RESUMEN
Currently much attention is focused on glutathione S transferase (GST)-induced suppression of apoptosis. The objective of our studies was therefore to see if GST isoenzymes rescue photoreceptors in retinal explants from rd1/rd1 mice, in which photoreceptors degenerate rapidly. Eyes from C3H rd1/rd1 and +/+ mice were collected at various time points between postnatal day (PN) 2 and PN28. Localization and content of alpha-GST and mu-GST was investigated by immunofluorescence and semi-quantitative Western blot analysis, respectively. In addition, PN2 and PN7 retinal explants were cultured till PN28, during which they were treated with 10 ng/ml alpha-GST or mu-GST. The spatiotemporal expression of both GST isoforms was closely similar: early presence in ganglion cell layer after which staining became restricted to Muller cells (particularly in the endfeet) and horizontal cell fibers in both rd1/rd1 and +/+. Doublets of alpha-GST and mu-GST were detected by Western blot analysis. Densitometry of these bands indicated steady reduction of alpha-GST content in rd1/rd1 retina starting from the second postnatal week. When alpha-GST and mu-GST were added exogenously to rd1/rd1 explants, photoreceptor rescue was produced that was more prominent in PN2 than in PN7 explants and more effective by alpha-GST than mu-GST. We propose that alpha-GST neuroprotection is mediated by reduction of tissue oxidative stress.
Asunto(s)
Glutatión Transferasa/metabolismo , Células Fotorreceptoras de Vertebrados/enzimología , Retina/enzimología , Degeneración Retiniana/enzimología , Animales , Western Blotting , Inmunohistoquímica , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos C3H , Técnicas de Cultivo de Órganos , Estrés Oxidativo/fisiología , Retina/citología , Retina/crecimiento & desarrollo , Degeneración Retiniana/genéticaRESUMEN
In neonatal retinal explants cultured long-term green cones are missing. Recently it was reported that thyroid hormone beta2 receptors (TR-beta2) are essential for these green cones to differentiate. Therefore transcript level of these receptors was investigated in our mouse retinal explants. However, thyroid receptors function as heterodimers with retinoid receptors (RR); so the fate of selected RRs was similarly analyzed using semi-quantitative RT-PCR. Loss of TR-beta2 and RR (RXR-gamma and ROR-beta2) mRNAs was observed after culturing the neonatal retina for 12 days. This indicates that these proteins are involved in determination of green cone identity. In addition, levels of the selected RR transcripts are differentially affected by short- or long-term culture. In the latter case an attached retinal pigment epithelium seems to play a protective role. Furthermore, divergent diurnal peaks of RR mRNAs are present in young as well as aged mouse retina and neocortex. This data might be relevant in the context of human ageing disorders.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Neocórtex/crecimiento & desarrollo , Receptores de Superficie Celular/genética , Receptores Citoplasmáticos y Nucleares , Receptores de Ácido Retinoico/genética , Receptores de Hormona Tiroidea/genética , Retina/crecimiento & desarrollo , Factores de Transcripción/genética , Envejecimiento/metabolismo , Animales , Diferenciación Celular/genética , Ritmo Circadiano/genética , Adaptación a la Oscuridad/genética , Regulación hacia Abajo/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C3H , Neocórtex/citología , Neocórtex/metabolismo , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Técnicas de Cultivo de Órganos/métodos , ARN Mensajero/metabolismo , Retina/citología , Retina/metabolismo , Receptor alfa de Ácido Retinoico , Receptores X Retinoide , Transcripción Genética/fisiología , Regulación hacia Arriba/genéticaRESUMEN
The anatomical substrates of the perception of motion have not yet been established in a detailed way on an individual level. The aim of this study was to develop a systematic procedure for mapping the visual cortex using Transcranial Magnetic Stimulation (TMS). The results showed that such an individual and detailed map of the spatial and temporal characteristics of motion perception can be constructed using TMS.
