Bernd Biedermann

A potassium channel-linked mechanism of glial cell swelling in the postischemic retina.

The cellular mechanisms underlying glial cell swelling, a central cause of edema formation in the brain and retina, are not yet known. Here, we show that glial cells in the postischemic rat retina, but not in control retina, swell upon hypotonic stress. Swelling of control cells could be evoked when their K(+) channels were blocked. After transient ischemia, glial cells strongly downregulated their K(+) conductance and their prominent Kir4.1 protein expression at blood vessels and the vitreous body. In contrast, the expression of the aquaporin-4 (AQP4) (water channel) protein was only slightly altered after ischemia. Activation of D(2) dopaminergic receptors prevents the hypotonic glial cell swelling. The present results elucidate the coupling of transmembraneous water fluxes to K(+) currents in glial cells and reveal the role of altered K(+) channel expression in the development of cytotoxic edema. We propose a mechanism of postischemic glial cell swelling where a downregulation of their K(+) conductance prevents the emission of intracellularly accumulated K(+) ions, resulting in osmotically driven water fluxes from the blood into the glial cells via aquaporins. Inhibition of these water fluxes may be beneficial to prevent ischemia-evoked glial cell swelling.

Kir potassium channel subunit expression in retinal glial cells: Implications for spatial potassium buffering†

To understand the role of different K+ channel subtypes in glial cell‐mediated spatial buffering of extracellular K+, immunohistochemical localization of inwardly rectifying K+ channel subunits (Kir2.1, Kir2.2, Kir2.3, Kir4.1, and Kir5.1) was performed in the retina of the mouse. Stainings were found for the weakly inward‐rectifying K+ channel subunit Kir4.1 and for the strongly inward‐rectifying K+ channel subunit Kir2.1. The most prominent labeling of the Kir4.1 protein was found in the endfoot membranes of Müller glial cells facing the vitreous body and surrounding retinal blood vessels. Discrete punctate label was observed throughout all retinal layers and at the outer limiting membrane. By contrast, Kir2.1 immunoreactivity was located predominantly in the membrane domains of Müller cells that contact retinal neurons, i.e., along the two stem processes, over the soma, and in the side branches extending into the synaptic layers. The results suggest a model in which the glial cell‐mediated transport of extracellular K+ away from excited neurons is mediated by the cooperation of different Kir channel subtypes. Weakly rectifying Kir channels (Kir4.1) are expressed predominantly in membrane domains where K+ currents leave the glial cells and enter extracellular “sinks,” whereas K+ influxes from neuronal “sources” into glial cells are mediated mainly by strongly rectifying Kir channels (Kir 2.1). The expression of strongly rectifying Kir channels along the “cables” for spatial buffering currents may prevent an unwarranted outward leak of K+, and, thus, avoid disturbances of neuronal information processing. GLIA 39:292–303, 2002.

Role of retinal glial cells in neurotransmitter uptake and metabolism

In addition to photoreceptors and neurons, glial cells (in particular Müller cells) contribute to the removal and metabolization of neurotransmitters in the neural retina. This review summarizes the present knowledge regarding the role of retinal glial cells in the uptake of glutamate, N-acetylaspartylglutamate, gamma-aminobutyric acid, glycine, and d-serine, as well as the degradation and removal of purinergic receptor agonists. Some major pathways of glutamate metabolism in Müller cells are described; these pathways are involved in the glutamate-glutamine cycle of the retina, in the defense against oxidative and nitrosative stress via the production of glutathione, and in the production of substrates for the neuronal energy metabolism. In addition, the developmental regulation of the major glial glutamate transporter, GLAST, and of the glia-specific enzyme glutamine synthetase is described, as well as the importance of a malfunction and even reversal of glial glutamate transporters, and a downregulation of the glutamine synthetase, as pathogenic factors in different retinopathies.

Role of glial K(+) channels in ontogeny and gliosis: a hypothesis based upon studies on Müller cells.

