Morphology and distribution of Müller cells in the retina of the toad Bufo marinus

We have previously shown that an antibody against neuron-specific enolase (NSE) selectively labels Müller cells (MCs) in the anuran retina (Wilhelm et al. 1992). In the present study the light- and electron-microscopic morphology of MCs and their distribution were described in the retina of the toad, Bufo marinus, using the above antibody. The somata of MCs were located in the proximal part of the inner nuclear layer and were interconnected with each other by their processes. The MCs were uniformly distributed across the retina with an average density of 1500 cells/mm². Processes of MCs encircled the somata of photoreceptor cells isolating them from each other by glial sheath, except for those of the double cones. Some of the photoreceptor pedicles remained free of glial sheath. Electron-microscopic observations confirmed that MC processes provide an extensive scaffolding across the neural retina. At the outer border of the ganglion cell layer these processes formed a non-continuous sheath. The MC processes traversed through the ganglion cell layer and spread beneath it between the neuronal somata and the underlying optic axons. These processes formed a continuous inner limiting membrane separating the optic fibre layer from the vitreous tissue. Neither astrocytic nor oligodendrocytic elements were found in the optic fibre layer. The significance of the uniform MC distribution and the functional implications of the observed pattern of MC scaffolding are discussed.     

Fine structure of the retino-optic nerve junction in the chicken

The aim of this study is to investigate a fine structure of the retino-optic nerve junction in the chicken. We especially focused on the myelin sheaths and astrocytes in the intraocular optic nerve (ION) and its adjoining parts. A part of the axons of retinal nerve fiber layer (NFL) were myelinated. Ganglion cell axons were ensheathed by loose myelin in the NFL and by a compact one in the ION and optic nerve (ON). Myelin structure changed from loose type to a compact one within the very narrow NFL-ION junction. Loose myelin forming cells are dark type of oligodendrocytes in the retina. From the most peripheral ON to the choroidal part of ION, astrocytes contained abundant microtubules. The optic nerve around the lamina cribrosa is exposed to mechanical force during eye movement. It is suggested that these microtubules may perform the cytoskeletal function. Astrocytes in the retinal part of ION had longer processes filled with abundant gliofilaments. They may provide the mechanical support for the ganglion cell axons, which are exposed directly to intraocular pressure. Although astrocytes in the retinal level of ION extended their processes into the retina, their soma was never found in the retina.     

The glial design of a teleost optic nerve head supporting continuous growth.

This study demonstrates the peculiarities of the glial organization of the optic nerve head (ONH) of a fish, the tench (Tinca tinca), by using immunohistochemistry and electron microscopy. We employed antibodies specific for the macroglial cells: glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), and S100. We also used the N518 antibody to label the new ganglion cells' axons, which are continuously added to the fish retina, and the anti-proliferating cell nuclear antigen (PCNA) antibody to specifically locate dividing cells. We demonstrate a specific regional adaptation of the GS-S100-positive Müller cells' vitreal processes around the optic disc, strongly labeled with the anti-GFAP antibody. In direct contact with these Müller cells' vitreal processes, there are S100-positive astrocytes and S100-negative cells ultrastructurally identified as microglial cells. Moreover, a population of PCNA-positive cells, characterized as glioblasts, forms the limit between the retina and the optic nerve in a region homologous to the Kuhnt intermediary tissue of mammals. Finally, in the intraocular portion of the optic nerve there are differentiating oligodendrocytes arranged in rows. Both the glioblasts and the rows of developing cells could serve as a pool of glial elements for the continuous growth of the visual system.     

Expression of glutamate transporters in human and rat retina and rat optic nerve

l-Glutamate is the major excitatory transmitter in the vertebrate retina and plays a central role in the transmission of the various retinal neurons. Glutamate is removed from the extracellular space by at least five different glutamate transporters. The cellular distribution of these has been studied so far mainly using immunocytochemistry. In the present study non-radioactive in situ hybridisation using complementary RNA probes was applied in order to identify the cell types of rat retina and optic nerve expressing generic GLT1, GLT1 variant (GLT1v or GLT1B), GLAST and EAAC1. The results were compared with immunocytochemical data achieved using affinity-purified antibodies against transporter peptides. In the immunohistochemical studies the human retina was included. The study showed that in the rat retina GLT1v and EAAC1 were coexpressed in various cell types, i.e. photoreceptor, bipolar, horizontal, amacrine, ganglion and Müller cells, whereas GLAST was only detected in Müller cells and astrocytes. In the rat optic nerve GLT1v and EAAC1 were preferentially expressed in oligodendrocytes, whereas GLAST was revealed to be present mainly in astrocytes. Generic GLT1 could not be detected in the retina or optic nerve. The cellular distribution of glutamate transporters (only immunocytochemistry) in the human retina was very similar to that of the rat retina. Remarkable results of our studies were that generic GLT1 was not detectable in the rat (and human) retina and that GLT1v and EAAC1 were demonstrable in most cell types of the retina (including photoreceptor cells and their terminals).     