Asunto(s)
Mapeo Encefálico/métodos , Percepción de Movimiento/fisiología , Corteza Visual/fisiología , Adulto , Femenino , Humanos , Estimulación Luminosa/métodos , Corteza Visual/anatomía & histología , Percepción Visual/fisiologíaRESUMEN
The neonatal mouse retina remains viable as an explant in serum-supplemented growth media for more than 4 weeks. Interpretation of drug effects on this tissue is compromised by the enigmatic composition of the serum. We sought to remove this ambiguity by culturing neonatal as well as late postnatal mouse retina in serum-free nutrient medium. In this study three important observations were made, (1) there is histotypic development of neonatal as well as preservation of late postnatal mouse retinal structure during long-term culture in serum-free medium, although the late postnatal tissue tends to show some loss of cells in the outer nuclear layer. (2) Protein expression in explant photoreceptor cells was similar to that in the litter-matched ones, except for green cone opsin and interphotoreceptor retinoid-binding protein, although mRNA of the latter is present at similar amounts as in age-matched in vivo controls. (3) Cells of the inner retina stained by antibodies to calcium-binding proteins display some novel sprouting of processes. The results show that the mouse retina can be cultured as an explant for more than 4 weeks in a serum-free medium. This represents an important step forward because, (1) the possibility of interference of drug effects by unknown serum factors has been eliminated; and (2) the spent culture medium can be analyzed to investigate biomolecules released by the retina in vitro.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Medio de Cultivo Libre de Suero/farmacología , Proteínas del Ojo , Células Fotorreceptoras Retinianas Conos/citología , Animales , Anticuerpos , Calbindina 2 , Calbindinas , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colorantes , Eosina Amarillenta-(YS) , Técnica del Anticuerpo Fluorescente , Expresión Génica , Hematoxilina , Ratones , Parvalbúminas/análisis , Parvalbúminas/genética , Parvalbúminas/inmunología , ARN Mensajero/análisis , Células Fotorreceptoras Retinianas Conos/química , Células Fotorreceptoras Retinianas Conos/inmunología , Proteínas de Unión al Retinol/análisis , Proteínas de Unión al Retinol/genética , Proteínas de Unión al Retinol/inmunología , Rodopsina/análisis , Rodopsina/inmunología , Proteína G de Unión al Calcio S100/análisis , Proteína G de Unión al Calcio S100/genética , Proteína G de Unión al Calcio S100/inmunologíaRESUMEN
Lens epithelium derived growth factor (LEDGF) has been shown to rescue embryonic chick photoreceptor cells from serum starvation and heat stress, light damaged photoreceptor cells in Lewis rats, and photoreceptor cells in RCS rats. The aim of our study is to study the rescue effect of LEDGF on photoreceptor cells in the rd/rd mouse using our long-term serum free organ culture. At the end of this culture period of 21-26 days LEDGF treated rd mouse retina showed an increased photoreceptor survival compared to the untreated controls. LEDGF has no effect on expression and localization of opsin and arrestin in the rod photoreceptor cells when RPE is present. The protective potency of LEDGF on the retinal photoreceptor cells is similar to that of BDNF. LEDGF is known to activate heat shock proteins (Hsps) and the elevated Hsps are also reported to suppress apoptosis.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Degeneración Nerviosa/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Células Fotorreceptoras/efectos de los fármacos , Degeneración Retiniana/tratamiento farmacológico , Envejecimiento/efectos de los fármacos , Envejecimiento/fisiología , Animales , Animales Recién Nacidos , Arrestina/metabolismo , Supervivencia Celular/fisiología , Modelos Animales de Enfermedad , Genotipo , Sustancias de Crecimiento/metabolismo , Inmunohistoquímica , Ratones , Ratones Mutantes Neurológicos , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Técnicas de Cultivo de Órganos , Células Fotorreceptoras/crecimiento & desarrollo , Células Fotorreceptoras/metabolismo , Degeneración Retiniana/genética , Degeneración Retiniana/fisiopatología , Opsinas de Bastones/metabolismoRESUMEN
PURPOSE: Heme oxygenase (HO)-1 immunoreactivity (IR) was examined in normal untreated retina and in retinal explants after in vitro treatment with stress agents. METHODS: Enucleated eyes from young adult C3H mice were immediately fixed and cryosectioned and the retina sections processed for immunocytochemistry with antibodies against HO-1 and glial fibrillary acidic protein (GFAP). From other eyes retinas were isolated and maintained in organ culture, either untreated for 4 days maximum or for 21 hours during which the explants were treated the first 3 hours with selected doses of sodium arsenate or hydrogen peroxide. Thereafter, the explants were processed identically with the normal tissue. RESULTS: In the normal retina, HO-1 and GFAP IR was very low. The culturing itself resulted in an increase in both HO-1 and GFAP immunolabeling in Müller cells of explanted retinas. Both sodium arsenate and hydrogen peroxide further induced strong HO-1 IR in Müller cells but not in other retinal cells. In contrast to HO-1, GFAP staining in Müller cells was not altered as a result of treatment, either by sodium arsenate or hydrogen peroxide at any concentration used. CONCLUSIONS: The results show for the first time that HO-1 can be induced in the retina in vitro by conditions of oxidative stress and that enzyme expression is confined exclusively to Müller cells.
