Cell Biology of the Müller Cell

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Chapter 17 Cell Biology of the Müller Cell

Andreas Reichenbach, Andreas Bringmann

Introduction

In addition to microglial cells, which are the resident immune cells of the retina, the retina contains two types of macroglial cell: astrocytes and Müller cells (Fig. 17.1A). Macroglial cells support the functioning and metabolism of retinal neurons. For this reason, they constitute an anatomical and functional link between these neurons and the compartments with which they need to exchange molecules (blood vessels, vitreous chamber, and subretinal space). Most nutrients, waste products, ions, water, and other molecules are transported through macroglial cells between the retinal vessels and neurons. As a peculiarity, astrocytes play a crucial role in retinal vascularization; the localization of these cells in the mature retina is restricted to the nerve fiber and ganglion cell layers.

Fig. 17.1 Müller cells (M) span the entire thickness of the neuroretina, and are arranged in a regular pattern. (A) Schematic drawing of the cellular constituents of a human retina. The perikarya of Müller cells are localized in the inner nuclear layer (INL). The funnel-shaped endfeet of Müller cells form the inner surface of the retina. In the outer (OPL) and inner plexiform layers (IPL), side branches which form perisynaptic membrane sheaths originate at the stem processes. Both astrocytes (AG) and Müller cells contact the superficial blood vessels (BV) and the inner surface of the retina. In the outer nuclear layer (ONL), the stem process of Müller cells forms membrane sheaths which envelop the perikarya of rods (R) and cones (C). Microvilli of Müller cells extend into the subretinal space which surrounds the photoreceptor segments (PRS). Microglial (MG) cells are located in both plexiform layers and the ganglion cell layer (GCL). A, amacrine cell; B, bipolar cell; G, ganglion cell; H, horizontal cell; P, pericyte; RPE, retinal pigment epithelium. (B–D) Confocal images of living guinea pig retina preparations. (B) Radial section, illustrating the tight package of the cellular elements shown in (A). The Müller cells are labeled with the vital dye, Mitotracker Orange (green); synapses and the outer segments of photoreceptor cells are counterlabeled with another vital dye, FM-43. (C, D) Optical horizontal sections through a flat-mounted retina, illustrating the regular pattern of Müller cell stem processes in the inner plexiform layer (D) and the almost total occupation of the ganglion cell layer by the Müller cell endfeet (green; M); only the somata of the ganglion cells (G) appear “empty” (C).

Morphology of müller cells

Heinrich Müller (1820−1864) in 1851 described the “radial fibers” of the retina which later became known as Müller cells. Müller cells are specialized radial glial cells which span the entire thickness of the neural retina, from the subretinal space to the vitreal surface (Fig. 17.1A, B). The somata of the cells are located in the inner nuclear layer, and two stem (trunk) processes radiate from the soma in opposite directions. The outer stem process draws towards the subretinal space; here, microvilli of Müller cells surround the photoreceptor inner segments. Zonulae adherentes between Müller and photoreceptor cells form the outer limiting membrane of the retina. The inner stem process projects towards the vitread surface of the retina; the end of this process is expanded into a funnel-shaped endfoot. Both the basement membrane at the inner retinal surface and the Müller cell endfoot membranes together constitute the inner limiting membrane. Lateral processes extend into the plexiform layers where they form elaborate sheaths around the neuronal synapses, as well as into the nuclear layers where they embed neuronal perikarya. Astrocytes and Müller cells ensheath the vessels of the superficial vascular plexus; the deep capillaries are ensheathed by en passant endfeet of Müller cells. Within the macula, Müller cells display a Z-shaped morphology because the outer processes accompany the centrifugally running cone axons in the Henle fiber layer (Figs 17.2 and 17.3C). The central fovea contains 20–30 atypical Müller cells. These cells are not associated with cone axons; instead, their outer process runs straight towards the inner retinal surface where the soma is located and where their large, flat endfeet contribute to the inner limiting membrane of the fovea proper.¹

Fig. 17.2 Müller cells of the primate macula are elongated and run in parallel with the Henle fibers. (A) Scanning electron microphotograph of the fovea of a macaque. The extremely high packing density of cone inner and outer segments in the photoreceptor segment layer (PSL), the complex centrifugal course of their axons crowded in the Henle fiber layer (HFL), and the absence of the ganglion cell (GCL), inner plexiform (IPL), inner nuclear (INL), and outer plexiform layers (OPL) are illustrated. The outer processes of Müller cells run among the Henle fibers but cannot be reliably identified. (B) When the Müller cell processes are visualized by vimentin immunohistochemistry (in brown; counterstaining of cell nuclei by hematoxylin and eosin; siamang retina), they can be traced along their path from the outer margin of the outer nuclear layer (ONL) up to their endfeet; it becomes obvious that they run in parallel with the Henle fibers.

Fig. 17.3 Müller cells form the cores of radial units of retinal neurons. Every Müller cell is surrounded by a distinct group of retinal neurons (A) with which it interacts specifically during development, in the mature functioning retina, and after injury of the retina. These repetitive radial units are almost identical throughout most regions of the retina (B) but differ in the (peri-)fovea (C) by the lack of rods, an increased number of neurons of the inner nuclear and ganglion cell layers, and by an elongation and Z-shaped course of the outer processes of Müller cells (cf. also Fig. 17.2). Blue, Müller cells; yellow, retinal neurons; orange, cones; brown, rods.

