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Leukemia inhibitory factor (LIF) is a cytokine that exerts different effects in the nervous system. It is involved in neuronal injuries and diseases and is assumed to be neuroprotective and to regulate reactive gliosis. In LIF-deficient (LIF-/-) mice, expression of glial fibrillary acidic protein in retinal Muller glial cells as a hallmark of reactive gliosis is suppressed during retinal degenerations. Here, we detected expression of LIF and its receptors in Muller cells of the murine retina. Moreover, electrophysiological alterations of Muller cells 7 days after transient retinal ischemia were studied by the patch-clamp technique. The amplitude of inward currents in Muller cells from the postischemic retina was reduced to 51% in wild type and to 70% in LIF-/- mice. This demonstrates that decrease of inward currents takes place in reactive Muller cells even in the absence of LIF.

It has been proposed that glutamate serves as a mediator between neurons and satellite glial cells (SGCs) in sensory ganglia and that SGCs release glutamate. Using a novel method, we studied glutamate release from SGCs from murine trigeminal ganglia. Sensory neurons with adhering SGCs were enzymatically isolated from wild type and transgenic mice in which vesicular exocytosis was suppressed in glial cells. Extracellular glutamate was detected by microfluorimetry. After loading the cells with a photolabile Ca(2+) chelator, the intracellular Ca(2+) concentration was raised in SGCs by a UV pulse, which resulted in glutamate release. The amount of released glutamate was decreased in cells with suppressed exocytosis and after pharmacological block of hemichannels. The data demonstrate that SGCs of the trigeminal ganglion release glutamate in a Ca(2+)-dependent manner.

Signalling of sphingosine-1-phosphate (S1P) via G-protein-coupled receptors of the Endothelial Differentiation Gene family differentially regulates cellular processes such as migration, proliferation and morphogenesis in a variety of cell types. Proliferation and migration of retinal Muller glial cells are involved in pathological events such as proliferative vitreoretinopathy and proliferative diabetic retinopathy. Investigation of possible functional roles of S1P receptors might thus open new insights into Muller cell pathophysiology. Here we show that cultured Muller cells from the guinea pig retina respond to application of S1P with an increase in the intracellular calcium content in a concentration-dependent manner (EC50 11 nM). This calcium increase consists of two components; an initial fast peak and a slow plateau component. The initial transient is caused by a release of calcium from intracellular stores and is suppressed by U-73122, a selective phospholipase C inhibitor. The slow plateau component is caused by a calcium influx. These results suggest that the S1P-induced calcium response in Muller cells partially involves signalling via G-protein-coupled receptors. Moreover, S1P slightly induced Muller cell migration but no proliferation. Thus, the data indicate that Muller cells might be involved in S1P signalling in the retina.

Excessive light causes damage to photoreceptor and pigment epithelial cells, and a local edema in the outer retina. Since Muller glial cells normally mediate the osmohomeostasis in the inner retina (mainly via channel-mediated transport of potassium and water), we determined whether retinal light injury causes an alteration in the retinal localization of glial water (aquaporin-4) and potassium (Kir4.1) channels, and in the potassium conductance of Muller cells. Mice were treated with bright white light (intensity, 15,000 lx) for 2 h. Light treatment results in Muller cell gliosis as indicated by the enhanced staining of the glial fibrillary acidic protein and an increase in the cell membrane area reflecting cellular hypertrophy. In light-injured retinas, the immunostaining of the photoreceptor water channel aquaporin-1 disappeared along with the degeneration of the outer retina, and the outer nuclear layer contained large spherical bodies representing photoreceptor nuclei which were fused together. The immunostainings of the aquaporin-4 and Kir4.1 proteins were increased in the outer retina after light treatment. Since the amplitude of the potassium currents of Muller cells remained largely unaltered, the increase in the Kir4.1 immunostaining is supposed to be caused by a redistribution of the channel protein. The data indicate that Muller glial cells respond to excessive light with an alteration in the localization of Kir4.1 and aquaporin-4 proteins; this alteration is thought to be a response to the edema in the outer retina and may support the resolution of edema.