Asunto(s)
Células del Tejido Conectivo/enzimología , Hemo Oxigenasa (Desciclizante)/biosíntesis , Estrés Oxidativo , Retina/enzimología , Animales , Arseniatos/farmacología , Células del Tejido Conectivo/efectos de los fármacos , Inducción Enzimática , Proteína Ácida Fibrilar de la Glía/metabolismo , Hemo-Oxigenasa 1 , Peróxido de Hidrógeno/farmacología , Técnicas para Inmunoenzimas , Proteínas de la Membrana , Ratones , Ratones Endogámicos C3H , Técnicas de Cultivo de Órganos , Retina/efectos de los fármacosRESUMEN
PURPOSE: To gather information regarding the combination of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF), compared with the individual factors when used as a treatment to retard photoreceptor cell loss in rd mouse retina explants and to investigate the observation that the retinal pigment epithelium (RPE) influences rod differentiation by this treatment. METHODS: Postnatal day (PN)2 or PN7 control and rd mouse retinas were grown with attached retinal pigment epithelium (RPE). The explants were kept in culture up to PN28. During this culture period CNTF, BDNF, CNTF+BDNF, or vehicle were continuously administered to the culture medium. The nontrophic factors cyclosporin A and N:-CBZ-aspartic acid-glutamic acid-valine-aspartic acid-fluoromethyl ketone (z-DEVD-fmk) were also used. The number of photoreceptor nuclei remaining in the outer nuclear layer (ONL) was analyzed in hematoxylin and eosin-stained sections. Rod- and cone-specific antibodies were used to determine identity and state of differentiation of the photoreceptors. RESULTS: Compared with vehicle treatment, BDNF or CNTF resulted in 1.4- or 2-fold more surviving cell rows in the ONL, respectively. However, when CNTF and BDNF were applied together, surviving ONL cell counts in the rd explants were approximately 3 times those in vehicle-treated explants. In the presence of CNTF or CNTF+BDNF, opsin and arrestin expression in rods was decreased compared with rods without attached RPE. Cyclosporin A and z-DEVD-fmk did not show rescue of rd photoreceptor cells. CONCLUSIONS: CNTF or BDNF treatment of rd retinal explants delays photoreceptor cell loss to some extent. However, when these agents are combined, photoreceptor rescue is much more effective. The quenching of opsin and arrestin expression caused by treatment suggests that simultaneous with rod rescue, rod differentiation is depressed. Regarding retinal degeneration, the results from the selective inhibitors of apoptosis rank the CNTF+BDNF combination treatment as the most consistent and effective experimental pharmacologic intervention currently available.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Factor Neurotrófico Ciliar/uso terapéutico , Epitelio Pigmentado Ocular/patología , Degeneración Retiniana/tratamiento farmacológico , Células Fotorreceptoras Retinianas Bastones/patología , Animales , Apoptosis/efectos de los fármacos , Arrestina/metabolismo , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Quimioterapia Combinada , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos C3H , Técnicas de Cultivo de Órganos , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Opsinas de Bastones/metabolismoRESUMEN
Retinoic acid is one of the active metabolites of vitamin A and has profound effects on the development of the CNS including retina. Previously, we have shown that rod-specific apoptosis is induced in retinal explants from neonatal mice by exposure to 9-cis-retinoic acid (9CRA) when the retinal pigment epithelium (RPE) is present. In explants lacking RPE, it instead has a differentiation-promoting effect seen as an accelerated opsin expression on postnatal day 3. To investigate the long-term effect of 9CRA exposure, we have explanted retinas from neonatal C3H mice with or without RPE attached and placed in organ culture. After 19 or 48 h in culture or 7, 8 or 13 days in culture, the