The electrophysiological properties of Müller cells, the principal glial cells of the retina, are determined by several types of K+ conductances. Both the absolute and the relative activities of the individual types of K+ channels undergo important changes in the course of ontogenetic development and during gliosis. Although immature Müller cells express inwardly rectifying K+ (KIR) currents at a very low density, the membrane of normal mature Müller cells is predominated by the KIR conductance. The KIR channels mediate spatial buffering K+ currents and maintain a stable hyperpolarized membrane potential necessary for various glial‐neuronal interactions. During “conservative” (i.e., non‐proliferative) reactive gliosis, the KIR conductance of Müller cells is moderately reduced and the cell membrane is slightly depolarized; however, when gliotic Müller cells become proliferative, their KIR conductances are dramatically down‐regulated; this is accompanied by an increased activity of Ca2+‐activated K+ channels and by a conspicuous unstability of their membrane potential. The resultant variations of the membrane potential may increase the activity of depolarization‐activated K+, Na+ and Ca2+ channels. It is concluded that in respect to their K+ current pattern, mature Müller cells pass through a process of dedifferentiation before proliferative activity is initiated. GLIA 29:35–44, 2000.

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 MΩ, compared to 279 ± 174 MΩ 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. GLIA 20:210–218, 1997.

Expression of potassium channels during postnatal differentiation of rabbit Müller glial cells

The postnatal maturation of Müller glial cells from immature radial glial cells is accompanied by specific changes in the activity of distinct types of K+ channels, as shown by whole‐cell and cell‐attached records on freshly isolated cells from retinae of young (postnatal days 1–30, P1–P30) and adult rabbits. (i) The density of inwardly rectifying currents, providing the main K+ conductance in adult Müller cells, was very low (0.8 pA/pF) from P1 to P6 but increased rapidly thereafter until a relatively stable level of 11.0 pA/pF was established at P17. (ii) Transient (A‐type) K+ currents were expressed in all immature cells at a high density (9.6 pA/pF). After P12, both the percentage of cells with A‐type currents and the peak amplitudes of the currents (2.8 pA/pF) declined. (iii) Delayed rectifying K+ currents developed slowly until after P30. (iv) The postnatal maturation of radial glial cells was accompanied by a strong decrease in the activity of large‐conductance, Ca2+‐activated K+ channels, the open probability of which (measured at the resting membrane potential) decreased from 0.69 at P2–4 to 0.06 at P13–14. The developmental decrease of the activity of Ca2+‐activated K+ channels is assumed to be mainly caused by alteration of the resting membrane potential which developed from low values (–49 mV) at P1–6 to high adult values (–84 mV) after P13. The activity of each distinct type of K+ channel investigated is differently modulated by developmental regulation. This may reflect different functional requirements of immature and mature Müller cells.

Altered membrane physiology in Müller glial cells after transient ischemia of the rat retina.

Inwardly rectifying K+ (Kir) channels have been implicated in the mediation of retinal K+ homeostasis by Müller glial cells. To assess possible involvement of altered glial K+ channel expression in ischemia‐reperfusion injury, transient retinal ischemia was induced in rat eyes. Acutely isolated Müller cells from postischemic retinae displayed a fast downregulation of their Kir currents, which began within 1 day and reached a maximum at 3 days of reperfusion, with a peak decrease to 20% as compared with control. This strong decrease of Kir currents was accompanied by an increase of the incidence of cells which displayed depolarization‐evoked fast transient (A‐type) K+ currents. While no cell from untreated control rats expressed A‐type K+ currents, all cells investigated from 3‐ and 7‐day postischemic retinae displayed such currents. An increased incidence of cells displaying fast transient Na+ currents was observed at 7 days after ischemia. These results suggest a role of altered glial Kir channel expression in postischemic neuronal degeneration via disturbance of retinal K+ siphoning.

Activation of P2Y receptors stimulates potassium and cation currents in acutely isolated human Müller (glial) cells