Müller cells constitute the cores of functional retinal columns

The human retina contains 8–10 million regularly arranged Müller cells (Figs 17.1D and 17.4C). Each Müller cell constitutes the core of a column of retinal neurons which represents the smallest functional unit for “forward information processing.”² Such a column contains one cone per Müller cell (Figs 17.3B, C and 17.4B, C) and up to 10 rods, as well as 6 (fovea) and 4 (periphery) inner nuclear layer neurons, and 2.5 (fovea) and 0.3 (periphery) ganglion cell layer neurons, respectively.¹

Fig. 17.4 Müller cells are “optical fibers” guiding the incoming light to the photoreceptor cells. (A) Artist’s view of the optical path through the retina. Light arrives at the inner retinal surface where it hits the Müller cell endfeet. Then it propagates through the Müller cell stem processes (thus bypassing the light-scattering retinal structures, particularly in the two plexiform layers) until it arrives at the photoreceptor cells. Every Müller cell illuminates a distinct group of photoreceptors, about 10 rods and one cone. (B, C) Vertical (B) and horizontal (C) sections through the guinea pig retina. Müller cells were immunolabeled for vimentin (green), and cones were labeled by peanut agglutinin (red). (B) The cone inner segments in the photoreceptor segment layer (RSL) appear continuous with the outer processes of Müller cells which envelop the cone perikarya in the outer nuclear layer (ONL). (C) The focus of the image is on the outer plexiform layer (OPL). The number of cone pedicles (red) roughly equals that of Müller cell processes (green); often the two elements appear as pairs. Thus, each cone receives its part of the image of the outside world by its “individual” light-guiding Müller cell.

Müller cells interact with the neurons of their columns in a kind of symbiotic relationship. They are responsible for all functional and metabolic support of their associated neurons. Müller cells provide trophic substances to neurons, and remove metabolic waste. They play a critical role in regulation of the extracellular space volume, ion and water homeostasis, and in the maintenance of the inner blood–retinal barrier. They release gliotransmitters and other neuroactive substances, and impact synaptic activity by neurotransmitter recycling which involves the supply of neurons with precursors of neurotransmitters. All of these functions directly or indirectly modify the neuronal activity. Müller cells support the survival of photoreceptors and neurons, are responsible for the structural stabilization of the retina, and are modulators of immune and inflammatory responses. They guide the light to the photoreceptors and buffer mechanical tissue deformations. Müller cells become activated upon virtually all pathogenic stimuli. Reactive Müller cells are neuroprotective but may also stop supporting the neurons, and rather contribute to neuronal degeneration. Currently, the many roles of Müller cells in the regulation of retinal function are still not fully resolved, and are the subject of ongoing intensive research. Noteworthy, much of the current knowledge about Müller cell (dys-)function was obtained in animal models and thus awaits confirmation on human cells.

Light guidance

Because the retina is inverted, light has to pass the entire depth of the neuroretina before it arrives at the photoreceptors. In the retina as in any other organ, all cells and their processes and organelles are phase objects which scatter the incoming light. In particular, the synapses in the plexiform layers have dimensions close to 500 nm, i.e., within the wavelength range of visible light; this makes them light-scattering structures.³ The backscattering of light from the retinal cell structures is clinically used by optical coherence tomography. However, light scattering should reduce visual sensitivity and acuity, particularly outside the fovea (where the inner layers are shifted aside, and the light directly hits the cones).

To reduce light scattering, nonfoveal Müller cells act as living optical fibers that guide the light through the inner retinal layers towards the photoreceptors (Fig. 17.4A).³ The funnel-shaped endfeet of Müller cells act as light collectors at the vitread surface of the retina. The refractory index of Müller cells is slightly lower than that of photoreceptor segments (which are also light-guiding fibers) but higher than that of the surrounding retinal tissue.³ On the other hand, the refractory index of Müller cell endfeet is rather low, about halfway between that of Müller cell stem processes and that of the vitreous.³ This allows a “soft coupling” of the light path between the vitreous and the retina, and reduces light reflection at the inner retinal surface. By light guidance, Müller cells transport an image (like a fiberoptic plate) with minimal loss of light intensity from the inner retinal surface to the photoreceptors; this image is resolved in pixels corresponding to individual Müller cells (Fig. 17.4A). Because the local densities of cones and Müller cells are roughly equal (Fig. 17.4C), every cone has its “private” Müller cell which delivers its part of the image, while several (up to 10) rods are illuminated by one Müller cell (Fig. 17.4A).

Recycling of cone photopigments

Müller cells support the function of photoreceptors by other mechanisms, as well. There exist two cycles in the retina that regenerate the photopigments, the rod and the cone visual cycle.⁴ In the photoreceptor outer segments, all-trans retinal is reduced to all-trans retinol. Rod-derived all-trans retinol is regenerated to 11-cis retinal in the pigment epithelium while cone-derived all-trans retinol is processed by Müller cells. Müller cells convert all-trans retinol to 11-cis retinol which is subsequently oxidized to 11-cis retinal by a retinol dehydrogenase, and released into the extracellular space for uptake by the cone photoreceptors. For the transport of the retinoids, Müller cells express cellular retinol-binding protein and cellular retinal-binding protein (CRALBP).⁵ Furthermore, Müller cells contribute to the assembly of the photoreceptor outer segments,⁶ in part by the release of lactose and pigment epithelium-derived factor (PEDF),^7,⁸ and phagocytose outer-segment discs shed from cones.⁹ Docosahexaenoic acid is required for outer-segment renewal and for the preservation of mitochondria.¹⁰ Müller cells incorporate docosahexaenoic acid into phospholipids, and channel it to the photoreceptors.¹⁰

Regulation of the synaptic activity by neurotransmitter uptake

Precise shaping (i.e., control in time and space) of synaptic activity depends upon the kinetics of the presynaptic neurotransmitter release and of the (re-)uptake of the transmitter molecules into the cells. In the neuroretina, photoreceptor cells, neurons, and macroglial cells express high-affinity transporters for neurotransmitters. Müller cells possess uptake and exchange systems for various neurotransmitters, particularly for glutamate and γ-aminobutyric acid (GABA) (Fig. 17.5).

