The Müller (glial) cell in normal and diseased retina: a case for single-cell electrophysiology

In the retina of most vertebrates there exists only one type of macroglia, the Müller cell. Müller cells express voltage-gated ion channels, neurotransmitter receptors and various uptake carrier systems. These properties enable the Müller cells to control the activity of retinal neurons by regulating the extracellular concentration of neuroactive substances such as K+, GABA and glutamate. We show here how electrophysiological recordings from enzymatically dissociated mammalian Müller cells can be used to study these mechanisms. Müller cells from various species have Na(+)-dependent GABA uptake carriers, but only cells from primates have additional GABA receptors that activate Cl- channels. Application of glutamate analogues causes enhanced membrane currents recorded from Müller cells in situ but not from isolated cells. We show that mammalian Müller cells have no ionotropic glutamate receptors but respond to increased K+ release from glutamate-stimulated retinal neurons. This response is involved in extracellular K+ clearance and is mediated by voltage-gated (inwardly rectifying) K+ channels which are abundantly expressed by healthy Müller cells. In various cases of human retinal pathology, currents through these channels are strongly reduced or even extinguished. Another type of voltage-gated ion channels, observed in Müller cells from many mammalian species, are Na+ channels. In Müller cells from diseased human retinae, voltage-dependent Na+ currents were significantly increased in comparison to cells from control donors. Thus, the expression of glial ion channels seems to be controlled by neuronal signals. This interaction may be involved in the pathogenesis of retinal gliosis which inevitably accompanies any degeneration of retinal neurons. In particular, Müller cell proliferation may be triggered by mechanisms requiring the activation of Ca(2+)-dependent K+ channels. Ca(2+)-dependent K+ currents are easily elicitable in Müller cells from degenerating retinae and can be blocked by 1 mM TEA (tetraethylammonium). In purified Müller cell cultures, the application of 1 mM TEA greatly reduces the proliferative activity of the cells. These data clearly show that Müller cells are altered in cases of neuronal degeneration and may be crucially involved in pathogenetic mechanisms of the retina.     

Loss of inwardly rectifying potassium currents by human retinal glial cells in diseases of the eye

We compared the inward K+ currents of Müller glial cells from healthy and pathologically changed human retinas. To this purpose, the whole-cell voltage-clamp technique was performed on noncultured Müller cells acutely isolated from human retinas. Cells originated from retinas of four healthy organ donors and of 24 patients suffering from different vitreoretinal and chorioretinal diseases. Müller cells from organ donors displayed inward K+ currents in the whole-cell mode similar to those found in other species. In contrast, this pattern was clearly changed in the Müller cells from patient retinas. In whole-cell recordings many Müller cells had strongly decreased inward K+ current amplitudes or lost these currents completely. Thus, the mean input resistance of Müller cells from patients was significantly increased to 1,129 +/- 812 M omega, compared to 279 +/- 174 M omega in Müller cells from healthy organ donor retinas. Accordingly, since the membrane potential is mainly determined by the K+ inward conductance in healthy Müller cells, a large amount of Müller cells from patient retinas had a membrane potential which was significantly lower than that of Müller cells from control eyes. The mean membrane potentials were -37 +/- 24 mV and -63 +/- 25 mV for patient and donor Müller cells, respectively. The newly described membrane characteristic changes of Müller cells from patient eyes are assumed to interfere severely with normal retinal function: (1) the retinal K+ homeostasis, which is partly regulated by the Müller cell-mediated spatial buffering, should be disturbed, and (2) the diminished membrane potential should influence voltage-dependent transporter systems of the Müller cells, e.g., the Na(+)-dependent glutamate uptake.     