Immunocytochemical localization of S-100 protein in astrocytes and Müller cells in the rabbit retina

Summary The localization of S-100 protein was studied in histological sections of retinae from adult rabbits. By use of double-immunolabeling techniques it was shown that most but not all radially oriented vimentin-positive Müller cells were co-labeled by an antiserum to S-100 protein. Glial fibrillary acidic protein-positive astrocytes, which in the rabbit retina are restricted to the medullary rays formed by myelinated optic nerve fibers, consistently showed S-100 protein immunoreactivity. The present report shows that, with respect to S-100 protein staining, Müller cells represent a heterogeneous population of glial elements.     

Uptake of circulating hyaluronic acid by the rat liver

Summary The present study deals with the localization and development of S-100 protein-like immunoreactivity in the retina, ciliary body and iris of human fetuses. In the retina, numerous astrocytes, densely distributed in the nerve-fiber layer and ganglion-cell layer, were stained strongly with the S-100 antiserum. The first immunoreactive astrocytes occurred at the posterior pole of the retina and spread gradually outward and toward the ora serrata with increasing age. Müller cells were not immunoreactive for S-100 during development, except in the retina of the latest fetus examined. S-100 immunoreactivity was also found in the nonpigmented ciliary epithelium and posterior epithelium of the iris, both of which are developed from the inner wall of the optic cup. On the other hand, the pigmented epithelium extending from retina to iris, derived from the outer layer of the optic cup, was free of S-100 immunoreactivity.     

Expression of αB-Crystallin in the Peripapillary Glial Cells of the Developing Chick Retina

Peripapillary glial cells (PPGCs) are a peculiar macroglia in avian species, located in the central retina adjacent to the optic nerve head. PPGCs have a similar shape and orientation to Müller cells, which traverse the entire layer of the retina; however, there are differences in protein expression between the two cell types. In the present study, we first demonstrated that PPGCs expressed αB-crystallin, which is not expressed in Müller cells, during retinal development. αB-crystallin was first faintly expressed in PPGCs of the E5 retina, adjacent to the optic nerve head. Further, αB-crystallin was exclusively expressed in PPGCs up to E14. The shape of these cells was bipolar with vitread and ventricular processes. The vitread processes of αB-crystallin+ PPGCs became finer at E18. Double labeling analysis clearly demonstrated that only vimentin+ or GFAP+ astrocytes were located in the optic nerve head and were demarcated from the retina by αB-crystallin+ PPGCs. Furthermore, we determined that αB-crystallin+ PPGCs, with a number of processes, completely wrapped the optic nerve head and were densely located in the junction of the optic nerve head and the retina in a whole mount preparation and in vertical-sectioned retinae. The results of present study, together with reports that retinal astrocytes migrate from the optic nerve head, suggest that PPGCs prevent astrocytes from migrating into the retina in avian species.     

In-vivo labeling of (3H)D-aspartate uptake sites in monkey retina

Summary Following prolonged topical application of (³H)D-aspartate in vivo, selective labeling of three distinct cell classes was observed in light-microscopic radioautographs from squirrel monkey retina. Müller (glial) cell bodies and their processes were intensely and consistently labeled in all preparations. Moderately labeled perikarya were occasionally detected in the area of bipolar cells, within the inner nuclear layer. These were particularly numerous in sections from the central retina where an intense diffuse labeling of the inner plexiform layer was also prominent. Finally, moderate to dense accumulations of label were observed over the cell bodies, internal segments and fiber processes of cone photoreceptors. These results strongly suggest that cones, as well as a sub-population of bipolar cells, use glutamate and/or aspartate as neurotransmitter(s) in monkey retina.     

Morphological differentiation of the Müller cell: Golgi and electron microscopy study in the chick retina

The sequence of morphological differentiation of Müller cells in the chick retina was investigated in relation to the differentiation of the retinal neurons using the Golgi method. From the beginning of differentiation, the Müller cell develops spurs and lateral processes. Some of these glial processes become transformed into accessory prolongations of the Müller cell. From the 17th or 18th day of incubation, the morphology of the Müller cells is similar to that of the adult retina. On the basis of their inner prolongation, two types of Müller cells were identified. The first type, with diffuse and abundant descending processes, is identical to that described classically. The second type is a cell characterized by sparse and scanty inner ramifications. This report also describes electron microscopic observations of Müller cells and their enwrapping relationship with the axons of the optic nerve fiber layer.     

Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma

Glaucoma is one of the leading eye diseases resulting in blindness due to the death of retinal ganglion cells. This study aimed to develop novel protocol to promote the differentiation of retinal Müller cells into ganglion cells in vivo in a rat model of glaucoma. The stem cells dedifferentiated from rat retinal Müller cells were randomized to receive transfection with empty lentivirus PGC-FU-GFP or lentivirus PGC-FU-Atoh7-GFP, or no transfection. The stem cells were induced further to differentiate. Ocular hypertension was induced using laser photocoagulation. The eyes were injected with Atoh7 expression vector lentivirus PGC-FU-Atoh7-GFP. Eyeball frozen sections, immunohistochemistry, RT-PCR, Western bolt, and apoptosis assay were performed. We found that the proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of the other two groups. The mean intraocular pressure of glaucomatous eyes was elevated significantly compared with those of contralateral eyes. Some retinal Müller cells in the inner nuclear layer entered the mitotic cell cycle in rat chronic ocular hypertension glaucoma model. Atoh7 contributes to the differentiation of retinal Müller cells into retinal ganglion cells in rat model of glaucoma. In conclusion, Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma, thus opening up a new avenue for gene therapy and optic nerve regeneration in glaucoma.     

Distribution of gelsolin in the retina of the developing rabbit

Summary The distribution of gelsolin, a calcium-dependent actin-severing and capping protein, in the retina of the developing and adult rabbit was studied. Gelsolin immunoreactivity was found in the photoreceptors and ganglion cells, where it may have a role in neuronal morphogenesis. Only the inner segment of the photoreceptors retained a high gelsolin content in the adult retina, perhaps because the attached outer segment is continuously renewed throughout life. Gelsolin, which is a major component of the rabbit brain oligodendrocytes, was also found in the myelin of the medullary ray region of the rabbit retina. Müller cells in all regions of the rabbit retina also contain gelsolin from early in development to adulthood. Since one of the functions of these cells is to ensheath neuronal elements in the inner plexiform and optic fiber layers, we suggest that gelsolin may play the same role in Müller cells as it does in oligodendrocytes, i.e., sheath formation via its calcium-dependent action on the actin microfilament networks.     

Astrocytes as gate-keepers in optic nerve regeneration--a mini-review

Animals that develop without extra-embryonic membranes (anamniotes--fish, amphibians) have impressive regenerative capacity, even to the extent of replacing entire limbs. In contrast, animals that develop within extra-embryonic membranes (amniotes--reptiles, birds, mammals) have limited capacity for regeneration as adults, particularly in the central nervous system (CNS). Much is known about the process of nerve development in fish and mammals and about regeneration after lesions in the CNS in fish and mammals. Because the retina of the eye and optic nerve are functionally part of the brain and are accessible in fish, frogs, and mice, optic nerve lesion and regeneration (ONR) has been extensively used as a model system for study of CNS nerve regeneration. When the optic nerve of a mouse is severed, the axons leading into the brain degenerate. Initially, the cut end of the axons on the proximal, eye-side of the injury sprout neurites which begin to grow into the lesion. Simultaneously, astrocytes of the optic nerve become activated to initiate wound repair as a first step in reestablishing the structural integrity of the optic nerve. This activation appears to initiate a cascade of molecular signals resulting in apoptotic cell death of the retinal ganglion cells axons of which make up the neural component of the optic nerve; regeneration fails and the injury is permanent. Evidence specifically implicating astrocytes comes from studies showing selective poisoning of astrocytes at the optic nerve lesion, along with activation of a gene whose product blocks apoptosis in retinal ganglion cells, creates conditions favorable to neurites sprouting from the cut proximal stump, growing through the lesion and into the distal portion of the injured nerve, eventually reaching appropriate targets in the brain. In anamniotes, astrocytes ostensibly present no such obstacle since optic nerve regeneration occurs without intervention; however, no systematic study of glial involvement has been done. In fish, vigorously growing neurites sprout from the cut axons and within a few days begin to re-enervate the brain. This review offers a new perspective on the role of glia, particularly astrocytes, as "gate-keepers;" i.e., as being permissive or inhibitory, by comparison between fish and mammals of glial function during ONR.     

Neuron-specific enolase-like immunoreactivity in the vertebrate retina: selective labelling of Müller cells in Anura

Summary Neuron-specific enolase (NSE) immunocytochemistry was carried out in retinae of goldfish, axolotl, clawed frog, cane toad, lizard, chick, guinea-pig, rabbit, rat, cat and human. With the exception of Anura, strong immunoreactivity was seen in the large ganglion, amacrine cells and horizontal cells of the retina in all of the other species. Photoreceptors were found to be labelled in the rat and human retina and only one cone type in rabbit. Photoreceptor pedicles and ellipsoids were stained in the goldfish and the somata and inner segments of some photoreceptors in axolotl. In the axolotl retina, besides neurons, Müller cells (MCs) were also immunolabelled. In the retina of the cane toad and the clawed frog MCs were the only stained elements. Similarly in other parts of the central nervous system of the cane toad, glial elements of the optic tectum and spinal cord were immunoreactive. In contrast, in the peripheral nervous system, neurons of the 1st sympathetic ganglion and the 2nd dorsal root ganglion were labelled. In double-labelling experiments, glial fibrillary acidic protein and NSE showed colocalisation both in the glial elements of the optic tectum and spinal cord and in MCs of the retina of the cane toad.On leave of absence from Department of Zoology, Attila József University, Szeged, Hungary     