Fig. 17.5 Recycling of amino acid neurotransmitters in the outer plexiform layer. The ribbon synapse of a photoreceptor cell (PC) synthesizes glutamate which is released in the dark. The postsynaptic elements are dendrites of bipolar (BC) and horizontal cells (HC). Horizontal cells release γ-aminobutyric acid (GABA) which is formed from glutamate. The synaptic complexes are surrounded by Müller cell sheets. The right side shows neurotransmitter uptake systems and some metabolic pathways within Müller cells. Glutamate, GABA, and ammonia (NH₄⁺) are transported into Müller cells and transformed to glutamine, alanine, and α-ketoglutarate (α-KG). These products are released from Müller cells, and taken up by neurons. Glutamine serves as a precursor for the transmitter synthesis in neurons (glutamate–glutamine cycle). Lactate, alanine, α-ketoglutarate, and glutamine are utilized by neurons as substrates for their energy metabolism. Glutamate is also used for the production of glutathione (GSH), which is an antioxidant, released from Müller cells and taken up by neurons under oxidative stress conditions. ALAT, alanine aminotransferase; AAT, aspartate aminotransferase; CGA, cystine-glutamate antiporter; CyT, cystine transporter; EAAC1, excitatory amino acid carrier 1; EAAT5, excitatory amino acid transporter 5; GABA-T, GABA transaminase; GAD, glutamic acid decarboxylase; AGC, aspartate-glutamate carrier; GAT, GABA transporter; GDH, glutamate dehydrogenase; GLAST, glutamate-aspartate transporter; GLT-1, glutamate transporter-1; GlyT, glycine transporter; GP, glutathione peroxidase; GR, glutathione reductase; GS, glutamine synthetase; GSH, glutathione; GSSG, glutathione disulfide; LDH, lactate dehydrogenase; MAS, malate-aspartate shuttle; OAA, oxaloacetate; PAG, phosphate-activated glutaminase; R•, free radicals; SSA, succinate semialdehyde; TCA, tricarboxylic acid cycle.

The major glutamate uptake carrier of Müller cells is the glutamate aspartate transporter (GLAST, or excitatory amino acid transporter-1, EAAT1).¹¹^–¹³ In addition, EAAT2 (GLT1) and EAAT3 were found in human Müller cells.^14,¹⁵ EAATs mediate the cotransport of three sodium ions and one proton, and the countertransport of one potassium ion, with each glutamate anion.¹⁶ The transport of an excess of sodium into the cell generates an inward current (the transporter is “electrogenic”; Figs 17.6A and 17.7A, B) and cellular depolarization. The amplitude of the glutamate transporter currents is voltage-dependent (Fig. 17.6A); a very negative membrane potential is essential for an efficient uptake of glutamate.¹⁷ Sodium-dependent carriers allow uphill transport of substrates into the cells against a concentration gradient. The driving force for the transport is the electrochemical sodium gradient across the plasma membrane, generated by the Na⁺,K⁺-ATPase. Other transporters, e.g., for GABA, are also electrogenic (Figs 17.6B and 17.7F). The subcellular distribution of the GABA transporter currents (Fig. 17.6B) corresponds with the observation that GABA is taken up by amacrine and Müller cells in the inner retina but exclusively by Müller cells in the outer retina.¹⁸

Fig. 17.6 The main glutamate and γ-aminobutyric acid (GABA) uptake carriers of Müller cells are electrogenic transporters. The membrane currents were recorded in whole Müller cells. (A) Administration of glutamate (1 mM) to a rabbit Müller cell evokes inward currents at negative membrane potentials (left). The current-voltage relation of the glutamate transporter currents in guinea pig Müller cells (right) shows that the efficiency of the glutamate transport increases with increasing (i.e., more negative) membrane potentials. (B) Subcellular distribution of the GABA transporter currents in a guinea pig Müller cell. The distribution of the currents was determined by focal ejections of GABA (1 mM) on to the following membrane domains of the cell: endfoot, inner stem process, soma, and inner and outer parts of the outer stem process.

Fig. 17.7 Neurotransmitters modify the membrane conductance of Müller cells. (A) Administration of glutamate (100 µM) to a rat Müller cell evoked inward currents through electrogenic glutamate transporters, while the glutamate receptor agonists, kainate (500 µM) and N-methyl-d-aspartate (NMDA) (100 µM, in the presence of 10 µM glycine and absence of magnesium) did not evoke membrane currents. This suggests that rat Müller cells express metabotropic but no functional ionotropic glutamate receptors. The current traces were recorded in whole-cell records at a membrane potential of –80 mV. (B) Time-dependent record of the whole-cell currents in a human Müller cell. The currents at +120 mV were mainly mediated by big-conductance potassium (BK) channels. The BK currents were transiently increased in response to extracellular adenosine triphosphate (ATP) (500 µM). Extracellular glutamate (500 µM) evoked a delayed transient increase in BK currents. The increase in the currents at –60 and –100 mV during the exposure of glutamate (arrow) reflects the activation of the electrogenic glutamate transporters. The arrowheads indicate transient activation of calcium-evoked cation channels. (C) Activation of metabotropic P2Y and ionotropic P2X₇ receptors in a human Müller cell. Activation of P2Y receptors by ATP (100 µM) evoked repetitive transient calcium-evoked activation of BK currents (that were recorded at the potential of +120 mV; left). Activation of P2X₇ receptors by 2’-/3’-O-(4-benzoylbenzoyl)-ATP (BzATP; 50 µM) evoked a sustained calcium-evoked activation of BK currents, as well as cation currents through P2X₇ receptor channels (right). (D) Immunostaining of two isolated human Müller cells against the P2X₇ receptor protein using two different antibodies. Arrows, perikarya; arrowheads, cell endfeet. Bars, 20 µm. (E) Extracellular glutamate (200 µM) increased the activity of a single BK channel that was recorded in a membrane patch localized at the perikaryum of a human Müller cell. Above, Time dependency of the opening probability of the channel. Below, Original records of the channel activity. The pipette potential was 0 mV (i.e., the recording was made near the resting membrane potential of the cell). C, closed-state current level; 1, open-state current level. (F) γ-aminobutyric acid (GABA) (100 µM) evoked two currents in a human Müller cell, a transient, rapidly inactivating current mediated by GABA_A receptor channels (arrow), and a sustained current mediated by electrogenic GABA transporters (asterisk).

