Changes in the relative number of bipolar-like cells in the retina of Pleurodeles waltl as a function of age and as a result of light exposure

The study of the population of bipolar-like cells (displaced bipolars) was continued in order to establish their role in development and regeneration of the retina in lower vertebrates. The size of the population of these cells was estimated on serial semithin sections in the retina of normal eyes in adult and young newt Pleurodeles waltl, as well as in adult newts subjected to long-term bright illumination. The population of displaced bipolars was significantly increased with reference to all cells of the outer nuclear layer. In young and illuminated animals, their numbers were approximately 1.3 and 1.4 times that in the adult animals not exposed to constant light. The results obtained favor the earlier suggestion of the involvement of the displaced bipolars in growth and restoration of the outer nuclear layer in the retina of newts during development and after trauma.     

An Immunohistochemical Study of Localization of the Calcium-Binding Protein Recoverin in Retina of the Newt Pleurodeles waltl

The presence and localization of the calcium-binding protein recoverin, initially found in photoreceptors of the bovine eye, were immunochemically studied in retina of the new Pleurodeles waltl. Polyclonal monospecific antibodies against recoverin were raised and the methods of immunoblotting and indirect immunofluorescence were used. A protein with an apparent molecular mass of 26 kDa was found in the retina extract, which was specifically stained by the antibodies against recoverin. Localization of recoverin was studied on the retina sections: an intense reaction was found in the inner segments and a weak reaction was found in the basal part of the outer segments of photoreceptors and in Landolt's clubs of displaced bipolars. The results we obtained suggest for the first time the presence of recoverin in the retina of a representative of the Urodeles and indicate to interspecific conservativeness of this protein and differences of its localization in the retina photoreceptors in different species. The data obtained open a possibility of using recoverin as a marker protein of photoreceptors and displaced bipolars in studies of retina regeneration in newts.     

Early patterns of neuronal degeneration in the retina of the goldfish after optic nerve sectioning

The early patterns of retinal degeneration were studied in the goldfish after optic nerve sectioning by l.m. and e.m. Beginning on the 2nd postsurgical day there was an initial degeneration of neurons in the ganglion cell and inner nuclear layers of the central retina. Massive ganglion cell degeneration in the whole retina (60%) as well as degeneration of neurons in inner and outer nuclear layer of the peripheral retina was evident around the 7th postsurgical day. The early degenerating cells appeared to be cones and cone bipolars.     

Localization of glutamate in the human retina during early prenatal development

In this study we have localized glutamate (GLU) in fetal (14–25 weeks gestation, Wg) human retinas by immunohistochemistry. At 14 Wg, GLU-immunoreactivity (IR) was localized only in the central part of retina, showing a prominently labelled nerve fiber layero A few ganglion cells and displaced amacrine cells were very weakly labelled. At 17 Wg, GLU was localized conspicuously in many ganglion cells, displaced amacrine cells, some amacrine cells and the prospective photoreceptor cell bodies in the neuroepithelial layero With progressive development at 20 and 25 Wg, the IR for GLU was found additionally in the Müller cell endfeet, some bipolar cells as well as in the horizontal cells that were aligned in a row along the outer border of the inner nuclear layer of the central retinao The photoreceptor cell bodies in the outer nuclear layer were also prominently immunopositive for GLU. The developmental distribution of GLU in the human retina tends to indicate that it plays an important role in the differentiation and maturation of retinal neurons.     

Morphology of calretinin and tyrosine hydroxylase-immunoreactive neurons in the pig retina