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We produced the monoclonal antibody RT10F7, characterized its antigenic specificity and expression in the adult and developing retina, in cultured retinal cells and in other parts of the central nervous system. In metabolically-labelled retinal cultures RT10F7 immunoprecipitated a protein of approximately 36,000 mol. wt. In the adult, RT10F7 stained endfeet of Müller cells in the ganglion cell layer, four horizontal bands in the inner plexiform layer, and radial fibres in the outer plexiform layer which terminated at the outer limiting membrane. In the inner nuclear layer, most somata were underlined by Müller processes that wrapped around them, but some cell bodies were immunoreactive for RT10F7 in the cytoplasm. During development, postnatal day 21 was the first age at which the adult pattern of immunoreactivity was present, although a fourth band in the inner plexiform layer was less clear than for the adult. By 14 and eight days after birth, the pattern of RT10F7 immunoreactivity approximated that of the adult; however, only three bands and one band were present, respectively, in the inner plexiform layer. At earlier ages, postnatal days 4, 1 and embryonic ages 19 and 15, the monoclonal antibody stained Müller cell endfeet and radial fibres, from the inner plexiform layer through the neuroblastic layer to the outer limiting membrane. At these ages, the immunoreactivity was more prominent at the level of Müller cell endfeet. The monoclonal antibody stained glia in preparations of dissociated retinal cells maintained in culture but not astrocytes or oligodendrocytes from optic nerve cultures. In brain sections, tanycytes exhibited RT10F7 immunoreactivity. The monoclonal antibody RT10F7 recognized a specific cell type in the retina, the Müller cell. In the adult and developing retina, RT10F7 recognized an antigen that is present primarily in Müller cell processes. This feature allowed us to follow the maturation of the Müller cell and correlate it with developmental events in the retina. RT10F7 is a specific marker for Müller cells in vivo and in vitro and may be useful for studies of function of Müller cells after ablation or after injuries that are known to activate Müller cells.     

Rapid increase in cytosolic calcium ion concentration mediated by acetylcholine receptors in cultured retinal neurons and Müller cells

PURPOSE: This study was conducted to detect the presence of muscarinic or nicotinic receptors in cultured retinal neurons and Müller cells. METHODS: Pure Müller cell cultures and cocultures of retinal neurons and Müller cells were used; the former, obtained from adult rabbit retinas, and the latter, retinal neurons from neonatal rats, were cocultured with Müller cells. Intracellular calcium ion concentration ([Ca2+]i) following the administration of acetylcholine, a cholinesterase inhibitor (trichlorfon), nicotine or muscarinic agonist with or without a receptor antagonist was monitored using the calcium ion indicator, fura-2. RESULTS: Acetylcholine and trichlorfon induced rapid increase in [Ca2+]i in half of either cell type. Trichlorfon induced positive response in coculture but not in the pure Müller cell cultures. This positive response was blocked only partially in the presence of atropine. Approximately 30-40% of neurons responded to nicotine at 5 microM, which was significantly blocked by alpha-bungarotoxin at 50 nM. No response to nicotine could be detected in Müller cells. Approximately 50% of neurons responded to muscarine at 50 microM, but 500 microM was required for the formation of calcium transients in 50% of Müller cells. The muscarine inducement of rapid increase in [Ca2+]i was blocked by atropine. The agonist of M1 (a muscarinic receptor subtype), McN-A-343, at 0.5 microM induced the most significant and rapid increase in [Ca2+]i both in neurons and Müller cells. McN-A-343 administration at 0.05 microM induced positive response in half the neurons, but only in approximately 10% of Müller cells. Such positive response was not observed following preincubation with the M1 antagonist, pirenzepine, at 50 microM. CONCLUSIONS: Cocultured retinal neurons enhance the release of acetylcholine following anticholinesterase administration, and approximately half the neurons were found to possess muscarinic and nicotinic receptors. However, Müller cells appeared to possess only the less sensitive muscarinic receptor. Muscarinic receptor subtypes on either type of cell contained at least M1.     

Müller glia cells and their possible roles during retina differentiation in vivo and in vitro

Müller cells are astrocyte-like radial glia cells which are formed exclusively in the retina. Here we present evidence that Müller cells are crucially involved in the development of the retina's architecture and circuitry. There is increasing evidence that Müller cells are present from the very early beginning of retinogenesis. We postulate the "gradual maturation hypothesis of Müller cells". According to this hypothesis, Müller cells are continuously generated by a gradual transition of neuroepithelial stem cells into mature Müller cells. This process may be partly reversible. Müller cells, or their immature precursors, are able to subserve different functions. They are primary candidates for stabilizing the complex retinal architecture and for providing an orientation scaffold. Thereby, they introduce a reference system for the migration and correct allocation of neurons. Moreover, they may provide spatial information and microenvironmental cues for differentiating neurons, and may also be important for the segregation of cell and fibre layers. Additionally, they seem to be involved in the guidance of axonal fibres both in radial and in lateral directions, as they are involved in the support and stabilization of synapses.     