In the outer plexiform layer, the photoreceptor-derived glutamate is mainly removed by photoreceptor, horizontal, and bipolar cells.^12,¹⁹ whereas Müller cells prevent the lateral spread of neurotransmitters beyond the synapse(s) where they are released, thus ensuring visual resolution (Fig. 17.5). In the inner plexiform layer, Müller cell-provided glutamate uptake takes a more active part in shaping the synaptic responses. The rapid termination of synaptic glutamate action in nonspiking inner retinal neurons and in retinal ganglion cells is predominantly mediated by the uptake of glutamate into Müller cells.^20,²¹ Generally, Müller cells remove the bulk of extracellular glutamate in the inner retina.^13,^22,²³

Malfunction of glial glutamate uptake contributes to glutamate toxicity

Glutamate toxicity is a major cause of neuronal loss in many retinal disorders, including glaucoma, ischemia, diabetes, and inherited photoreceptor degeneration. A malfunction and/or downregulation of GLAST may cause, or contribute to, a rise in extracellular glutamate towards excitotoxic levels.²³^–²⁵ Hypoxia facilitates the formation of free radicals in mitochondria; the resulting lipid peroxidation disrupts glutamate transport into Müller cells.²⁶ This mechanism is probably implicated in the neuronal dysfunction in diabetic retinopathy²⁷ and in ganglion cell death in Leber hereditary optic neuropathy.²⁸ Inflammatory lipids such as arachidonic acid, a reduction in extracellular pH as occurs in ischemia, and zinc ions released from photoreceptors²⁹ all inhibit glial glutamate uptake.^30,³¹

Because glutamate transport is voltage-dependent (Fig. 17.6A), depolarization decreases the efficiency of glial glutamate uptake.^17,³² Depolarization of Müller cells occurs in response to pathological rises in extracellular potassium (as occurs in ischemia and glaucoma), to the opening of cation channels in Müller cells (Fig. 17.7C), and to a decrease in the potassium permeability of glial membranes due to inactivation and/or downregulation of inwardly rectifying potassium (Kir) channels (Fig. 17.8A, B). An inactivation of Kir channels was observed in animal models of various retinopathies (see below). Depolarization of Müller cells can also be evoked by inflammatory lipids such as arachidonic acid and prostaglandins which potently inhibit Na⁺,K⁺-ATPase. Depolarization of Müller cells may even cause a reversal of the glutamate transport.³³ A nonvesicular release of glutamate and aspartate via reversed operation of glial glutamate transporters was implicated in the excitotoxic damage of retinal ganglion cells.^34,³⁵ A release of glial glutamate might also be mediated by the cystine glutamate antiporter (which is activated under oxidative stress conditions when an elevated production of glutathione requires an increased uptake of cystine; Fig. 17.5) and by exocytosis of glutamate-containing secretory vesicles.^1,³⁶ Under various pathological conditions including ischemia, glaucoma, retinal detachment, and proliferative retinopathies, an enhanced expression or activity of GLAST was observed.^11,³⁷ This might counterbalance the depolarization-induced malfunction of glial glutamate uptake.

Fig. 17.8 In the course of retinal ischemia–reperfusion, Müller cells of the rat change their membrane conductance(s) and their osmotic swelling properties. Transient retinal ischemia was induced by elevation of intraocular pressure above systolic blood pressure for 1 hour. (A) Examples of the whole-cell potassium currents of Müller cells isolated from a control retina, and from retinas 3 and 7 days after transient retinal ischemia. (B) The amplitude of the Kir currents decreases time-dependently after transient retinal ischemia. Simultaneously, the resting membrane potential (RMP) of the cells decreases and the whole-cell capacitance (that is proportional to the cell membrane area) increases, indicating cellular hypertrophy. The parameters were measured at different periods (3 hours to 3 days) after transient ischemia. (C) Under hypo-osmotic stress, Müller cells in slices of retinas 3 days postischemia display cellular swelling which is not observed in Müller cells in slices of control retinas. The time-dependent alteration in the cross-sectional area of Müller cell somata is shown. The images display Müller cell somata in a postischemic retina, before (above) and after (below) hypotonic challenge. Bar, 5 µm.

Production of neurotransmitter precursors

To sustain the mechanism of transmitter recycling at the synapses, the Müller cells are endowed with specific enzymes. After GABA has been taken up by Müller cells, it is readily converted to glutamate by the GABA transaminase, and glutamate is converted to glutamine by the glutamine synthetase (Fig. 17.5). Glutamine synthetase is exclusively localized to glial cells.³⁸ Glutamine is released from Müller cells, and taken up by neurons as a precursor for the resynthesis of glutamate and GABA (glutamate–glutamine cycle; Fig. 17.5).³⁹ In the photoreceptor cells, this cycle accounts only for a part of glutamate supply; they directly take up glutamate from the synaptic cleft, and, in addition, synthesize glutamate from α-ketoglutarate as well as from Müller cell-derived glutamine.^19,⁴⁰ By contrast, the production of glutamate in bipolar and ganglion cells almost entirely depends on Müller cell-derived glutamine.⁴⁰ Pharmacological blockade of the glutamine synthetase in Müller cells causes a rapid loss of the glutamate content of bipolar and ganglion cells, and (within 2 minutes) the animals become functionally blind.⁴⁰ Likewise, a significant amount of GABA in amacrine cells is synthesized from Müller cell-derived glutamine.⁴⁰

Downregulation of the glial glutamine synthetase contributes to neuronal dysfunction and glutamate toxicity in inherited photoreceptor degeneration and retinal detachment.^41,⁴² The downregulation is mediated (at least in part) by the basic fibroblast growth factor (bFGF).⁴³ bFGF is rapidly released within the retina after detachment, and its expression level increases under ischemic conditions, after light injury, and in response to inherited photoreceptor degeneration and mechanical injury.¹ Though bFGF is a neurotrophic factor which supports the survival of photoreceptors and neurons,⁴⁴^–⁴⁶ the bFGF-induced downregulation of glutamine synthetase might rather aggravate neuronal degeneration. Under ischemic conditions, the GABA transaminase activity is decreased, and GABA accumulates in Müller cells.^47,⁴⁸ This impairs the efficiency of glial GABA uptake due to the decrease in the extra- to intracellular gradient as a driving force.