Creatine transporter localization in developing and adult retina: importance of creatine to retinal function

Creatine and phosphocreatine are required to maintain ATP needed for normal retinal function and development. The aim of the present study was to determine the distribution of the creatine transporter (CRT) to gain insight to how creatine is transported into the retina. An affinity-purified antibody raised against the CRT was applied to adult vertebrate retinas and to mouse retina during development. Confocal microscopy was used to identify the localization pattern as well as co-localization patterns with a range of retinal neurochemical markers. Strong labeling of the CRT was seen in the photoreceptor inner segments in all species studied and labeling of a variety of inner neuronal cells (amacrine, bipolar, and ganglion cells), the retinal nerve fibers and sites of creatine transport into the retina (retinal pigment epithelium, inner retinal blood vessels, and perivascular astrocytes). The CRT was not expressed in Müller cells of any of the species studied. The lack of labeling of Müller cells suggests that neurons are independent of this glial cell in accumulating creatine. During mouse retinal development, expression of the CRT progressively increased throughout the retina until approximately postnatal day 10, with a subsequent decrease. Comparison of the distribution patterns of the CRT in vascular and avascular vertebrate retinas and studies of the mouse retina during development indicate that creatine and phosphocreatine are important for ATP homeostasis. photoreceptor; development; glutamine synthetase; neurochemistry     

Differential expression and distribution of Kir5.1 and Kir4.1 inwardly rectifying K+ channels in retina

Kir5.1 is an inwardly rectifying K⁺ channel subunit whose functional role has not been fully elucidated. Expression and distribution of Kir5.1 in retina were examined with a specific polyclonal antibody. Kir5.1 immunoreactivity was detected in glial Müller cells and in some retinal neurons. In the Kir5.1-positive neurons the expression of glutamic acid decarboxylase (GAD₆₅) was detected, suggesting that they may be GABAergic-amacrine cells. In Müller cells, spots of Kir5.1 immunoreactivity distributed diffusely at the cell body and in the distal portions, where Kir4.1 immunoreactivity largely overlapped. In addition, Kir4.1 immunoreactivity without Kir5.1 was strongly concentrated at the endfoot of Müller cells facing the vitreous surface or in the processes surrounding vessels. The immunoprecipitant obtained from retina with anti-Kir4.1 antibody contained Kir5.1. These results suggest that heterotetrameric Kir4.1/Kir5.1 channels may exist in the cell body and distal portion of Müller cells, whereas homomeric Kir4.1 channels are clustered in the endfeet and surrounding vessels. It is possible that homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels play different functional roles in the K⁺-buffering action of Müller cells. inwardly rectifying potassium channel; heteromerization; glial Müller cells; amacrine cells; potassium siphoning     

Neurolin, a cell surface glycoprotein on growing retinal axons in the goldfish visual system, is reexpressed during retinal axonal regeneration.

The mAb E 21 recognizes a cell surface glycoprotein selectively associated with fish retinal ganglion cell axons that are in a state of growth. All retinal axons and ganglion cells in goldfish embryos stained for E 21. In adult fish, however, E 21 immunoreactivity exhibited a patterned distribution in ganglion cells in the marginal growth zone of the continuously enlarging fish retina and the new axons emerging from these cells in the retina, optic nerve, and optic tract. The E 21 antigen was absent from older axons, except the terminal arbor layer in the tectum, the Stratum fibrosum et griseum superficiale where it was uniformly distributed. Upon optic nerve transection, the previously unlabeled axons reacquired E 21 positivity as they regenerated throughout their path to the tectum. Several months after ONS, however, E 21 staining disappeared from the regenerated axons over most of their lengths but reappeared as in normal fish in the terminal arbor layer. The immunoaffinity-purified E 21 antigen, called Neurolin, has an apparent molecular mass of 86 kD and contains the HNK1/L2 carbohydrate moiety, like several members of the class of cell adhesion molecules of the Ig superfamily. The NH2-terminal amino acid sequence has homologies to the cell adhesion molecule DM-Grasp recently described in the chicken. Thus, retinal ganglion cell axons express Neurolin during their development and are able to reexpress this candidate cell adhesion molecule during axonal regeneration, suggesting that Neurolin is functionally important for fish retinal axon growth.