explants were either fixed for histochemical examination or frozen for assay of DEVDase activity. We found that long-term exposure to 9CRA caused a decrease in the number of cell layers in the outer nuclear layer (ONL) only in explants with the RPE attached. When explants with RPE attached were exposed to 9CRA only during the second postnatal week, neither an increase in DEVDase activity, TUNEL-positive cells, nor a decrease in cell layers of the ONL could be demonstrated, indicating that the retina was insensitive to the apoptosis-inducing effect of 9CRA after the first postnatal week. The absence of RPE in control explants resulted in a higher number of rosettes and the extrusion of cells into the subretinal space.
Asunto(s)
Envejecimiento/fisiología , Animales Recién Nacidos/fisiología , Apoptosis/fisiología , Epitelio Pigmentado Ocular/fisiología , Retina/fisiología , Tretinoina/farmacología , Alitretinoína , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Recuento de Células , Ratones , Ratones Endogámicos C3H , Técnicas de Cultivo de Órganos , Péptido Hidrolasas/metabolismo , Retina/citología , Retina/enzimologíaRESUMEN
The retina of mammals contains various amounts of cone photoreceptors that are relatively evenly distributed and display a radially or horizontally oriented area of peak density. In most mammalian species two spectrally different classes of cone can be distinguished with various histochemical and physiological methods. These cone classes occur in a relatively constant ratio, middle-to-longwave sensitive cones being predominant over short-wave cones. Recent observations do not support the idea that each cone subpopulation is uniformly distributed across the retina. With appropriate type-specific markers, unexpected patterns of colour cone topography have been revealed in certain species. In the mouse and the rabbit, the "standard" uniform pattern was found to be confined exclusively to the dorsal retina. In a ventral zone of variable width all cones express short-wave pigment, a phenomenon whose biological significance is not known yet. Dorso-ventral asymmetries have been described in lower vertebrates, matching the spectral distribution of light reaching the retina from various sectors of the visual field. It is not clear, however, whether the retinal cone fields in mammals carry out a function similar to that of their counterparts in fish and amphibians. Since in a number of mammalian species short-wave cones are the first to differentiate, and the expression of the short-wave pigment seems to be the default pathway of cone differentiation, we suggest that the short-wave sensitive cone fields are rudimentary areas conserving an ancestral stage of the photopigment evolution.
Asunto(s)
Retina/citología , Células Fotorreceptoras Retinianas Conos/citología , Animales , Humanos , Inmunohistoquímica , Ratones , Conejos , Retina/fisiología , Células Fotorreceptoras Retinianas Conos/fisiologíaRESUMEN
The dorsal cap of Kooy of the inferior olive (DC) is involved in compensatory eye movements. The rat DC receives a prominent input from the nucleus prepositus hypoglossi (NPH); part of these axon terminals are immunoreactive for choline acetyltransferase (ChAT) and part of them are GABAergic. In the present study we investigated the fine distribution of cholinergic terminals in the rat DC, and the possible coexistence of ChAT and GABA. ChAT-positive terminals were observed throughout the entire neuropil of the rat DC contacting both extraglomerular and intraglomerular dendrites. Twenty nine percent of these terminals also contained GABA. The ChAT/GABA double-labelled terminals showed the same morphological characteristics as terminals traced from the NPH. The present data demonstrate colocalization of ChAT and GABA in axon terminals of the rat DC and strongly suggest that neurons in the NPH are the source of these profiles.