Glial cells control the water homeostasis in the neural retina, in part via water transport through aquaporin (AQP) water channels. We investigated whether the immunolocalization of two water channels, AQP1 and AQP4, alters in the rat retina during experimental diabetes. Wistar rats were rendered diabetic by a single dose of streptozotocin, and retinal tissues were immunostained following 4 and 6 months. In control tissues, immunoreactive AQP4 was expressed by glial cells (Müller cells and astrocytes) predominantly in the inner retina, and AQP1 was expressed in the outer retina and by distinct amacrine cells. In diabetic retinas, additional strong expression of AQP1 was found in glial cells located in the innermost retinal layers. The superficial retinal vessels were surrounded by AQP4 in control retinas, and by AQP1 in diabetic retinas. A similar alteration in the localization of AQP1 and AQP4 has been described in the rat retina after transient ischemia. The data suggest that the glial cell-mediated water transport in the retina of diabetic animals is altered especially at the superficial vessel plexus.

Water accumulation in retinal glial (Muller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Sigma (σ) receptor activation is known to have neuroprotective effects in the retina. Here, we show that the nonselective σ receptor agonist ditolylguanidine, and the selective σ1 receptor agonist PRE-084, inhibit the osmotic swelling of Muller cell somata induced by superfusion of rat retinal slices with a hypoosmotic solution containing barium ions. In contrast, PRE-084 did not inhibit the osmotic swelling of bipolar cell somata. The effects of σ receptor agonists on the Muller cell swelling were abrogated in the presence of blockers of metabotropic glutamate and purinergic P2Y1 receptors, respectively, suggesting that σ receptor activation triggers activation of a glutamatergic-purinergic signaling cascade which is known to prevent the osmotic Muller cell swelling. The swelling-inhibitory effect of 17β-estradiol was prevented by the σ1 receptor antagonist BD1047, suggesting that the effect is mediated by σ1 receptor activation. The data may suggest that the neuroprotective effect of σ receptor activation in the retina is in part mediated by prevention of the cytotoxic swelling of retinal glial cells.

Glial cells control the retinal osmohomeostasis, in part via mediation of water fluxes through aquaporin (AQP) water channels. By using immunohistochemical staining, we investigated whether ischemia-reperfusion of the rat retina causes alterations in the distribution of AQP1 and AQP4 proteins. Transient ischemia was induced in retinas of Long-Evans rats by elevation of the intraocular pressure for 60 min. In control retinas, immunoreactive AQP1 was expressed in the outer retina and by distinct amacrine cells, and AQP4 was expressed by glial cells (Müller cells and astrocytes) predominantly in the inner retina. After ischemia, retinal glial cells in the nerve fiber/ganglion cell layers strongly expressed AQP1. The perivascular staining around the superficial vessels altered from AQP4 in control retinas to AQP1 in postischemic retinas. The data suggest that the glial cell-mediated water transport in the retina is altered after ischemia especially at the superficial vessel plexus.

Aquaporins are involved in the maintenance of ionic and osmotic balance in the central nervous system and in the eye. Whereas the expression of aquaporin-9 immunoreactivity in the brain has been described for catecholaminergic neurons and glial cells, the expression of aquaporin-9 in the neural retina is unclear. We examined the distribution of aquaporin-1 and -9 immunoreactivities in retinas of the rat. Aquaporin-9 immunoreactivity was expressed exclusively by tyrosine hydroxylase (TH) positive amacrine cells, while aquaporin-1 immunoreactivity was expressed by photoreceptor cells and by TH negative amacrine cells. The staining pattern of aquaporin-9 did not change up to 4 weeks after pressure-induced transient retinal ischemia. It is concluded that catecholaminergic, putatively dopaminergic, amacrine cells of the retina express aquaporin-9.