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Gap junctional communication between glial cells is thought to play a role in K+ spatial buffering, in the propagation of inter-astrocytic Ca2+ waves, and in glial-neuronal signaling. In the present study, we characterize dye coupling between astrocytes, and between astrocytes and Müller cells, in the isolated rat retina. Whole-cell patch recordings were obtained from retinal astrocytes and Müller cells and the cells filled with Lucifer Yellow and neurobiotin. Spread of Lucifer Yellow to two to ten neighboring astrocytes occurred in 90% of the astrocyte recordings. After fixation and incubation of the retina with fluorescent conjugated streptavidin, neurobiotin was seen to label clusters of 13-88 astrocytes, as well as > 100 Müller cells. In contrast, when Müller cells were filled with Lucifer Yellow and neurobiotin, both tracers were confined solely to the recorded Müller cell. The uncoupling agents octanol, halothane, and doxyl-stearic acid were tested for their ability to uncouple retinal glia in situ. All three agents eliminated the visible spread of Lucifer Yellow from the injected astrocyte and the spread of neurobiotin into Müller cells. However, only doxyl-stearic acid combined with octanol eliminated the spread of neurobiotin between astrocytes. These results demonstrate that astrocytes in the rat retina are coupled to each other and to Müller cells. The astrocyte-to-Müller cell coupling is asymmetric, allowing transfer of the tracer in the forward direction only. In addition, astrocyte-to-Müller cell coupling is more sensitive to the uncoupling agents tested than is astrocyte-to-astrocyte coupling.     

Distribution of mitochondria within Müller cells--I. Correlation with retinal vascularization in different mammalian species

The distribution of mitochondria within retinal glial (Müller) cells and neurons was studied by electron microscopy, by confocal microscopy of a mitochondrial dye and by immunocytochemical demonstration of the mitochondrial enzyme GABA transaminase (GABA-T). We studied sections and enzymatically dissociated cells from adult vascularized (human, pig and rat) and avascular or pseudangiotic (guinea-pig and rabbit) mammalian retinae. The following main observations were made. (1) Müller cells in adult euangiotic (totally vascularized) retinae contain mitochondria throughout their length. (2) Müller cells from the periphery of avascular retinae display mitochondria only within the sclerad-most end of Müller cell processes. (3) Müller cells from the vascularized retinal rim around the optic nerve head in guinea-pigs contain mitochondria throughout their length. (4) Müller cells from the peripapillar myelinated region ('medullary rays') of the pseudangiotic rabbit retina contain mitochondria up to their soma. In living dissociated Müller cells from guinea-pig retina, there was no indication of low intracellular pH where the mitochondria were clustered. These data support the hypothesis that Müller cells display mitochondria only at locations of their cytoplasm where the local O2 pressure (pO2) exceeds a certain threshold. In contrast, retinal ganglion cells of guinea-pig and rabbit retinae display many mitochondria although the local pO2 in the inner (vitread) retinal layers has been reported to be extremely low. It is probable that the alignment of mitochondria and the expression of mitochondrial enzymes are regulated by different mechanisms in various types of retinal neurons and glial cells.     

Regulation of insulin-like growth factors I and II and their binding proteins in human bone marrow stromal cells by dexamethasone

Müller cells are highly permeable to potassium ions and play a major role in maintaining potassium homeostasis in the vertebrate retina during light-evoked neuronal activity. Potassium fluxes across the Müller cell's membrane are believed to underlie the light-evoked responses of these cells. We studied the potassium currents of turtle Müller cells in the retinal slice and in dissociated cell preparations and their role in the genesis of the light-evoked responses of these cells. In either preparation, the I-V curve, measured under voltage-clamp conditions, consisted of inward and outward currents. A mixture of cesium ions, TEA, and 4-AP blocked the inward current but had no effect on the outward current. Extracellular cesium ions alone blocked the inward current but exerted no effect on the photoresponses. Extracellular barium ions blocked both inward and outward currents, induced substantial depolarization, and augmented the light-evoked responses, especially the OFF component. Exposing isolated Müller cells to a high potassium concentration did not cause any current or voltage responses when barium ions were present. In contrast, application of glutamate in the presence of barium ions induced a small inward current that was associated with a substantially augmented depolarizing wave relative to that observed under control conditions. This observation suggests a role for an electrogenic glutamate transporter in generating the OFF component of the turtle Müller cell photoresponse.     

