vertebrados/ultraestrutura , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/metabolismo , Opsinas de Bastonetes/metabolismoRESUMO
PURPOSE: To measure the concentrations of polyamines, determine their cellular and subcellular localization, and analyze effects of their depletion in developing rabbit retina. METHODS: Isolated retinas at different developmental stages were analyzed for polyamine content by high-performance liquid chromatography (HPLC). An antibody against polyamines was used to localize endogenous stores in both freshly harvested retinas and neonatal retinal explants. To determine the effects of polyamine depletion on immature retina, neonatal explants were cultured in the presence or absence of alpha-difluoromethylornithine (DFMO), an inhibitor of the polyamine synthetic enzyme ornithine decarboxylase (ODC). Similar studies were also performed on dissociated cell cultures. Tissue was assessed using standard histologic stains as well as cell-specific markers (peanut agglutinin for cone photoreceptors and calbindin for horizontal cells). RESULTS: Retinal polyamine content was highest at birth, remained relatively high during the first postnatal week, and then steadily decreased to adult levels. At all ages analyzed, spermine concentration was higher than putrescine or spermidine; however, the differential was greatest in the adult. Polyamine immunoreactivity was localized to distal processes of both rods and cones during development. Strong immunoreactivity was maintained in adult cone inner and outer segments; comparatively weak staining was observed in the adult rods. Heavy staining of ganglion cells was present throughout development but was localized in the cytoplasm in immature cells and in the nucleus in the adult. Amacrine cells stained only in the adult. Polyamine depletion caused a disruption of immature cones, evident in the loss of their somata in the outer nuclear layer, in their processes in the outer plexiform layer in retinal explants, and in their decreased association with horizontal cells in dissociated cell culture. CONCLUSIONS: The relatively high concentrations of polyamines in neonatal retina and their discrete localization in developing photoreceptor outer segments and ganglion cells suggests an important role for these compounds in development. The disruption of cone-specific markers in polyamine-depleted retinas indicates a specific reliance on polyamines for expression of normal cone morphology or morphologic development. These developmental effects may involve polyamine-sensitive ion channels, which are known to exist in retina, or direct interactions with specialized cytoskeletal elements within outer segments.
Assuntos
Poliaminas Biogênicas/fisiologia , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Poliaminas Biogênicas/análise , Calbindinas , Técnicas de Cultura de Células , Cromatografia Líquida de Alta Pressão , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Técnicas Imunoenzimáticas , Inibidores da Ornitina Descarboxilase , Coelhos , Retina/química , Retina/efeitos dos fármacos , Retina/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/química , Proteína G de Ligação ao Cálcio S100/metabolismo , Frações SubcelularesRESUMO
PURPOSE: To characterize the spatial and temporal expression of key structural and phototransduction cascade proteins in the monkey cone outer segment (OS). METHODS: Retinas from Macaca monkeys from ages fetal day (Fd) 89 through adulthood were double labeled using immunofluorescence for short (S) or long/medium (L/M) wavelength-sensitive cone opsin and either a structural protein (peripherin) or a phototransduction cascade protein (alpha-transducin [alpha-T], phosphodiesterase [PDE], or rhodopsin kinase [RK]). The spatial and temporal patterns of expression for each protein at each age were determined and graphed as a percentage of retinal coverage. RESULTS: In both cone types, opsins and phototransduction proteins appear first in the fovea and last at the retinal edge. Peripherin appears concomitantly with opsin in both S and L/M cones, but S cones express peripherin and opsin 1 to 3 weeks before neighboring L/M cones. Alpha-T, PDE, and RK are expressed together in the L/M cone OS shortly after L/M opsin appears. Phototransduction proteins are not expressed in S cones until 1 to 3 weeks after the appearance of S opsin and at the same time that neighboring cones are expressing both L/M opsin and phototransduction proteins. CONCLUSIONS: The concomitant appearance of opsin and peripherin strongly suggests roles in promoting the structural integrity of the developing OS. Phototransduction cascade proteins appear in the developing OS at the same time as one another, but after opsin. The significant lag between their expression and that of S cone opsin indicates that phototransduction proteins are not essential for OS formation, nor does opsin expression trigger their expression. The different temporal but similar spatial expression patterns of phototransduction proteins within S and L/M cones suggests that some local signal(s) coordinates their appearance.
Assuntos
Animais Recém-Nascidos/metabolismo , Proteínas do Olho/metabolismo , Feto/metabolismo , Macaca/embriologia , Macaca/crescimento & desenvolvimento , Glicoproteínas de Membrana , Células Fotorreceptoras Retinianas Cones/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Técnica Indireta de Fluorescência para Anticorpo , Receptor Quinase 1 Acoplada a Proteína G , Proteínas de Filamentos Intermediários/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Periferinas , Proteínas Quinases/metabolismo , Células Fotorreceptoras Retinianas Cones/embriologia , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Opsinas de Bastonetes/metabolismo , Transducina/metabolismo , Visão OcularRESUMO
PURPOSE: Normal function of the retina relies on the orderly stereotyped organization of different neurons and their synaptic connections. How such neural organization is patterned during development remains poorly understood due to the paucity of adequate developmental markers. This study was to examine the spatial organization and development of cone photoreceptors quantitatively in the mouse retina. METHODS: A transgenic approach was used to generate a living cone cell marker by driving GFP expression in mouse cones with the human red/green opsin gene 5' sequences. The spatial organization and development of the cones in the mouse retinas were examined quantitatively with epifluorescence and scanning laser confocal microscopy. Cone specific GFP expression in the developing retinas was verified with peanut agglutinin (PNA) staining. Developmental expression of mouse cone opsin genes was determined with RT-PCR. RESULTS: The fluorescent retinal cells expressing GFP can be visualized as early as on embryonic day E15. Following up morphological differentiation of these cells revealed features that were consistent with the typical morphology of the mouse cones. Double labeling with cone specific PNA showed that these cells were co-labeled starting from postnatal day P1, and that a subpopulation of PNA positive cones expressed the GFP. The fluorescent cell densities had a similar ventral and dorsal distribution from E15 to P2, increased dramatically in the ventral by P6, and in the dorsal from P7. Nearest neighbor distance analysis demonstrated that this subpopulation of cones was organized into a regular mosaic pattern with a regularity index of 4.82 in the central and 3.55 in the peripheral retina. Quantitative pattern assessment of the developing cones revealed that the fluorescent cells appeared to be distributed in a non-random array before birth. The regularity of the cone array began to rise on P7, in parallel with the onset of mouse green opsin gene expression and the development of cone pedicles. The regular pattern of cone mosaic organization was basically formed by P10, coinciding with the timing of the cone pedicle maturation. CONCLUSIONS: The cones in the mouse retina are organized in a regular mosaic pattern. Patterning the cone mosaic appears to follow a two phase developmental process involving regulated opsin gene expression and cone pedicle maturation: an early phase where a non-random array emerges during cone differentiation, and a late phase where the regular mosaic pattern is mature at the time when cone synaptic contacts are being formed.