Aquaporin water channels play a crucial role in the maintenance of ionic and osmotic homeostasis in the neural tissue. In the sensory retina, aquaporin-4 is expressed by Müller glial cells, predominantly in the inner retina, while aquaporin-1 is expressed mainly in the outer retina. However, it is unknown whether aquaporin-1 expression occurs in Müller cells or photoreceptor cells. By using immunohistochemical staining of retinal slices from rds mice, we show that the immunoreactivity for aquaporin-1 disappears along with the photoreceptor cell degeneration. In suspensions of dissociated retinal cells from control mice, photoreceptor cells expressed aquaporin-1 immunoreactivity while Müller cells were largely devoid of staining. The data suggest that photoreceptor cells, but not Müller cells, express aquaporin-1 in the murine retina.

Endothelin-1 (ET-1) is a potent vasoconstrictor that causes hypoperfusion of the neurosensory retina. We investigated immunohistochemically the expression of the receptors for ET-1, ETA and ETB, in control and locally detached retinas of the pig. Immunoreactivity for ETA was expressed in the innermost retinal layers and in the outer plexiform layer in control retinas, and was additionally strongly expressed by retinal blood vessels at 7 days after detachment of the sensory retina from the pigment epithelium. Immunoreactivity for ETB was expressed by the innermost retinal layers, by ganglion cell somata, and by Muller glial cells in the control tissue, and was not altered in its expression after detachment. The vascular expression of ETA may suggest a hypoperfusion of the retina after detachment.

Although spatial buffering of excess extracellular K+ by K+ channels is a main function of retinal glial (Müller) cells, there are severe limitations to long distance K+-spatial buffering that have been predicted for (immature) glial cells: (i) a lack of inwardly rectifying K+ (Kir) channels [Glia 21(1997) 46]; and (ii) high internal resistance of outgrowing (cable like) processes [W. Rall, Handbook of Physiology, Section 1, vol. 1, Part 1, American Physiological Society, Bethesda, 1977, pp. 39-97]. In order to determine if changes in developing Müller cells improve or worsen their capability of carrying K+ spatial buffering currents, we compared the whole-cell currents of acutely isolated Müller cells at 5, 11 and 28 postnatal days of rabbits. Both K+-spatial buffer limitations described above were found in early postnatal stage (5 days), however, the cells overcome these limitations shortly after 11 days. During the period of 11-28 days, rabbit Müller cells simultaneously increase stalk axial conductance and express Kir channels. Both processes take place during the critical stage of retinal maturation, and should dramatically improve "cable" K+-spatial buffering.

To investigate injury-induced alterations of purinergic P2Y receptor-mediated calcium responses in glial (Müller) cells of the rabbit retina, neural retinae were experimentally detached from the pigment epithelium. The ATP-evoked calcium responses were recorded in the endfeet of glial cells at the vitread surface of retinal wholemounts. In control retinae, approximately 7% of the glial cells investigated showed ATP-evoked calcium responses. Within 24 h of detachment, significantly more retinal glial cells (42%) showed calcium responses, and glial ATP responsiveness increased further in retinae which were detached for 48 (44%) or for 72 h (64%). The results indicate that in the detached retina, glial cells upregulate their responsiveness to extracellular ATP within 24 h of injury. Thus, P2Y receptor-mediated signalling may be involved in the early steps of glial response to retinal injury.

Whole-cell voltage-clamp recordings were used to detect voltage-gated Ca(2+) channels in freshly isolated retinal glial (Müller) cells of the toad (Bufo marinus). Using Ca(2+) ions (2 mM) as charge carriers (in the presence of 1 mM Mg(2+)), no inwardly directed currents could be observed during the application of depolarizing voltage steps. However, after omitting the divalent cations from the bath solution, large-amplitude inwardly directed currents were evoked that were carried by Na(+) ions, and were mediated by at least two different kinds of Ca(2+) channels, transient low voltage-activated (LVA) channels and sustained high voltage-activated (HVA) channels. While the LVA currents activated at potentials positive to -90 mV and peaked at -40 mV, the HVA currents activated positive to -60 mV and peaked at -20 mV. It is concluded that Müller glial cells of the toad express distinct types of voltage-gated Ca(2+) channels that may be activated, under certain conditions, close to physiological membrane potentials.