Müller cells in the retina of the cane toad, Bufo marinus, express neuropeptide Y-like immunoreactivity

We have used light- and electron-microscopic immunohistochemistry to identify the presence of immunoreactivity to neuropeptide Y (NPY) within Müller cells in the retina of the cane toad, Bufo marinus. Müller cells containing NPY-like immunoreactivity (NPY-LI) were identified at the light-microscopic level by the coexistence with immunoreactivity to glial fibrillary acidic protein (GFAP) and at the ultrastructural level by their characteristic relationship to neuron cell bodies and processes. At the light-microscopic level, those cells which contained both NPY-LI and GFAP-LI usually had small cell bodies in the inner nuclear layer, while those cells which contained only NPY-LI were identified as large and small amacrine cells. The radially oriented primary processes in the inner plexiform layer and the vitreal end feet of GFAP-LI Müller cells also expressed NPY-LI. At the ultrastructural level, thin lamellar processes of Müller cells with NPY-LI enclosed some amacrine cell bodies in the inner nuclear layer and amacrine cell dendrites in the inner plexiform layer. These observations suggest that NPY-LI is localized in Müller cells in addition to two types of amacrine cells previously identified in the Bufo retina. This study provides the first evidence that glial elements in the vertebrate retina express NPY-LI.     

Obliterative angiopathy of placental stem villi (author''s transl)]

Fixed retinae of chick embryos and chicks of the first week after hatching were fractured and examined with the scanning electron microscope. The matrix cells of the retina proliferate up to the beginning of the second week. The migrating cells are oriented in cell cords. This columnar organizaion prevails up to the development of the plexiform layers formed as a consequence of the outgrowth of the dendritic and axonal cell processes. Special attention was paid to the differentiation of the ganglion, bipolar and receptor cells, and the radial fibers (Müller cells). Two main morphological patterns are significant for the organization of the retina during neurogenesis: a)the cell to cell contacts of migrating cells and b)the spatial arrangement of Müller cells which could provide guidelines for migration of neuronal elements.     

Alterations in the Ha-ras-1 and the p53 pathway genes in the progression of N-methyl-N-nitrosourea-induced rat mammary tumors

Glutamate is the most prominent excitatory neurotransmitter in the retina and brain. It has become clear that the physiology of many glial cells, including retinal Müller cells, is modified by a host of neurotransmitters, including glutamate. The experiments presented here demonstrate that Müller cells isolated from the tiger salamander retina have metabotropic glutamate receptors that, when activated, lead to the release of calcium ions (Ca2+) from intracellular stores. The Ca2+-sensitive fluorescent dye, Fura-2, and video imaging microscopy were used to monitor changes in cytosolic calcium ion concentration ([Ca2+]i) evoked by glutamate (30-50 microM), (1S,3R)-ACPD (50-200 microM), quisqualate (10-50 microM), and L-AP4 (5-100 microM). Bath application of each of these metabotropic receptor agonists in the absence of extracellular Ca2+ resulted in an increase in [Ca2+]i that often began in the distal end of the cell and occurred later in the endfoot. This wavelike increase in [Ca2+]i is reminiscent of the Ca2+ waves evoked in these cells by other Ca2+ releasing agents such as ryanodine and caffeine. Extracellular application ofATP also evoked increases in [Ca2+] in Müller cells. The presence on Müller cells of receptors for retinal neurotransmitters, such as glutamate and ATP, demonstrates that these glial cells can respond to changes in the retinal extracellular environment and hence neuronal activity. Since Müller cells span almost all layers of the retina, they are likely to be exposed to most retinal neurotransmitters. The Ca2+ waves evoked in Müller cells by neurotransmitters could represent a form of signaling from the outer retinal layers to the inner ones.     