Trophic support of photoreceptors and neurons

The uptake of glutamate and GABA by Müller cells links neuronal excitation with the release of metabolic substrates, as well as with the defense against oxidative stress. Müller cells produce various substrates of the oxidative neuronal metabolism such as glutamine, lactate, alanine, and α-ketoglutarate (Fig. 17.5)⁴⁹; these substrates are used by photoreceptors and neurons in periods of metabolic stress in the dark. The metabolic support is regulated by neuron-derived glutamate (which stimulates the uptake of glucose and the production of lactate) and potassium (which induces hydrolyzation of glycogen in Müller cells).^1,⁵⁰

Antioxidative support of photoreceptors and neurons

Glutamate in Müller cells is also utilized for the production of the antioxidant, glutathione (Fig. 17.5).³⁹ The retina has a high need of antioxidant protection; this results from light exposure together with high oxygen consumption and the presence of high polyunsaturated fatty acid levels in photoreceptors. In addition, the outer retina underlies circadian periods of hypoxia (dark) and hyperoxia (light)⁵¹ which both increase oxidative stress. Usually, retinal glutathione is confined to glial and horizontal cells.^39,⁵² In Müller cells, glutathione constitutes about 2% of the total protein.¹ In response to oxidative stress such as during ischemia, glutathione is rapidly released from Müller cells and provided to the neurons,⁵² where it acts as cofactor of the enzyme glutathione peroxidase. The glial release of antioxidants like glutathione, and of neuroprotective factors like bFGF and adenosine, is implicated in the protection of photoreceptors from the harmful effects of circadian light exposure.

Müller cells from aged animals contain reduced levels of glutathione; this is associated with mitochondrial damage, membrane depolarization, and reduced cell viability.⁵³ An age-dependent decrease in retinal glutathione may accelerate the pathogenesis of retinopathies in the elderly. In diabetic retinopathy, the decreased glutamate uptake of Müller cells results in reduced glutathione synthesis^54,⁵⁵ and in an upregulation of glutaredoxin which catalyzes the deglutathionylation of proteins; the latter mechanism induces nuclear translocation of nuclear factor-κB and upregulation of proinflammatory factors.⁵⁶ Other antioxidants provided by Müller cells include pyruvate, α-ketoglutarate, metallothionein, lysozyme, ceruloplasmin, heme oxygenase, and reduced ascorbate.¹

Removal of carbon dioxide

Photoreceptor cells display the highest rate of oxidative metabolism of all cells in the body. This high metabolic activity is associated with high oxygen and glucose demands. The active glucose metabolism, in turn, generates much carbon dioxide and water. Via transcellular transport, Müller cells redistribute metabolic waste into the blood and vitreous. Carbon dioxide is cleared by a process called carbon dioxide siphoning ⁵⁷; it is rapidly hydrated to bicarbonate and protons by carbonic anhydrases.⁵⁸ Carbonic anhydrase II is localized within Müller and amacrine cells, whereas the membrane-bound carbonic anhydrase XIV is present in glial cells and vascular endothelia.^57,^59,⁶⁰ Bicarbonate is transported to the blood and vitreous by sodium bicarbonate cotransporters and anion exchangers, both enriched in glial endfoot membranes.⁵⁷

Regulation of the extracellular pH

By the removal of carbon dioxide, Müller cells also regulate the extracellular pH. Light-evoked neuronal activity is associated with extracellular alkalinization.⁶¹ The pH shift is balanced by an efflux of protons from photoreceptors⁶² and by the consecutive action of carbonic anhydrases and glial sodium bicarbonate cotransporters.⁶³ Because sodium bicarbonate transporters are electrogenic, depolarization of Müller cells by potassium released from active neurons causes an influx of bicarbonate into the cells.⁶³ The carbonic anhydrase-generated protons remain outside the cells and cause an extracellular acidification.⁶³ Because extracellular acidification inhibits synaptic transmission,⁶⁴ Müller cell-mediated pH regulation may protect neurons against overexcitation.

Spatial potassium buffering

Neuronal activity is associated with rapid ion shifts between the intra- and extracellular spaces. Sodium, chloride, and calcium ions flow into active neurons, and potassium ions are released from neurons. Light onset causes increases in extracellular potassium in the plexiform layers, and a decrease in the subretinal space.^65,⁶⁶ If not corrected, increased potassium will cause neuronal depolarization and hyperexcitation. Müller cells buffer imbalances in the extracellular potassium concentration via permission of transcellular potassium currents, a process termed “spatial potassium buffering” or “potassium siphoning.”^67,⁶⁸ They take up excess potassium from the extracellular fluid, and release equal amounts of potassium into fluid-filled spaces outside the neuroretina where the potassium concentration is constant or decreased, i.e., into blood vessels, the vitreous, and the subretinal space (Fig. 17.9C).

Fig. 17.9 The subcellular localization of Kir channel subtypes determines the direction of spatial buffering potassium currents through Müller cells. (A) Current-voltage (I-V) relations of Kir4.1 and Kir2.1 channels. Kir4.1 channels mediate inward and outward potassium currents with similar amplitudes, whereas Kir2.1 channels mediate inward currents but almost no outward currents. (B) Immunolocalization of glial Kir channel proteins in the normal and postischemic rat retina. The Kir4.1 protein is predominantly localized at the limiting membranes of the neuroretina (arrowheads) and around the blood vessels (arrows). The Kir2.1 protein is localized in the inner retina in membrane domains of Müller cells that abut neuronal compartments such as processes that traverse the inner plexiform layer (IPL) (arrowheads). Seven days after a 1-hour transient retinal ischemia, the expression of Kir4.1 protein is largely downregulated whereas the localization of the Kir2.1 protein is not altered. Note the decrease in the thickness of the inner retina which is a characteristic feature of retinal ischemia–reperfusion injury. (C) Schematic drawing of the potassium buffering currents flowing through Müller cells during neuronal activation. Activated neurons release potassium which is absorbed by Müller cells through Kir2.1 and Kir5.1/4.1 channels, and distributed into the blood vessels, the vitreous, and the subretinal space through Kir4.1 channels. Kir4.1 channels mediate in- and outward currents and, thus, contribute to the osmohomeostasis between the neuroretina and extraretinal fluid-filled spaces. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Bars, 20 µm.