The morphology of calretinin- and tyrosine hydroxylase-immunoreactive (IR) neurons in adult pig retina was studied. These neurons were identified using antibody immunocytochemistry. Calretinin immunoreactivity was found in numerous cell bodies in the ganglion cell layer. Large ganglion cells, however, were not labeled. In the inner nuclear layer, the regular distribution of calretinin-IR neurons, the inner marginal location of their cell bodies in the inner nuclear layer, and the distinctive bilaminar morphologies of their dendritic arbors in the inner plexiform layer suggested that these calretinin-IR cells were AII amacrine cells. Calretinin immunoreactivity was observed in both A-and B-type horizontal cells. Neurons in the photoreceptor cell layer were not labeled by this antibody. The great majority of tyrosine hydroxylase-IR neurons were located at the innermost border of the inner nuclear layer (conventional amacrines). The processes were monostratified and ran laterally within layer 1 of the inner plexiform layer. Some of the tyrosine hydroxylase-IR neurons were located in the ganglion cell layer (displaced amacrines). The processes of displaced tyrosine hydroxylase-IR amacrine cells were also located within layer 1 of the inner plexiform layer. Some processes of a few neurons were located in the outer plexiform layer. A very low density of neurons had additional bands of tyrosine hydroxylase-IR processes in the middle and deep layers of the inner plexiform layer. The processes of tyrosine hydroxylase-IR neurons extended radially over a wide area and formed large, moderately branched dendritic fields. These processes occasionally had varicosities and formed "dendritic rings". These results indicate that calretinin- and tyrosine hydroxylase-IR neurons represent specific neuronal cell types in the pig retina.     

Changes of lysosomal enzymes during hereditary degeneration and histogenesis of retina in mice

Summary During the post-natal development of the retina in mice, macrophages which are selectively stained for N-Acetyl--glucosaminidase enter the retina through the vascular route. Most of these cells finally occupy the outer and the inner levels of the inner nuclear layer adjoining the plexiform layers and are transformed into very small cells which persist in the adult retina without further change.In mice with hereditary retinal degeneration (rd rd) these -glucosaminidase positive macrophages enter the outer nuclear layer of the retina, soon after the onset of degeneration undergo extensive hypertrophy and rapidly phagocytize the degenerating photoreceptor cells. After the digestion of the ingested materials the enzyme activity is very much reduced and the cells become smaller in size. They eventually acquire the morphological features seen in the normal retina.     

SOMATOSTATIN-LIKE IMMUNOREACTIVE CELLS IN THE GROUND SQUIRREL RETINA

Immunocytochemical techniques were employed to locate somatostatin (SS)-containing cells in the retina of the 13-lined ground squirrel (Spermophilus tridecemlineatus). In normal retinas immunostain was limited to neuronal processes, yet distinctly labeled somata were detected in retinas of animals pretreated with colchicine. Labeled cell bodies were located in the outermost and innermost portions of the inner nuclear layer (INL) and in the ganglion cell layer (GCL). The largest population of SS-like immunoreactive neurons was found in the innermost INL. These cells were identified as small and medium sized amacrine cells whose soma diameters ranged from 4 to 14μm. A smaller population of immunoreactive cells was observed in the outermost region of the INL. These cells, presumptive horizontal cells, were found mainly in peripheral regions of the retina. Immunoreactive cells in the GCL were of two types: displaced amacrines, and retinal ganglion cells. SS-positive axons in the optic fiber layer suggest that some of the immunoreactive GCL neurons were ganglion cells, and it is our opinion that these cells belong to a class of associational ganglion cells previously identified in other species.     

Defects in the outer limiting membrane are associated with rosette development in the Nrl-/- retina

The neural retinal leucine zipper (Nrl) knockout mouse is a widely used model to study cone photoreceptor development, physiology, and molecular biology in the absence of rods. In the Nrl(-/-) retina, rods are converted into functional cone-like cells. The Nrl(-/-) retina is characterized by large undulations of the outer nuclear layer (ONL) commonly known as rosettes. Here we explore the mechanism of rosette development in the Nrl(-/-) retina. We report that rosettes first appear at postnatal day (P)8, and that the structure of nascent rosettes is morphologically distinct from what is seen in the adult retina. The lumen of these nascent rosettes contains a population of aberrant cells protruding into the subretinal space that induce infolding of the ONL. Morphologically adult rosettes do not contain any cell bodies and are first detected at P15. The cells found in nascent rosettes are photoreceptors in origin but lack inner and outer segments. We show that the adherens junctions between photoreceptors and Müller glia which comprise the retinal outer limiting membrane (OLM) are not uniformly formed in the Nrl(-/-) retina and thus allow protrusion of a population of developing photoreceptors into the subretinal space where their maturation becomes delayed. These data suggest that the rosettes of the Nrl(-/-) retina arise due to defects in the OLM and delayed maturation of a subset of photoreceptors, and that rods may play an important role in the proper formation of the OLM.     