Prolonged delivery of brain-derived neurotrophic factor by adenovirus-infected Müller cells temporarily rescues injured retinal ganglion cells

In this study, we demonstrate that: (i) injection of an adenovirus (Ad) vector containing the brain-derived neurotrophic factor (BDNF) gene (Ad.BDNF) into the vitreous chamber of adult rats results in selective transgene expression by Müller cells; (ii) in vitro, Müller cells infected with Ad.BDNF secrete BDNF that enhances neuronal survival; (iii) in vivo, Ad-mediated expression of functional BDNF by Müller cells, temporarily extends the survival of axotomized retinal ganglion cells (RGCs); 16 days after axotomy, injured retinas treated with Ad.BDNF showed a 4.5-fold increase in surviving RGCs compared with control retinas; (iv) the transient expression of the BDNF transgene, which lasted approximately 10 days, can be prolonged with immunosuppression for at least 30 days, and such Ad-mediated BDNF remains biologically active, (v) persistent expression of BDNF by infected Müller cells does not further enhance the survival of injured RGCs, indicating that the effect of this neurotrophin on RGC survival is limited by changes induced by the lesion within 10-16 days after optic nerve transection rather than the availability of BDNF. Thus, Ad-transduced Müller cells are a novel pathway for sustained delivery of BDNF to acutely-injured RGCs. Because these cells span the entire thickness of the retina, Ad-mediated gene delivery to Müller cells may also be useful to influence photoreceptors and other retinal neurons.     

Late proliferation of retinal Müller cell progenitors facilitates preferential targeting with retroviral vectors in vitro

During vertebrate neural retina development, the relationship between mitotic activity in progenitor cells and the acquisition of a mature cell phenotype remains an area of controversy. The Müller glial cell has long been recognized as one of the last cell types of the retina to mature, which occurs under the influence of cell-cell interactions. In this report we examine the acquisition of the Müller cell phenotype in relation to mitotic activity. Using immunohistochemical markers, we demonstrate that a gene product characteristic of mature Müller cells, the 2M6 antigen, is expressed in mitotically active cells, even after all the major retina architectural features have been laid down. Furthermore, we show that retroviral infection, a process that requires mitotically active cells, preferentially targets Müller cell progenitors when late embryonic retina is infected in vitro. The two lines of evidence are consistent with a model for Müller cell differentiation that includes a mitotically active progenitor that has already begun to express specific differentiation gene products.     

Tractional force generation by porcine Müller cells. Development and differential stimulation by growth factors

PURPOSE: To assess the ability of retinal Müller cells to generate tractional forces during dedifferentiation in culture and to assess their responsiveness to contraction-stimulating growth factors. METHODS: Müller cells were isolated from papain-DNase-digested porcine retina. The identity of the isolated cells was confirmed by immunodetection of carbonic anhydrase II (CA-II), cellular retinaldehyde-binding protein (CRALBP), glial fibrillary acidic protein (GFAP), vimentin, and alpha smooth muscle actin (alpha SMA). Tractional force generation was assessed as a function of Müller cell contraction of collagenous extracellular matrices in vitro. The effects of potential promoters were assessed by addition directly to culture medium. The contributions of specific promoting to the contraction-promoting activity in serum were assessed by adding neutralizing antibodies and measuring loss of stimulatory activity. RESULTS: Freshly isolated Müller cells did not generate substantial matrix contraction. However, this activity increased 150-fold within 12 days in culture and continued to increase during the next 21 days. Development of the capacity for extracellular matrix contraction coincided with the acquisition of immunodetectable alpha SMA and loss of GFAP. Matrix contraction by Müller cells was stimulated in a dose-dependent fashion by human serum, platelet-derived growth factor (PDGF), and insulin-like growth factor-I (IGF-I). Müller cells were not stimulated by transforming growth factor beta 1 (TGF beta 1), transforming growth factor beta 2 (TGF beta 2), or endothelin-1 (E1). Neutralizing antibodies against PDGF and IGF-I reduced the activity in human serum by 37% and 58%, respectively, and 87% when added together. CONCLUSIONS: Porcine Müller cells in culture acquire the ability to contract extracellular matrices and thus generate tractional forces. Acquisition of this activity coincides with alpha SMA expression and loss of GFAP. Further, this activity is dependent on the presence of exogenous promoters, including PDGF or IGF-I.     