Asunto(s)
Colina O-Acetiltransferasa/análisis , Neuronas/química , Neuronas/enzimología , Núcleo Olivar/citología , Ácido gamma-Aminobutírico/análisis , Animales , Fibras Colinérgicas/química , Fibras Colinérgicas/enzimología , Microscopía Electrónica , Neuronas/ultraestructura , Terminales Presinápticos/química , Terminales Presinápticos/ultraestructura , Ratas , Ratas Wistar , Aglutinina del Germen de Trigo-Peroxidasa de Rábano Silvestre ConjugadaRESUMEN
Acetylcholine (ACh) plays an important role in pituitary gland function. Little is known, however, about the source and trajectory of pituitary ACh, the location of pituitary cholinergic receptors, and the pathways along which the release of pituitary ACh is controlled. Therefore choline acetyltransferase (CHAT) immunoreactive profiles have been investigated in the rat median eminence and pituitary. Furthermore, both muscarinic- (mAChRp-L) and nicotinic receptor proteinlike (nAChRp-L) immunoreactivity have been examined in the rat, rabbit, and cat pituitary. The results have demonstrated that the rat pituitary ChAT network is composed of neurons in the hypothalamic arcuate nucleus and a great number of terminals in the median eminence. In the pituitary, ChAT immunolabeled profiles were virtually absent. This suggests that much of the ACh acting on pituitary cells is released as a humoral factor from the median eminence. All the examined animals expressed mAChRp-L immunostained endocrine cells in the intermediate lobe. Apart from this, marked species differences in AChRp-L immunolabeled profiles have been found. In addition, strong mAChRp-L immunoreactive rod to cone-shaped bodies were detected associated with blood vessels of the anterior and intermediate lobes in the rat and rabbit, but not in the cat. The immunolabeling was present in particles on the body plasma membrane. These characteristics suggest that the function of these structures might be to sense pituitary blood ACh levels. Consequently the name blood acetylcholine reading bodies (BARBs) was adopted to indicate these structures. It is proposed that the BARBs may play a role in the feedback control of ACh release from the median eminence.
Asunto(s)
Acetilcolina/sangre , Fibras Colinérgicas/ultraestructura , Hipófisis/inervación , Acetilcolina/fisiología , Animales , Gatos , Colina O-Acetiltransferasa/metabolismo , Eminencia Media/metabolismo , Hipófisis/fisiología , Conejos , Ratas , Receptores Colinérgicos/metabolismoRESUMEN
The developmental, genetic, and biochemical similarities that have been observed between the cerebellum and retina form the basis for ongoing investigations into retinal expression of cerebellar-specific proteins. We have examined the mouse, rat, rabbit, and human retina for expression of a protein that is present in parasagittal Purkinje cell strips and that is recognized by the antibody Zebrin-II. This protein has recently been identified as a member of the aldolase C isoenzymes. Western blotting and immunocytochemistry have been used. The monoclonal antibody Zebrin-II recognized a prominent 36 kDa protein band on immunoblots of both the cerebellum and the retina of the examined species. Immunocytochemistry showed that, in the three nonhuman species, cells were stained in the ganglion cell layer (GCL). In addition, in the mouse and rabbit, cells in the inner nuclear layer (INL) were also labeled. Except for the visual streak, there were more immunopositive cells in the rabbit GCL and INL than in corresponding areas of the mouse retina. In the human, in contrast to the other species, the photoreceptor cell layer was strongly aldolase C immunoreactive. In all species except for the rat, the photoreceptor inner segments also displayed a weak labeling. The results show that this aldolase C isoenzyme is another protein that is selectively expressed by the cerebellum and retina. Furthermore, the retinal expression is species specific, and this pattern seems to show a good correlation with the oxygenation level of the individual compartments. The indication that this aldolase C isoenzyme has specific developmental functions in the retina provides additional clues for our understanding of cerebellar organization.
Asunto(s)
Ratones/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células de Purkinje/metabolismo , Conejos/metabolismo , Ratas/metabolismo , Animales , Western Blotting , Femenino , Peces/metabolismo , Humanos , Inmunohistoquímica , Masculino , Ratones Endogámicos BALB C , Distribución TisularRESUMEN
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.