Calmodulin gene expression in the neural retina of the adult rat

Calmodulin (CaM) mRNAs are expressed with low abundancy in the adult rat neural retina. However, when digoxigenin (DIG)-labeled cRNA probes specific for each CaM mRNA population were hybridized at slightly alkaline pH (pH 8.0), the widespread distribution of CaM mRNA-expressing cells was revealed, with similar abundance for all three CaM genes. The CaM genes displayed a uniquely similar, layer-specific expression throughout the retina, and no significant differences were found in the distribution patterns of the CaM mRNA populations or the labeled cell types. The strongest signal for all CaM mRNAs was demonstrated in the ganglion cell layer and the inner nuclear layer, where the highest signal intensity was found within the inner sublamina. Similarly intermediate signal intensities for all CaM genes were detected in the inner and outer plexiform layers, within the vicinity of the outer limiting membrane and in the retinal pigment epithelium. A very low specific signal was characteristic in the outer nuclear layer and the photoreceptor inner segment layer, while no specific hybridization signal was observed in the photoreceptor outer segment layer. In summary, all CaM genes exhibited a similar and a characteristically layer-specific expression pattern in the adult rat retina.     

Morphology of the retina in deep‐water fish Nezumia sclerorhynchus (Valenciennes, 1838) (Gadiformes: Macrouridae)

Using light microscopy, we examined the retina of benthopelagic fish Nezumia sclerorhynchus. Although the retina is typical of other vertebrates, having three nuclear and two synaptic layers, it presents some features associated with the animal's deep‐sea habitat. A stratum argenteum containing iridescent crystals is located in the choroid. The pigment cell layer shows bulky cells filled with melanin granules but without the typical apical processes. The visual cells, consisting of a big population of rods, are arranged in several banks. No cones were observed. The outer segments are very long and cylindrical, and the inner segments are constituted by a small ellipsoid at the proximal end. The outer nuclear layer contains several rows of oval nuclei, and the spherules in the outer plexiform layer have less regular outlines than nuclei. The inner retina is characterized by very large horizontal cells, and presumable bipolar and amacrine cells separated by large spaces that are occupied by neuronal processes. Finally, the low density of ganglion cells produces a thin nerve fibre layer. The results of this study suggest that the retina of Nezumia sclerorhyncus exhibits high visual sensitivity and that vision is a sense that plays an important role in its behaviour.     

GABA neurones in retinas of different species and their postnatal development in situ and in culture in the rabbit retina

Summary The localisation of GABA immunoreactive neurones in retinas of a variety of animals was examined. Immunoreactivity was associated with specific populations of amacrine neurones in all species examined, viz. rat, rabbit, goldfish, frog, pigeon and guinea-pig. All species, with the exception of the frog, possessed immunoreactive perikarya in their retinal ganglion cell layers. These perikarya are probably displaced amacrine cells because GABA immunoreactivity was absent from the optic nerves and destruction of the rat optic nerve did not result in degeneration of these cells. GABA immunoreactivity was also associated with the outer plexiform layers of all the retinas studied; these processes are derived from GABA-positive horizontal cells in rat, rabbit, frog, pigeon and goldfish retinas, from bipolar-like cells in the frog, and probably from interplexiform cells in the guinea-pig retina.The development of GABA-positive neurones in the rabbit retina was also analysed. Immunoreactivity was clearly associated with subpopulations of amacrine and horizontal cells on the second postnatal day. The immunoreactivity at this stage is strong, and fairly well developed processes are apparent. The intensity of the immunoreactivity increases with development in the case of the amacrine cells. The immunoreactive neurones appear fully developed at about the 8th postnatal day, although the immunoreactivity in the inner plexiform layer becomes more dispersed as development proceeds. The immunoreactive horizontal cells become less apparent as development proceeds, but they can still be seen in the adult retina.The GABA immunoreactive cells in rabbit retinas can be maintained in culture. Cultures of retinal cells derived from 2-day-old animals can be maintained for up to 20 days and show the presence of GABA-positive cells at all stages. In one-day-old cultures the GABA immunoreactive cells lacked processes but within three days had clearly defined processes. After maintenance for 10 days a meshwork of GABA-positive fibres could also be seen in the cultures.     