Trafficking of molecules and metabolic signals in the retina

Photoreceptors need the support of pigment epithelial (PE) and Müller glial cells in order to maintain visual sensitivity and neurotransmitter resynthesis. In rod outer segments (ROS), all-trans-retinal is transformed to all-trans-retinol by retinol dehydrogenase using NADPH. NADPH is restored in ROS by the pentose phosphate pathway utilizing high amounts of glucose supplied by choriocapillaries. The retinal formed is transported to PE cells where regeneration of 11-cis-retinal occurs. Müller cells take up and metabolize glucose predominantly to lactate which is massively released into the extracellular space (ES). Lactate is taken up by photoreceptors, where it is transformed to pyruvate which, in turn, enters the Krebs cycle in mitochondria of the inner segment. Stimulation of neurotransmitter release by darkness induces 130% rise in the amount of glutamate released into ES. Glutamate is transported into Müller cells where it is predominantly transformed to glutamine. Stimulation of photoreceptors induces an eightfold increase in glutamine formation. It appears, therefore, that there is a signaling function in the transfer of amino acids from Müller cells to photoreceptors. Work on the model-system of the honeybee retina demonstrated that photoreceptors release NH4+ and glutamate in a stimulus-dependent manner which, in turn, contribute to the biosynthesis of alanine in glia. Alanine released into the extracellular space is taken up and used by photoreceptors. Glial cells take glutamate by high-affinity transporters. This uptake induces a transient change in glial cell metabolism. The transformation of glutamate to glutamine is possibly also controlled by the uptake of NH4+ which directly affects cellular metabolism.     

Diffuse lamellar keratitis. A new syndrome in lamellar refractive surgery

PURPOSE: Alpha2-adrenergic agonists have specific and selective effects on the retina to induce expression of basic fibroblast growth factor and to protect photoreceptors. This work explores the signaling pathway that mediates these effects. METHODS: Alpha2-adrenergic agonists xylazine and clonidine were administered systemically to male adult Sprague-Dawley rats. The activation state of extracellular signal-regulated kinases (ERKs) in the retina was assessed by immunoblot analysis, using antibodies that specifically recognize the dually phosphorylated forms of p44/p42 ERKs. Localization of phosphorylated ERKs was determined by immunocytochemistry. RESULTS: Intramuscular injection of 6 mg/kg xylazine induced an increase in ERK phosphorylation in the retina within 30 minutes that lasted 3 hours. Xylazine induced ERK phosphorylation at 1 mg/kg and reached a maximum at 10 mg/kg. Injection of clonidine also induced ERK phosphorylation in the retina. Yohimbine, a specific alpha2-adrenergic antagonist, completely prevented the induction of ERK phosphorylation. Immunocytochemical studies showed that the increase in ERK phosphorylation occurred mainly in Müller cells. In the brain, xylazine injection resulted in a decrease in ERK phosphorylation. CONCLUSIONS: Our results indicate that systemically administered alpha2-adrenergic agonists selectively activate ERKs in retinal Müller cells. The induced activation of ERKs in Müller cells is probably one of the early events that result in photoreceptor protection. These results also indicate that Müller cells are unique in response to alpha2-adrenergic agonists and imply a role for Müller cells in alpha2-adrenergic agonist-induced photoreceptor protection.     

NADPH diaphorase activity in mammalian retinas is modulated by the state of visual adaptation

NADPH diaphorase histochemistry is commonly used to identify cells containing nitric oxide synthase (NOS), the enzyme catalyzing the production of nitric oxide from L-arginine. NADPH diaphorase activity and NOS immunostaining was demonstrated in different cells of the vertebrate retina; photoreceptors, horizontal cells, amacrine cells, ganglion cells, and Müller cells. However, the physiological role of nitric oxide (NO) in the retina has yet to be elucidated. In this study, we tested the assumption that NADPH diaphorase activity in the retinas of rabbits and rats depended on the state of visual adaptation. In the rabbit, light adaptation enhanced NADPH diaphorase activity in amacrine cells and practically eliminated it in horizontal cells. Dark adaptation induced the opposite effects; the NADPH diaphorase activity was reduced in amacrine cells and enhanced in horizontal cells. Retinas from eyes that were injected intravitreally with L-glutamate exhibited a pattern of NADPH diaphorase activity that was similar to that seen in dark-adapted retinas. In rats, the NADPH diaphorase activity of amacrine and horizontal cells exhibited adaptation dependency similar to that of the rabbit retina. But, the most pronounced effect of dark adaptation in the rat's retina was an enhancement of NADPH diaphorase activity in Müller cells, especially of the endfoot region. Assuming that NADPH diaphorase activity is a marker for NOS, these findings suggest that NO production in the mammalian retina is modulated by the level of ambient illumination and support the notion that NO plays a physiological role in the retina.     