Because Müller cell membranes are highly permeable to potassium, Müller cells have a very negative resting membrane potential, close to the potassium equilibrium potential, around –80 mV.^69,⁷⁰ The potassium permeability is mainly mediated by special potassium channels belonging to the Kir subfamily of K⁺ channels.⁷¹ In particular, Kir4.1 channels (which mediate bidirectional potassium currents; Fig. 17.9A) and Kir2.1 channels (which mediate inward potassium currents) are implicated in potassium siphoning.^72,⁷³ The channel proteins are localized in a polarized fashion in the Müller cell membrane (Fig. 17.9B). Homomeric Kir4.1 channels are mainly localized in membrane domains across which the cells extrude potassium into spaces outside the neuroretina, i.e., in perivascular and vitread endfoot membranes, and in the microvilli that extend into the subretinal space.^72,⁷⁴ Membranes which abut neuronal cell structures contain Kir channels which mediate inward potassium currents, i.e., Kir2.1 (Fig. 17.9B) and heterotetrameric Kir4.1/Kir5.1 channels.^73,⁷⁵ Thus, the direction of the transcellular potassium currents is determined by the subcellular localization of different Kir channel subtypes. Local increases in extracellular potassium cause the driving force for passive currents through Kir channels. Müller cells also express other types of potassium channels which may contribute to potassium buffering, including voltage-gated potassium (K_A, K_DR), calcium-activated big-conductance potassium (BK) channels (Figs 17.7E and 17.10A), and tandem-pore domain potassium channels.⁷⁶ In addition, the Na⁺,K⁺-ATPase and transporter molecules, e.g., the K/Na/2Cl cotransporter, contribute to Müller cell-mediated potassium homeostasis.¹

Fig. 17.10 Age- and disease-related alterations in the membrane conductance of human Müller cells. (A) Four different types of potassium currents can be recorded in Müller cells from postmortem donors without apparent eye diseases (left): BK, currents mediated by big-conductance potassium channels; K_A, fast transient (A-type) potassium currents; K_DR, delayed rectifying potassium currents; Kir, inwardly rectifying potassium currents. Müller cells obtained from patients with proliferative vitreo- and diabetic retinopathies (right) display an almost complete absence of Kir currents. The inward potassium currents (evoked by membrane hyperpolarization from a holding potential of –80 mV) are depicted downwardly. The outward potassium currents (evoked by membrane depolarization) are depicted upwardly. The arrow indicates transient inward currents through voltage-gated sodium channels. (B) Single-action potential-like discharges (arrow) can be evoked in the current-clamp mode by large depolarizing current steps in a Müller cell of a patient with proliferative vitreoretinopathy. Depolarizing currents from 40 to 200 pA (increment, 20 pA) were applied after administration of a hyperpolarizing current of 250 pA (resulting in a membrane potential between –100 and –125 mV). (C) Age-dependent alterations in the densities of Kir and L-type voltage-gated calcium channel (VGCC) currents in human Müller cells. While the Kir currents display an age-dependent decrease, the currents through L-type calcium channels increase in the course of aging.

Many of the homeostatic functions of Müller cells, including potassium siphoning and neurotransmitter uptake, depend on the very negative membrane potential constituted by the Kir4.1 channels.⁷² Kir4.1 channels are also involved in cell volume regulation which compensates for changes in the osmotic conditions.^1,⁷⁷ Under various pathological conditions, Kir4.1 channels of Müller cells are downregulated and/or dislocated (Fig. 17.9B); this is associated with functional inactivation and results in a decrease of the potassium currents through Müller cells (Fig. 17.8A, B). Such alterations have been observed in animal models of various ischemic-hypoxic and inflammatory diseases, including ischemia-reperfusion, ocular inflammation, diabetic retinopathy, blue-light injury, retinal detachment, retinal vein occlusion, and proliferative vitreoretinopathy, as well as in Müller cells from patients with proliferative retinopathies (Fig. 17.10A).^1,⁷⁷ The impaired transcellular potassium transport and the depolarization of the cells (which impairs the glutamate uptake) contribute to neuronal hyperexcitation and glutamate toxicity.³² Human Müller cells display an age-dependent decrease in Kir currents, on average by 50% between the ages of 40 and 80 years (Fig. 17.10C). This may enhance the susceptibility for age-dependent retinopathies because dysfunctional Müller cells cannot efficiently buffer pathological complications such as neuronal hyperexcitation due to hypoxia caused by diabetic alterations of the vessels.

Water clearance

Usually, water accumulates in the retinal tissue due to various mechanisms, including a water influx from the blood coupled to the glucose uptake, and metabolic water production by the oxidative degradation of glucose. The accumulation of metabolic water is especially abundant in macular tissue which displays a high metabolic activity and a high density of cells. This generates the necessity for a substantial constitutive efflux of water out of the retina into the blood. In addition, the transmembrane ion shifts associated with neuronal activity (in particular, with ionotropic glutamatergic signaling) needs, for osmotic reasons, to be buffered by rapid water transport across glial membranes.

Retinal water clearance is mediated by an osmotically driven transcellular water transport coupled to transport of osmolytes, and in particular to potassium transport.^1,⁷⁴ The Müller cell-mediated clearance of the retinal tissue from excess potassium will simultaneously redistribute metabolic water out of the neuroretina. The transmembrane water transport is facilitated by water-selective channels, the aquaporins. Aquaporin-4 is mainly localized in glial membranes within the inner retina and the outer plexiform layer,⁷⁴ while aquaporin-1 is present in photoreceptor and pigment epithelial cells (Fig. 17.11).^1,⁷⁸ In Müller cell membranes that contact fluid-filled spaces outside the neuroretina, aquaporin-4 (Fig. 17.11) and Kir4.1 channels (Fig. 17.9B) are colocalized.⁷⁴ This suggests that osmotic gradients between the neuroretina on the one hand and the blood, vitreal fluid, and subretinal fluid on the other hand are compensated by bidirectional potassium and water flux across Müller cell membranes. In addition, Müller cells express aquaporin-4 in their perisynaptic membranes within the plexiform layers. Because the plexiform layers are high-resistance barriers for paracellular fluid movement,⁷⁹ any water flowing into the neurons (associated with ion flux through ionotropic receptors) will be delivered from Müller cells through aquaporin-4.¹ Aquaporin-4 knockout mice display reduced electroretinogram b-waves,⁸⁰ suggesting that Müller cell-mediated water transport is implicated in the maintenance of regular neuronal activity. Ischemia-reperfusion results in a decreased thickness of the inner retina (Fig. 17.9B) due to neuronal degeneration caused by reactive oxygen radicals and glutamate toxicity.⁸¹ Deletion of aquaporin-4 in mice protects against ischemic injury of the retina,⁸² suggesting that Müller cell-mediated water transport is implicated in the pathomechanisms of neuronal cell death after ischemia.