Pax-6 expression during retinal regeneration in the adult newt

The present study examined the expression of Pax-6 during retinal regeneration in adult newts using in situ hybridization. In a normal retina, Pax-6 is expressed in the ciliary marginal zone, the inner part of the inner nuclear layer, and the ganglion cell layer. After surgical removal of the neural retina, retinal pigment epithelial cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, Pax-6 was expressed in all retinal precursor cells. As regeneration proceeded, differentiating cells appeared at the scleral and vitreal margins of the regenerating retina, which had no distinct plexiform layers. In this stage, the expression of Pax-6 was localized in a strip of cells along the vitreal margin of the regenerating retina. In the late stage of regeneration, when the layer structure was completed, the expression pattern of Pax-6 became similar to that of a normal retina. It was found that Pax-6 is expressed in the retinal precursor cells in the early regenerating retina and that the expression pattern of Pax-6 changed as cell differentiation proceeded during retinal regeneration.     

根田鼠视网膜的胚后发育

视觉对动物的生活习性尤其是取食具有重要意义。本文对根田鼠视网膜的胚后发育进行了研究,结果表明:出生3d内根田鼠视网膜分化程度较低,神经节母细胞层尚未分化,占据了视网膜层的一半以上;5日龄时,外网层开始出现;6日龄时,外网层开始清晰,外核层与内核层更加清晰;18日龄时,视网膜结构与成年根田鼠结构相似,各层结构清晰可见。测量了神经节细胞层和外核层的细胞密度以及核层厚度,结果表明:随着个体发育,外核层细胞层厚度及细胞密度不断增加;而神经节细胞层厚度及细胞密度不断减少。与褐家鼠、黑线姬鼠、大仓鼠、棕色田鼠、甘肃鼢鼠、达乌尔黄鼠、岩松鼠视网膜相比,根田鼠视网膜结构介于夜行性与昼行性鼠类之间[动物学报52(2):376-382,2006]。     

Embryonic appearance of rod opsin in the urodele amphibian eye

Notophthalmus (Triturus) viridescens, a urodele amphibian (newt) common to the Eastern United States, is a promising subject for developmental and regeneration studies. We have available a monoclonal antibody shown to be specific in many vertebrates for rod opsin, the membrane apoprotein of the visual pigment rhodopsin. This antibody to an N-terminal epitope, by rigorous biochemical and immunological criteria, recognizes only rod photoreceptor cells of the retina in light-and electron-microscopic immunocytochemistry. To determine the ontogeny and localization of rhodopsin in developing rods as an indicator of function in the embryonic urodele retina, we have utilized this antibody in the immunofluorescence technique on sections of developing N. viridescens. It was applied to serial sections of the eye region of Harrison stage 28 (optic vesicle) through stage 43 (most adult retina histology complete) embryos, and subsequently visualized with biotinylated species antibody followed by extravidin fluorescein isothiocyanate. The first positive reaction to rhodopsin could be detected in two to four cells (total) of the stage 37 embryonic eye, in the region of the central retinal primordium where the photoreceptors will be found. Some indications of retinal outer nuclear and inner plexiform layers could be seen at this time. Later embryonic stages demonstrated increasing numbers of positive cells in the future photoreceptor outer nuclear layer and outer and inner segments, spreading even to the peripheral retina. Nevertheless, by stale 43, no positive cells could be found at the dorsal or ventral retinal margins. Thus, biochemical differentiation of a photoreceptor population in the urodele retina occurs at a stage before retinal histogenesis is complete. The total maturation of retinal rods occurs topographically over a long period until the adult distribution is achieved. Correspondence to: D.S. McDevitt     