The ability of various neurotransmitters/neuroactive substances to induce fast, transient rises of Ca2+‐activated K+ currents (IBK) caused by release of Ca2+ from intracellular stores was investigated in Müller glial cells of the human retina. Müller cells were enzymatically isolated from retinas of healthy donors or of patients with proliferative vitreoretinopathy, and the transmembrane ionic currents were recorded using the whole‐cell and the cell‐attached patch‐clamp techniques. The results of the screening experiments indicate that human Müller cells express, in addition to GABAA and perhaps glutamatergic and cholinergic receptors, predominantly P2 receptors. ATP and other nucleotides exerted two effects on membrane currents: repetitive transient increases of the IBK amplitude and, in a subpopulation of cells investigated, the appearance of a transient cation conductance at negative potentials. ATP and UTP increased dose‐dependently the IBK amplitude with half‐maximal effects at 0.33 and 0.50 μM, respectively. Since several different P2 receptor agonists increased the IBK, it is assumed that human Müller cells express a mixture of different types of P2Y receptors. In cell‐attached patches, extracellular application of ATP or UTP transiently increased the open probability of single putative BK channels. The increase of IBK and the appearance of the cation conductance in whole‐cell records were abolished when intracellular Ca2+ was buffered by a high‐EGTA pipette solution or when IP3 was included in the pipette solution. The expression of agonist‐evoked transient cation currents was found to be stronger in cells from patients as compared to cells from healthy donors. It is concluded that human Müller glial cells express P2Y receptors that, via IP3 formation, cause intracellular Ca2+ release. The increased intracellular Ca2+ concentration stimulates the activity of BK channels and may induce opening of cation channels. Both the ATP‐induced activity of BK channels and the increased expression of Ca2+‐gated cation channels may be important in respect to proliferative Müller cell gliosis. GLIA 37:139–152, 2002.

High-affinity GABA uptake in retinal glial (Müller) cells of the guinea pig: electrophysiological characterization, immunohistochemical localization, and modeling of efficiency.

Glial cells may act as important modulators of neuronal information processing, in particular, via fast uptake of neuronally released transmitters. Here, we characterize the electrogenic γ‐aminobutyric acid (GABA) transporters present in the plasma membranes of Müller (glial) cells of the guinea pig retina and present an estimate of their functional efficiency. The GABA‐evoked whole‐cell currents are voltage‐dependent, with increasing amplitudes and decreasing affinity constants at more negative membrane potentials. The transmembranal GABA transport is concentration‐dependent, with near‐maximal currents at 100 μM GABA, and is dependent on extracellular sodium and chloride ions; the stoichiometry is 1 GABA/2 Na+/1 Cl−. Immunohistochemical labeling and whole‐cell voltage‐clamp records reveal that Müller cells express both GAT‐1 and GAT‐3 (but not GAT‐2), and that the transporter proteins are expressed predominantly at plasma membrane sites that, in situ, are localized in the outer retina where GABA uptake is performed exclusively by Müller cells. When extracellular GABA enters the cell interior, it evokes, via activation of the GABA transaminase, an NAD(P)H fluorescence signal selectively in the distal region of the Müller cells where their mitochondria are located. Using our experimental data, we simulated the GABA clearance from the extracellular space surrounding one Müller cell; these estimates show that a pulse of 100 μM extracellular GABA is fully cleared after 70 ms. It is suggested that Müller cells may be involved in the regulation of GABAergic transmission within the retina by providing a fast termination of GABAergic signaling via their highly efficient GABA uptake. GLIA 39:217–228, 2002.

Comparative studies on mammalian Muller (retinal glial) cells

Muller cells from 22 mammalian species were subjected to morphological and electrophysiological studies. In the ‘mid-periphery’ of retinae immunocytochemically labeled for vimentin, estimates of Muller cell densities per unit retinal surface area, and of neuron-to-(Muller) glia indices were performed. Muller cell densities were strikingly similar among the species studied (around 8000–11000 mm−2) with the extremes of the horse (≤5000 mm−2) and the tree shrew (≥20000 mm−2). By contrast, the number of neurons per Muller cell varied widely, being clustered at 6–8 (in retinae with many cones), at about 16, and at up to more than 30 (in strongly rod-dominated retinae). Isolated Muller cell volumes were estimated morphometrically, and cell surface areas were calculated from membrane capacities. Muller cells isolated from thick vascularized retinae (carnivores,rats, mice, ungulates) were longer and thinner, and had smaller volumes but higher surface-to-volume ratios than cells from thin paurangiotic (i.e. with blood vessels only near the optic disc) or avascular retinae (rabbits, guinea pigs, horses, zebras). In whole-cell voltage-clamp studies, Muller cells from all mammals studied displayed two dominant K+ conductances, inwardly rectifying currents and delayed rectifier currents. TTX-sensitive Na+ currents were recorded only in some species. Based on these data, the following hypotheses are presented, (a) neuron-to-(Muller) glia indices are determined by precursor cell proliferation rather than by metabolic demands; (b) Muller cell volumes depend on available space rather than on the number of supported neurons; and (c) it follows that, the specific metabolic activities of Muller cells must differ greatly between species, a difference that may contribute to distinct patterns of retinal vascularization.