Fig. 17.11 Müller cells mediate the osmohomeostasis predominantly of the inner retina. Water fluxes through the retina are shown. Under normal conditions, water accumulates in the neural retina and subretinal space (blue arrows) due to an influx from the blood (coupled to the uptake of nutrients such as glucose) and vitreous chamber (due to the intraocular pressure), and due to the oxidative synthesis of adenosine 5’-triphosphate (ATP) in the mitochondria that generates carbon dioxide and water. The excess water is redistributed into the blood by a transcellular water transport through Müller cells and the retinal pigment epithelium (RPE). The water transport across cell membranes is facilitated by aquaporin (AQP) water channels. RPE cells express aquaporin-1, while Müller cells express aquaporin-4. The transcellular water transport is osmotically coupled to the transport of osmolytes, especially potassium (orange arrows) and chloride ions (yellow arrow). The ion fluxes across the cell membranes are facilitated by transporter molecules and ion channels. In Müller cells, the Kir4.1 potassium channel is colocalized with AQP4 in membranes that surround the vessels, and at both limiting membranes of the retina.

Contribution to edema development and resolution

Müller cells in animal models of retinopathies, which are known to be associated with the development of edema in the human retina, undergo an altered expression of aquaporins.¹ In response to outer retinal and/or subretinal edema caused by blue-light illumination, Müller cells strongly increase their expression of aquaporin-4 in the outer nuclear layer (Fig. 17.12B). In ischemia and diabetes, Müller cells and astrocytes express aquaporin-1 in membranes which contact the vitreous and the innermost blood vessels.¹ These alterations might be helpful for edema resolution. On the other hand, dysfunctional Müller cells might also contribute to edema development. Edema is characterized by a thickening of the retinal tissue, caused by water accumulation due to vascular leakage and/or by a swelling of neuronal and glial cells. Whereas neuronal swelling is observed early after ischemia-hypoxia, and is mainly caused by an overexcitation of ionotropic glutamate receptors,⁸¹ Müller cell swelling occurs within days of hypoxia.⁸³ Because the water transport through Müller cells is coupled to the potassium currents,⁷⁴ downregulation or functional inactivation of Kir4.1 channels observed under pathological conditions (Fig. 17.8A, B) should also disturb the transcellular water transport. Indeed, Müller cell bodies in slices of diseased retinas swell upon hypo-osmotic challenge which is not observed in control tissues (Fig. 17.8C). The swelling of Müller cells reflects an alteration in the osmotically driven water transport across Müller cell membranes. In the absence of functional Kir4.1 channels, the cells are not capable of rapidly releasing potassium and, thus, to compensate the osmotic gradient between the Müller cell interior and the hypo-osmotic environment. This results in water influx and cellular swelling. Because Müller cells are still capable of taking up excess potassium from the extracellular space through Kir2.1 channels which are not altered in their expression after ischemia (Fig. 17.9B), the impairment in the release of potassium from Müller cells will result in a potassium accumulation within the cells and, thus, in increased intracellular osmotic pressure.⁷⁷ The increased osmotic pressure draws water from fluid-filled spaces outside the neuroretina (blood, vitreous) into the perivascular and endfeet regions of Müller cells, resulting in Müller cell swelling. The water influx from blood and vitreous is supported by aquaporin-4 water channels which are not altered, or even increased, in their expression (Fig. 17.12).⁸³ Thus, dysfunctional water clearance, and possibly Müller cell swelling, contribute to the development of edema.

Fig. 17.12 Upregulation of glial fibrillary acidic protein (GFAP) is a characteristic marker of Müller cell gliosis under pathological conditions. (A) Retinal slices from 6-month diabetic (right) and age-matched control rats (left) were immunostained against GFAP (green) and the glial water channel, aquaporin-4 (AQP4; red). Cell nuclei were labeled with Hoechst 33258 (blue). In the control retina, only astrocytes at the inner margin of the retina contain GFAP. In the retina of the diabetic animal, hypertrophied Müller cell fibers, traversing the whole neuroretina, display strong labeling for GFAP. (B) Retinal slices of a rat were stained 3 days after illumination of a circumscribed area of the retina with blue light which resulted in apoptotic death of photoreceptor cells. The light-injured retinal area is shown on the right (asterisk); the uninjured area is shown on the left. The strong upregulation of AQP4 in Müller cell processes within the outer nuclear layer (ONL) is likely a response to the outer retinal edema which developed due to the light-induced injury of the retinal pigment epithelium (resulting in leakage of the outer blood–retinal barrier) and the volume decrease of apoptotic cells which is mediated by an efflux of osmolytes (especially of potassium and chloride) and water. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer. Bars, 20 µm.