人胎视网膜神经肽Y免疫反应神经元的发育

本文用免疫细胞化学ＡＢＣ法,研究１５—３８周龄人胎视网膜神经肽Ｙ免疫反应（ＮｅｕｒｏｐｅｐｔｉｄｅＹｉｍｍｕｎｏｒｅｃｔｉｖｅ,ＮＰＹ－ＩＲ）神经元（以下称ＮＰＹ－ＩＲ细胞）的发育。结果表明：①胎龄１５周视网膜中央部已出现不同类型的ＮＰＹ－ＩＲ细胞：位于黄斑及其周围外核层的为ＮＰＹ－ＩＲ视锥细胞;位于内核层最内一列的为ＮＰＹ－ＩＲ无长突细胞位于节细胞层的可能为ＮＰＹ－ＩＲ移位无长突细胞或节细胞;内核层和节细胞层的ＮＰＹ－ＩＲ细胞的突起均分布在内网层的第１亚层。②胎龄２４周后,ＮＰＹ－ＩＲ视锥细胞完全消失。③随着视网膜的发育,内核层和节细胞层的ＮＰＹ－ＩＲ细胞数量增多,突起增粗增长,胞体分布由中央部扩展到周边部,其中内核层ＮＰＹ－ＩＲ细胞的密度呈现从中央部向周边部逐渐降低的分布方式,节细胞层ＮＰＹ－ＩＲ细胞则多数集中分布在视网膜的边缘和黄斑之间,形成较高密度的环状区。     

Single cell shape and population densities of indoleamine-accumulating and displaced bipolar cells in Reeves' turtle retina

Two types of bipolar cell in the Geoclemys reevesii retina were studied quantitatively by means of specific cell labelling with an indoleamine derivative (5,6-dihydroxytryptamine, 5,6-DHT), a nucleic acid stain (4,6-diamidino-2-phenylindole, DAPI) and Lucifer yellow CH. Indoleamine-accumulating (IA) bipolar cells were selectively labelled with 5,6-DHT applied intraocularly. After the cells accumulated 5,6-DHT, the indoleamine fluorescence was photoconverted to diaminobenzidine products to allow observation of morphological details. Close examination of many cells (cell number; n = 120) showed that the IA bipolar cells consist of a single morphological type whose axon collaterals ramify sublaminae 1, 4 and 5 respectively. This terminal branching pattern corresponds to cells that hyperpolarize when their receptive field centres are illuminated (Weiler 1981). The density of IA bipolar cells was highest in the visual streak (4130 cells mm-2) and lowest at the peripheral margin (1970 cells mm-2). By applying a small amount of DAPI to the eye, nuclei located in the most proximal row of the outer nuclear layer were labelled selectively. By using selective intracellular dye injection into DAPI-labelled cells under fluorescence microscope (Tauchi & Masland 1984, 1985), these cells were found to have Landolt's clubs and single descending axons. Dye injections into more than fifty DAPI-labelled somata showed that they belonged exclusively to displaced bipolar cells. These comprised at least two subtypes that differ in the ramification pattern of their axon terminals within the inner plexiform layer: one was monostratified, whereas the other was bistratified. The displaced bipolar cell density was as high as 9400 cells mm-2 in the central retina, falling to 2000 cells mm-2 in the superior margin. In vitro Lucifer labelling revealed that the overall bipolar cell density in the central retina was as high as 39,300 cells mm-2. Both the conventionally located and displaced bipolar cells were included in this population. About 11% of the total bipolar cell population consisted of IA bipolar cells. Assuming that one half of the conventionally located bipolar cells are the centre-hyperpolarizing type, IA bipolar cells represent approximately 28% of the total. As displaced bipolar cells represent almost one quarter of the total bipolar population, the dislocation of their somata stands out morphologically, inviting investigation of possible functional correlates.     

Onset and time course of apoptosis in the developing zebrafish retina

In mammalian development, apoptosis spreads over the retina in consecutive waves and induces a remarkable amount of cell loss. No evidence for such consecutive waves has been revealed in the fish retina so far. As the zebrafish is of growing importance as a model for retinal development and for degenerative retinal diseases, we examined the onset and time course of apoptosis in the developing zebrafish retina and in adult fish. We found that apoptosis peaked in the ganglion cell layer (GCL) and inner nuclear layer (INL) in early developmental stages (3-4 days post-fertilization; dpf) followed by a second, but clearly smaller wave at 6-7dpf. Apoptosis in the outer nuclear layer (ONL) started at 5dpf and peaked at 7dpf. This late-onset high peak of apoptosis of photoreceptors is different from that of all other species examined to date. With 1.09% of cells in the GCL and 1.10% in the ONL being apoptotic, the rate of apoptosis in the developing zebrafish retina was conspicuously lower than that observed in other vertebrates (up to 50% in GCL). During development (2-21dpf), apoptotic waves were most obvious in the central retina, whereas in the periphery near the marginal zone (MZ), apoptosis was much lower; in adult animals, practically no apoptosis was present in the central retina but it still occurred near the MZ. Our data show that the onset and time course of apoptosis in the GCL and INL of the zebrafish is comparable with other vertebrates; however, the amount of apoptosis is clearly reduced. Thus, apoptosis in the zebrafish retina may serve more as a mechanism for the fine tuning of the retinal neuronal network after mitotic waves during development or in remaining mitotic areas than as a mechanism for eliminating large numbers of excess cells.     