Asunto(s)
Células Fotorreceptoras/crecimiento & desarrollo , Células Fotorreceptoras Retinianas Conos/crecimiento & desarrollo , Animales , Animales Recién Nacidos , Histocitoquímica , Ratones , Ratones Endogámicos C3H , Morfogénesis , Técnicas de Cultivo de Órganos , Células Fotorreceptoras/citología , Células Fotorreceptoras/metabolismo , Epitelio Pigmentado Ocular/fisiología , Retina/fisiología , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Conos/metabolismo , Pigmentos Retinianos/metabolismoRESUMEN
Cholinergic modulation of locus coeruleus (LC) neurons evokes a variety of neuronal and behavioural effects. In an attempt to understand the LC cholinergic circuit, several markers has been investigated and compared. (Immuno)-histochemical and autoradiographic methods have been used on rat, rabbit, and pig tissue. To identify the boundaries of the LC in each of these species, sections through the entire brainstem have been stained for tyrosine hydroxylase. The results indicate that the pig does not possess a LC proper that conforms to the accepted features of this cell group. However, in this location fusiform cells reminiscent of LC interneurons are still present. This group of fusiform neurons has been named the nucleus angularis grisea periventricularis (NAGP). LC cells of the rat and rabbit show strong acetylcholinesterase (AChE) activity. In the pig the NAGP is markedly free from AChE staining. Muscarinic binding sites are densely distributed over the rabbit LC and adjacent region. The rat and rabbit LC neurons synthesise both muscarinic (mAChR) and nicotinic receptor protein (nAChR). In the pig NAGP region mAChR and nAChR positive cell bodies are almost absent, while some nAChR immunoreactive dendrites are present. The light microscopic data in the rabbit have been confirmed by electron microscopic analysis. It is concluded that the general concept of a noradrenergic LC that is present throughout mammals is questionable. At present, choline acetyltransferase immunoreactive terminals that closely correspond to the other cholinergic components in the rat or rabbit LC have not been observed. However, in these species the cholinergic sensitivity of LC cells is mediated via both muscarinic and nicotinic receptors on somata and dendrites.
Asunto(s)
Acetilcolinesterasa/análisis , Interneuronas/química , Locus Coeruleus/química , Receptores Muscarínicos/análisis , Receptores Nicotínicos/análisis , Animales , Autorradiografía , Colina O-Acetiltransferasa , Dendritas/química , Dendritas/ultraestructura , Femenino , Inmunohistoquímica/métodos , Interneuronas/ultraestructura , Locus Coeruleus/ultraestructura , Masculino , Microscopía Electrónica , Conejos , Ratas , Ratas Wistar , Porcinos , Tirosina 3-MonooxigenasaRESUMEN
Recently, we have reported the peculiar topographic separation of shortwave- and middlewave-sensitive (S and M) cones in the retina of the common house mouse (Mus musculus) and in a number of inbred laboratory mouse strains derived from the same species. In an attempt to follow the phylogeny of the complementary cone fields, we have investigated the retina of other mouse-like rodents. Two monoclonal anti-visual pigment antibodies, OS-2 and COS-1, specific to the S and M cones, respectively, have been used to identify the two cone types. Immunocytochemistry on retinal sections and on whole-mount preparations have shown that, as in the house mouse, the two cone types in the mound builder mouse (Mus spicilegus) occupy opposite halves of the retina. In contrast, in the wood mouse (Apodemus sylvaticus), both cone types are scattered uniformly across the whole retinal surface. Another distinguishing feature between the two genera is the frequency of the S cones. Whereas their density in the Mus species is above 7,000/mm2 in the S-field, the maximum density of the S cones in A. sylvaticus is one order of magnitude smaller. In another species of this genus (the herb field mouse, A. microps), the S cones are completely missing.