Previous RT-PCR experiments revealed that the neural retina of the rat contains gene transcripts of numerous aquaporins (AQPs), including AQP6 (Tenckhoff et al., Neuroreport 16 (2005) 53-56). In the present study, we investigated the localization of AQP6 immunoreactivity in slices of the rat neural retina, and determined whether blue light injury of the retina affects the tissue distribution of this channel. AQP6 immunoreactivity was found to be selectively localized to the outer plexiform layer. Around the ribbon synapses in this layer, AQP6 labeling was co-localized with the glial water channel AQP4. AQP6 labeling was not colocalized with the marker of horizontal cells, calbindin, nor with the marker of rod bipolar cells, protein kinase Cα. Along with the degeneration of photoreceptor cells after blue light treatment of the retina, AQP6-labeled ribbon synapses disappeared, and a punctate AQP6 staining redistributed into the inner nuclear layer. The co-localization of AQP6 and the glial water channel AQP4 suggests a preferential localization of AQP6 in glial membranes that surround the ribbon synapses in the outer plexiform layer. AQP6 might be involved in the glia-mediated osmo and ion regulation of the extracellular space in this layer.

Retinal Muller glial cells are involved in K+ ion homeostasis of the tissue. Inwardly rectifying K(+) (Kir) channels play a decisive role in the process of spatial K+ buffering. It has been demonstrated that Kir-mediated currents of Muller cells are downregulated in various cases of retinal neurodegeneration. However, this has not yet been verified for any murine animal model. The aim of the present study was to investigate Muller cells after transient retinal ischemia in mice. High intraocular pressure was applied for 1h; the retina was analysed 1 week later. We studied protein expression in the tissue by immunohistochemistry, and membrane currents of isolated cells by patch-clamp experiments. We found the typical indicators of reactive gliosis such as upregulation of glial fibrillary acidic protein. Moreover, the membrane capacitance of isolated Muller cells was increased and the amplitudes of Kir-mediated currents were slightly, but significantly decreased. This murine high intraocular pressure model of transient retinal ischemia is proposed as a versatile tool for further studies on Muller cell functions in retinal degeneration.

Retinae of early postnatal rabbits were enzymatically dissociated and explanted in a culture system. The prospective myelinated region was discarded in order to avoid the presence of astrocytic or mesenchymal cells. After about 14 days in vitro (DIV), outgrowing glial (Muller) cells formed what light optically appeared to be confluent monolayers but by electron microscopy was shown to consist of flat epitheloid cells which overlapped considerably by extension of cytoplasmic tongues. Applying the freeze-fracture technique, apposed membranes of these cells were demonstrated to express infrequently but consistently both gap and tight junctions. This kind of junctions has never been observed on the membrane of rabbit Muller cells in situ. In comparison with Muller cell membranes in situ, the density of intramembrane particles was considerably reduced. Orthogonal arrays of particles which are characteristic elements of Muller cells in situ were not detected. Our results suggest that in homogeneous cell culture, Muller cells form some kind of epithelium-like specialized intercellular junctions. This situation resembles that of closely related glial cell types which form homogeneous layers in situ as e.g. retinal pigment epithelium cells expressing tight junctions, and marginal astrocytes being coupled by extensive gap junctions.