Developmental study of the expression of B50/GAP-43 in rat retina

B50/GAP-43 has been implicated in neural plasticity, development, and regeneration. Several studies of axonally transported proteins in the optic nerve have shown that this protein is synthesized by developing and regenerating retinal ganglion cells in mammals, amphibians, and fish. However, previous studies using immunohistochemistry to localize B50/GAP-43 in retina have shown that this protein is found in the inner plexiform layer in adults. Since the inner plexiform layer contains the processes of amacrine cells, ganglion cells, and bipolar cells to determine which cells in the retina express B50/GAP-43, we have now used in situ hybridization to localize the mRNA that codes for this protein in the developing rat retina. We have found that B50/GAP-43 is expressed primarily by cells in the retinal ganglion cell layer as early as embryonic day 15, and until 3 weeks postnatal. Some cells in the inner nuclear layer, possibly a subclass of amacrine cells, also express B50/GAP-43 protein and mRNA; however, the other retinal neurons–bipolar cells, photoreceptors, and horizontal cells express little, if any, B50/GAP-43 at any stage in their development. Early in development, the protein appears in the somata and axons of ganglion cells, while later in development, B50/GAP-43 becomes concentrated in the inner plexiform layer, where it continues to be expressed in adult animals. These results are discussed in terms of previous proposals as to the functions of this molecule. © 1993 John Wiley & Sons, Inc.     

Localization of Crystallins in Muller Cells of Common Frog Retina

Cell localization of 23 kDa- and 35 kDa-crystallins in the retina of adult common frogs Rana temporaria L. was studied using indirect immunofluorescence. Intense specific fluorescence of both crystallins was observed all over the retina, in both periphery and central area. It was localized in elongated radially oriented cells, whose bodies were located in the inner nuclear layer. These cells gave many fluorescing processes in the same layer and main processes in the outer nuclear and ganglion layers, one in each. The processes formed a strong network of fibers around the photoreceptor and ganglion cells. Intense fluorescence was also observed in the layer of nerve fibers and adjoining inner limiting membrane. The distribution and morphology of crystalline-containing cells mostly coincides with what is known for the Muller cells of vertebrate eye. The identity of the cells we described and Muller cells was also confirmed using the antiserum to glial fibrillary acidic protein.     

Human retinal development: ultrastructure of the inner retinal layers

Studies of the developing human retina from 6.5 to 18 weeks' gestational age (16–156 mm) by light and electron microscopy are concerned with the morphogenesis of neuroblast cells, plexiform layers, and inner limiting membrane. The transient layer of Chievitz is formed posteriorly by 20 mm (7 weeks), inner plexiform by 48 mm (9 weeks), outer plexiform layer by 83 mm (12 weeks), identifiable cones by 83 mm, and rods by 120 mm (15 weeks). Mitotic activity continues posteriorly until 120 mm and was seen in inner layers of the retina until 103 mm (13 weeks). Outer neuroblastic differentiation is marked by diversification from a uniform cell population to one containing at least three cell types differing in their nuclear shape, chromatin pattern, and cytoplasmic characteristics. Differentiating ganglion cells accumulate polysomes, rough endoplasmic reticulum, Golgi complexes, microtubules, and dense bodies. Müller cell bodies in ganglion and inner nuclear layers extend processes between ganglion cells, and radial fibers, containing extensive smooth endoplasmic reticulum, to the vitreal surface. Synapses appear in the inner and outer plexiform layers by 83 mm (12 weeks), and by 120 mm (15 weeks) demonstrate a variety of conventional and ribbon forms similar to those found in the adult. Synaptogenesis therefore begins considerably before the development of photoreceptor outer segments.