Asunto(s)
Muridae/anatomía & histología , Células Fotorreceptoras Retinianas Conos/anatomía & histología , Animales , Anticuerpos Monoclonales/inmunología , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Células Fotorreceptoras Retinianas Conos/química , Pigmentos Retinianos/análisis , Pigmentos Retinianos/inmunología , Especificidad de la EspecieRESUMEN
Previously we have presented the morphological features of a neonatal mouse retinal explant kept in culture for 3 to 4 weeks. To further evaluate the organotypic parameters of the tissue we have examined the presence of opsin, S-antigen, and interphotoreceptor retinoid-binding protein (IRBP) in the same experimental paradigm, using light microscopic immunocytochemistry. In vitro, opsin and S-antigen staining is found in photoreceptor somata from genetically normal explants and those derived from mice with the rd or the rds mutation. When present, inner and outer segments label more intensely. No IRBP staining has been found in cell bodies of any genotype. However, some labeling is found in the plexiform layers and in the inner segments. The results indicate that photoreceptor proteins are continuously produced in vitro. This further establishes the organotypic nature of the retinal explant in culture. The administration of growth factors to these explants has been investigated. Neither basic fibroblast growth factor nor nerve growth factor alone has affected the explants phenotype. However, the combination of these proteins has significantly retarded rd cell loss in vitro.
Asunto(s)
Proteínas del Ojo/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factores de Crecimiento Nervioso/farmacología , Células Fotorreceptoras/metabolismo , Retina/efectos de los fármacos , Degeneración Retiniana/metabolismo , Animales , Antígenos/metabolismo , Arrestina , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos C3H , Ratones Mutantes , Técnicas de Cultivo de Órganos , Retina/metabolismo , Degeneración Retiniana/prevención & control , Proteínas de Unión al Retinol/metabolismo , Opsinas de Bastones/metabolismoRESUMEN
We have found a complex eye disease in the SJL/N mouse. This animal is closely related to the SJL/J mouse, which is homozygous for retinal degeneration (rd) and which also suffers from extraocular reticulum cell sarcomas at around 200 days of age. In the SJL/N animal, a high incidence of subretinal tumor is present at 9 days after birth. Furthermore, we have observed an extensive neuroretinal hyperplasia, a phenomenon that is termed "hyperplastic neuroretinopathy", and that is probably the consequence of elevated levels of cytokines in the animals. In addition to these anomalies, the SJL/N mouse shows progressive dystrophy of the retinal pigment epithelium (RPE) from day 4 onwards, and accelerated photoreceptor cell degeneration is completed by day 16. The early RPE dystrophy appears to be a secondary autoimmune disease, since cells in this structure and in the choroid develop MHC class II antigens, whereas we suspect that the accelerated photoreceptor cell loss is induced by a soluble toxic agent. The F1 progeny derived from cross-breeding the SJL/N and Balb/c +/+ strains also shows a high incidence of subretinal tumor and hyperplastic neuroretinopathy, but neither the RPE dystrophy nor retinal degeneration.
Asunto(s)
Enfermedades Autoinmunes/patología , Neoplasias del Ojo/genética , Linfoma de Células B Grandes Difuso/genética , Ratones Endogámicos , Ratones Mutantes , Epitelio Pigmentado Ocular/patología , Degeneración Retiniana/patología , Factores de Edad , Animales , Cruzamientos Genéticos , Neoplasias del Ojo/patología , Femenino , Antígenos de Histocompatibilidad Clase II/análisis , Hiperplasia , Linfoma de Células B Grandes Difuso/patología , Masculino , Ratones , Ratones Endogámicos BALB C/genética , Ratones Endogámicos/genética , Ratones Mutantes/genética , Morfogénesis , Neoplasias Primarias Múltiples , Células Fotorreceptoras/ultraestructura , Epitelio Pigmentado Ocular/inmunología , Retina/patología , Degeneración Retiniana/genéticaRESUMEN
We have found two immunologically distinguishable cone types in the retina of the mouse, each localized to two opposite halves of the eye. One cone type was labelled by the monoclonal antibody COS-1 specific to the middle-to-long wave sensitive visual pigment of the mammals, while the other type was stained by the shortwave-specific monoclonal antibody (OS-2). These results were confirmed with other antibodies directed against specific sequences of the visual pigments. As a result of the uneven distribution of the two cone types the mouse retina is divided into two fields separated by an oblique meridional line. The middlewave sensitive cones were present exclusively in the dorsal half of the mouse retina (M-field). The overwhelming majority of the shortwave sensitive cones occupied the ventral half (S-field), and only a small number was scattered among the middlewave sensitive cones in the dorsal retina. The ratio of the two cone types in the M-field corresponds to what has been found in the retina of other mammals, including rodents such as the gerbil and the rat. The S-field represents an entirely unique area with the unusually great number of shortwave sensitive cones and with the complete lack of the middlewave sensitive ones. The present study provides the structural basis for dichromacy in a rodent species considered for a long time to be monochromat. In addition, it shows that the ventral retina, containing exclusively S-cones in a relatively high density, is a unique retinal field not present in other mammalian species studied so far.