Previous RT-PCR experiments revealed the expression of gene transcripts for a variety of aquaporins in the neural retina, including aquaporin-0. We investigated by immunohistochemistry and Western blotting whether the aquaporin-0 protein is expressed in the retina of the rat. In addition to the lens, immunoreactivity for aquaporin-0 was expressed in the neural retina, but was absent in the pigment epithelium, choroidea, and sclera. In the neural retina, aquaporin-0 immunoreactivity was expressed by the nuclei and the synaptic terminals of protein kinase alpha- and beta-expressing bipolar and amacrine cells, and by the nuclei of neuronal cells in the ganglion cell layer. The immunoreactivity for aquaporin-0 did not co-localize with calbindin, a marker of horizontal cells, or with aquaporin-4, the glial water channel. Transient retinal ischemia caused a slight decrease in the retinal content of aquaporin-0, likely by degeneration of protein kinase alpha-expressing bipolar cells. It is concluded that aquaporin-0 may be involved in the regulation of the activity of retinal second order neurons.

Ischemia–reperfusion of the rat retina causes gliosis of Muller cells that is associated with a decrease of their K + conductance. By using quantitative PCR and immunohistochemical staining of retinal slices, we investigated the effect of transient ischemia–reperfusion on retinal expression of two inward-rectifying K + (Kir) channels, Kir4.1 and Kir2.1. In control retinas, Muller cells prominently expressed both Kir4.1 and Kir2.1 proteins. At 7 days after reperfusion, the expression of Kir4.1 protein was strongly downregulated, while the Kir2.1 protein expression remained unaltered. The expression of Kir4.1 mRNA was reduced by 55% after ischemia while the expression of Kir2.1 mRNA was not altered. The data suggest that the glial expression of distinct Kir channels is differentially regulated after retinal ischemia, with deletarious consequences for K + ion and water homeostasis.

This study was aimed at investigating the postnatal differentiation of cone photoreceptors in the rabbit retina in an organotypic explant culture system. Both short wavelength (S) and middle wavelength (M) cone opsins were expressed in culture but M cones appeared only in retinal explants from the dorsal half of the eye. Stimulating the explants with retinal pigment epithelial cell (RPE) conditioned medium resulted in a suppression of opsin expression despite of an increase of the number of presumptive peanut agglutinin-labeled cones. These results suggest that at birth the immature cones are largely undetermined in terms of their final cone identity although some positional information ('dorsal' vs. 'ventral' retina) is present. Furthermore, factors from RPE may inhibit as well as stimulate different steps of cone cell differentiation.

The cell-attached configuration of the patch-clamp technique was used to characterize the outward currents in acutely isolated Muller cells from the guinea pig retina. Sixty-five of 353 patches displayed macroscopic, outwardly rectifying currents due to depolarizing voltage steps. Single channel transitions were found in only two patches. The remaining patches did not reveal any voltage-dependent currents. Tail current analysis revealed a reversal potential close to the resting membrane potential. The currents disappeared if internal K+ was replaced by Cs+ in inside-out patches. From these results we conclude that guinea pig Muller cells possess voltage-dependent K+ channels that are distributed in clusters.

We studied the antigenic expression of the aspartic proteinase cathepsin E in normal and pathologic human ocular tissues obtained from donors of different age. In the retina the enzyme was immunolocalized in neurons of outer and inner plexiform layers and in few ganglionic neurons. Muller cells were also sometimes immunoreactive for cathepsin E. An increase of neuronal enzyme immunoreactivity with age was evident. Immunocompetent blood cells invading the vitreous body were strongly immunostained for the enzyme. The enzyme is possibly involved in the retinal protein metabolism and might play immunological roles in certain pathologic events.

In retinal light damage, degeneration of photoreceptors may cause alterations of glial (Muller) cells. We performed immunocytochemical studies on Muller cells isolated from retinae of rats exposed to enhanced illumination for 24 months, a procedure which leads to complete loss of photoreceptor cells. One group of rats was fed daily with Ginkgo biloba extract (EGb 761, an established free radical-scavenger) during the last 8 months of life when the remaining photoreceptors (about 50%) die. We found that (1) Muller cells respond to photoreceptor damage by increased expression of glial fibrillary acidic protein, (2) Muller cells reduce expression of glutamine synthetase when the major glutamate-releasing neurons are lost, and (3) the application of exogenous free radical scavengers prevents the expression by Muller cells of the protooncogene protein Bcl-2, a molecule assumed to activate endogenous free radical-scavenging activities.