Retinal light damage vs. normal aging of rats: altered morphology, intermediate filament expression, and nuclear organization of Müller (glial) cells

In retinal light damage, degeneration of photoreceptors results in alterations of glial (Müller) cells. In particular, Müller cells show signs of gliosis such as thickening of their stem processes, and expression of glial fibrillary acidic protein (GFAP) which is normally not detectable by immunocytochemistry. We were interested in a quantification of these morphological alterations, and in possible effects of an application of free radical scavengers (Ginkgobiloba extract EGb 761). For this purpose, we studied Müller cells in retinae of albino rats exposed to enhanced illumination for 24 months, a procedure which causes a complete loss of photoreceptor cells. The cells were labeled by (i) bulk filling with the fluorescent dye, Procion yellow, and by (ii) immunocytochemical demonstration of vimentin and GFAP. One group of rats was fed daily with EGb 761 during the last 8 months of life when the remaining photoreceptors (about 50%) died. The retinae were compared with retinae from 3 months-old albino rats, serving as normal young controls, and with retinae from 24 month-old pigment rats, representing normal aging processes. As age-related changes of the ultrastructure of glial cell (astrocytic) nuclei have been described in the literature, the organization of Müller cell nuclei was also studied by an argyrophilic stain, and by electron microscopy. We found that in the thin light-damaged retinae, Müller cells were shorter but thicker than in age-matched control retinae. The volumes of their vitread stem processes were almost unchanged. Müller cells were GFAP-immunoreactive in the light-damaged retinae but not in the controls. The application of EGb 761 prevented the expression by Müller cells of (detectable levels of) GFAP. By contrast, in retinae from EGb 761-treated animals the volumes of the vitread stem processes were significantly increased in comparison to untreated animals. The number of nuclear organization regions was significantly enhanced in Müller cell nuclei from light-damaged untreated albino rats, as compared with the young controls. Application of EGb 761 prevented much of this increase. Thus, exogeneous free radical scavengers do not prevent the occurrence of an reactive hypertrophy but inhibit the expression of "pathological marker molecules", and the (accompanying) signs of enhanced nuclear activity.     

Eph family transmembrane ligands can mediate repulsive guidance of trunk neural crest migration and motor axon outgrowth

The establishment of cell and fibre layers and the specification of different cell types are crucial processes during development of the central nervous system. Here we investigated the developmental architecture of radial glia cells in these processes using so-called spheroids that arise from dissociated chicken embryonic neural cells in rotation culture. We were able to produce retinal, tectal, and telencephalic spheroids from E6 embryos and cerebellar spheroids from E10 embryos. Cell and fibre differentiation can be observed in all types of spheroids, however, it is most abundant in retinal spheroids. Moreover, only in retinal spheroids a histotypic organization can be detected. Using immunohistochemistry and electron microscopy, we assign this -at least partially- to the capacity of Müller cells to form radial scaffolds, since we observe a congruency between these radial scaffolds and the presence of rosettes formed by photoreceptor precursors and Müller cells. Tectal, telencephalic and cerebellar spheroids do not show organized radial glia scaffolds, instead, the radial glia cells are randomly arranged and the spheroids do not show histotypical organization. The application of the specific gliotoxin 6-aminonicotinamide to growing retinal spheroids leads to a significant decrease in the number and size of the rosettes. Concomitantly, the degree of histotypical organization is also drastically reduced. This organizing capacity of Müller cells in vitro now strongly suggests the presence of a comparable function also in vivo. Moreover, since non-retinal radial glia cells are not able to re-organize an histotypic organization in vitro, Müller cells seem to be qualitatively different from other radial glia cells. In future studies we want to untangle these differences.     

Na+ channels are expressed by mammalian retinal glial (Müller) cells

Müller cells constitute the principal glia of the vertebrate retina. Unlike other types of neuroglial cells such as astrocytes and Schwann cells, Müller cells have not yet been demonstrated to express Na+ channels. Here we present first evidence of Müller cell Na+ currents from voltage-clamp studies in enzymatically isolated cells. Some cells from retinae of cats and dogs, but none from rabbit or guinea-pig retinae, revealed fast and rapidly inactivating inward currents in response to depolarizing voltage steps. The currents reversibly disappeared in Na+ free solutions or under tetrodotoxin (TTX, 1 microM). Activation and inactivation characteristics of these currents were strikingly similar to those of neurone-type Na+ channels.