Asunto(s)
Células Fotorreceptoras/citología , Retina/citología , Animales , Anticuerpos Monoclonales/inmunología , Femenino , Inmunohistoquímica , Lectinas , Masculino , Ratones , Ratones Endogámicos , Aglutinina de Mani , Células Fotorreceptoras/inmunología , Células Fotorreceptoras/ultraestructura , Adhesión en Plástico , Retina/ultraestructura , Especificidad de la EspecieRESUMEN
It is now well established that extensive extrahypothalamic vasopressin (VP) systems exist in the rat, monkey and human brain. There are marked differences between species, but in each case VP nuclei provide dense afferents to the dorsal pontine tegmentum. Here VP may play a role in the mechanisms exerted by the locus coeruleus (LC) neurons, possibly both as a neurotransmitter and as a neuromodulator. Although we are aware of some properties of VP systems, e.g., gonadal steroid dependency in the rat, major gaps characterize our knowledge of its anatomy. With regard to the interaction of VP with the LC in the brainstem of mammals some of the questions which stand out are: (1) Is VP really being biosynthesized and transported by LC cells and, if not, what is its function within these cells? (2) Is there a structural difference between male and female LC neurons in the rat as a consequence of the sex-dimorphic VP innervation? (3) What is the origin of VP afferents in the dorsal pontine tegmentum of the (non)human primate and are these afferents also controlled by gonadal steroids? Research strategies to answer these questions will provide us with information to resolve some of the current inconsistencies about the anatomy and the function of the VP and LC systems in the brain.
Asunto(s)
Macaca fascicularis/anatomía & histología , Puente/fisiología , Ratas/anatomía & histología , Vasopresinas/fisiología , Vías Aferentes/fisiología , Animales , Mapeo Encefálico , Femenino , Humanos , Macaca fascicularis/fisiología , Masculino , Fibras Nerviosas/química , Fibras Nerviosas/fisiología , Núcleo Hipotalámico Paraventricular/química , Núcleo Hipotalámico Paraventricular/fisiología , Puente/anatomía & histología , Ratas/fisiología , Ratas Endogámicas , Caracteres Sexuales , Especificidad de la Especie , Núcleo Supraquiasmático/química , Núcleo Supraquiasmático/fisiologíaRESUMEN
Retinae from neonatal mice were explanted in toto, with or without the retinal pigment epithelium (RPE) and adjoining mesenchymal cells, and maintained in organ culture for up to 3 weeks. The explants remained flat, rosette formation was minimal and histogenetic changes followed in the normal sequence. After 11, 14 and 21 days in vitro the three cellular layers--the outer nuclear layer including well differentiated rod and cone perikarya, the inner nuclear layer and the ganglion cell layer--with the intervening plexiform layers were comparable to those of the in vivo eyes. Electron microscopic analysis revealed that in the explants without RPE the nuclear layers developed as in vivo, but receptor outer segments (ROS) were not formed. When the RPE was present, receptor inner segments appeared normal and ROS including profuse disc structures were developed. Presence of synaptic elements was also recognized. Mesenchymal cells, when present differentiated into choroidal and scleral tissues and appeared to play a supportive role for the RPE cells. The system is described in detail and its suitability for the analysis of various cellular and metabolic factors in the development of the retina is discussed.