In thick sections of Golgi-impregnated late fetal rat brains, radial glial cells were measured for both length and diameter of their main (basal) processes. The process diameter was found to decrease proportionally to the square root of cell length; thus, the cytoplasm volume remained fairly constant for cells in the range of lengths studied (100–2500 μm). The measured data were used for calculation of the cell's space constant λ in order to estimate their capability to carry spatial buffering K+ currents. These calculations show that long and slender cells are unable to perform sufficient K+ clearance by such currents. This supports the hypothesis that perinatally when the maturing neurons release enhanced K+ during electrical activity, such long thin cells are subject to long-lasting depolarizations and, thereby, forced to undergo mitotic cell division transforming them into multipolar astrocytes.

Muller (glial) cells from the rabbit retina were isolated by means of papaine and mechanical dissociation. Their Na + ,K + -adenosine triphosphatase (ATPase) activity was measured using a radiochemical method, and its K + dependence was determined. In contrast to that of photoreceptors (data from the literature), the Na + ,K + -ATPase of Muller cells could be shown to increase its activity greatly when the [K + ] was enhanced up to 10 mM. The functional implications of this behaviour for the K + clearance in the retina are discussed.

Radial glial (Muller) cells were isolated from postnatal rabbit retinae by enzymatic dissociation in papain-containing solution, air-dried, and submitted to Pappenheim's panoptic stain. Morphometric data of these cells were evaluated by light microscopy. During postnatal development, the cells become substantially thicker and shorter, their nuclei lose the rod shape and move more toward scleral layers, and the nucleus-cytoplasm volume relation decreases. Whereas the cell volume increases from birth on, substantial outgrowth of fine side branches within the plexiform layers fails to occur before electrical activity is established there, i.e. after postnatal day 9. A model is proposed relating the growth of sheath-bearing glial processes to local protein synthesis stimulated by external K+ accumulation due to neuronal activity. Early myelinated nerve fibers are suggested to bear mechanical resistance to growing radial glial processes thus causing a splitting of these processes when they enter developing nerve fiber layers.

Rabbit retinal Muller (glial) cells were isolated by means of papain and mechanical dissociation. In a special perfusion chamber, the cells were penetrated with a recording microelectrode. Membrane potential changes were recorded in response to extracellular application of both high-K+ solutions and of ouabain, and that during perfusion with normal and Na+-free solutions, respectively. In other Muller cell preparations, Na+,K+-adenosine triphosphatase (ATPase) activity was measured using a radiochemical method, and its Na+ dependence was determined. All results strongly suggest that the Muller cell's Na+,K+ pump can be activated in the presence of extremely low amounts of Na+. This provides additional evidence for significant differences between the glial and the neuronal enzyme.

The cellular localization of cathepsin B and D immunoreactivity was demonstrated at the light micro-scopic level in the retina of adult rabbits by use of the peroxidase-antiperoxidase technique. Antisera were raised against rat liver enzymes. Whereas cathepsin D immunoreactivity was confined to Muller (glial) cells, cathepsin B was demonstrated in some, but not all, neuronal cell types. It is proposed that the two enzymes might carry different functions within the neuronal versus glial compartment.

Muller (glial) cells were isolated from rabbit retinae by papaine and mechanical dissociation. In a special perfusion chamber, the cells were penetrated with a recording electrode. When high-K+ solutions were applied into the environment of the cells by means of a second micropipette, the cell membrane depolarized strongly. During prolonged application of high-K+ solutions, however, there occurred a marked repolarization, and after cessation of high-K+ application, a strong hyperpolarization was observed. Both effects disappeared under the influence of ouabain, suggesting the accumulation of intracellular K+ by an active membrane pump. The data were used for calculation of the membrane's Na+:K+ permeability ratio, the intracellular K+ concentration, the pump rate and the mean pump site density. The calculated values are in good agreement with published data from mammalian astrocytes and are compared with those from amphibian Muller cells.