Further factors which induce osmotic Müller cell swelling are reactive oxygen radicals and inflammatory lipids (arachidonic acid, prostaglandins).¹ Increased vascular permeability is associated with the extravasation of albumin; in the presence of a hypo-osmotic environment, albumin induces Müller cell swelling.⁸⁴ The effect of albumin is mediated by activation of transforming growth factor-ß receptor type II, oxidative stress, production of inflammatory lipids, and intracellular sodium overload.⁸⁴ Thus, extravasated albumin may represent one factor which links vascular leakage, Müller cell swelling, and dysregulation of retinal fluid clearance. Other blood-derived factors such as thrombin and glutamate (as a constituent of the plasma), which close Kir channels and depolarize Müller cells, might be involved.^85,⁸⁶ A reduced efficiency of glial glutamate uptake, due to cellular depolarization, may explain in part the clinical observation that the presence of hemorrhages is associated with an aggravated reduction in retinal function.⁸⁷

Regulation of the blood–retinal barrier

Vascular leakage caused by opening of the tight junctions between vascular endothelial cells and/or by increased vesicular transport of serum proteins across vascular endothelia is an important pathogenic mechanism of retinal edema.⁸⁸ Retinal capillaries are closely ensheathed by glial processes. Usually, Müller cells enhance the barrier function of vascular endothelia⁸⁹ by the secretion of factors such as PEDF, thrombospondin-1, and glial cell line-derived neurotrophic factor (GDNF).^90,⁹¹ However, in response to hypoxia, inflammation, or glucose deprivation, Müller cells produce factors such as vascular endothelial growth factor (VEGF) and tumor necrosis factor that increase vascular permeability.^90,^92,⁹³ Müller cells are also a source of matrix metalloproteinases^93,⁹⁴ which degrade the tight junction protein, occludin.⁹⁵

Mediation of neurovascular coupling

Changes in neuronal activity induce different responses in Müller cells, including alterations in their membrane potential and intracellular calcium responses triggered by activation of transmitter receptors.¹ Müller cells in the rat retina generate spontaneous calcium transients that start within the inner plexiform layer and propagate to the endfeet of the cells.⁹⁶ Flickering light increases the frequency of the calcium transients; this is likely mediated by adenosine 5’-triphosphate (ATP) released from neurons (amacrine and ganglion cells) and subsequent activation of metabotropic purinergic (P2Y) receptors on Müller cells.⁹⁶ Such purinergic neuron-to-glia signaling is involved in the activity-dependent regulation of the local retinal blood flow.⁹⁷ Calcium responses in Müller cells evoked by neuron-derived ATP (Fig. 17.13B, C) activate the calcium-dependent phospholipase A₂; arachidonic acid metabolites are then released from perivascular glial membranes which cause dilation (epoxyeicosatrienoic acids) or constriction (20-hydroxyeicosatetraenoic acid) of arterioles.⁹⁷ Whether vasodilating or vasoconstricting responses are produced depends on nitric oxide.⁹⁷ ATP and adenosine, which are released from both neurons and glial cells,⁹⁸ evoke constriction and relaxation, respectively, of pericytes.^99,¹⁰⁰

Fig. 17.13 Purinergic signaling in the inner retina involves Müller cells and blood vessels. (A-C) P2Y receptor-evoked calcium responses in Müller cell endfeet. (A) Images taken from a whole mount of a control porcine retina (left) and a porcine retina that was isolated 3 days after a transient retinal ischemia of 1 hour (right). The tissues were exposed to adenosine triphosphate (ATP) (200 µM) for 1 minute, and the peak calcium responses in Müller cell endfeet within the ganglion cell layer are shown. In the image from the control retinal tissue, only one Müller cell endfoot displayed a calcium response, whereas numerous endfeet displayed responses in the image from the postischemic retina. (B) Intracellular peak calcium responses in Müller cell endfeet at the vitreal surface of a rat retina evoked by ATP (500 µM). Note the constriction of the arteriole in response to ATP. (C) Time dependence of the calcium responses in three Müller cell endfeet marked by the circles in (B).

Regulation of the extracellular space volume

Neuronal activity is also associated with alterations in the volume of neuronal cell compartments (in particular, of synapses).¹ The ion flux through ionotropic receptors is (for osmotic reasons) coupled to a water flux. The ion flux into neurons (and of excess potassium into Müller cells) decreases the osmolarity of the extracellular fluid,¹⁰¹ and the influx of water into neurons results in a swelling of neuronal cell processes and, thus, in a decrease in the extracellular space volume.¹ To avoid a decrease in the extracellular space volume (which would cause neuronal hyperexcitation), Müller cells inhibit their swelling even if the extracellular fluid is hypo-osmotic, which rather favors a water influx. In addition, Müller cells elongate and reduce the thickness of their inner stem processes, at least in vitro.¹ The rapid, activity-dependent alterations in cell shape are supported by the viscoelastic properties of Müller cells (see below).

To regulate cellular volume, Müller cells possess a purinergic signaling mechanism which is triggered by glutamate released from neurons or Müller cells.¹ The final step of this autocrine cascade is the release of adenosine from Müller cells, which activates their adenosine A₁ receptors, resulting in the opening of potassium and chloride channels. The ion efflux compensates the osmotic gradient across the Müller cell membrane and thus prevents cellular swelling. This mechanism might also accelerate the clearance of excess fluid from the retina through Müller cells.¹ The anti-inflammatory corticosteroid triamcinolone acetonide, which is clinically used to induce a rapid clearance of retinal edema, was shown to inhibit the osmotic swelling of Müller cells, via inducing a release of endogenous adenosine.¹⁰² The adenosine-induced opening of ion channels may re-establish the ion and water transport through Müller cells under conditions when the Kir4.1 channels are inactivated.

Responses to mechanical stress

Neuronal activity is associated with rapid alterations in the size of neuronal synapses and cell bodies, in particular, in the plexiform layers which contain many ionotropic receptors. Müller cells respond by reshaping their processes; this is supported by the viscoelastic properties of the cells. Müller cells are softer than neurons; in particular, the processes within the plexiform layers are very soft.¹⁰³ Because growing neurites prefer soft substrates,¹⁰⁴ soft glial processes may support the growth of neuronal processes implicated in synaptic plasticity.¹⁰³ Gliotic Müller cells display an increase in their stiffness due to upregulation of intermediate filaments, particularly, of glial fibrillary acidic protein (GFAP) (Fig. 17.12); this will inhibit neurite growth and might be one reason for the aberrant tissue repair after retinal injury.¹⁰⁵ The increased stiffness of gliotic Müller cells might be also responsible for the fact that, in retinal neovascularization, new blood vessels grow towards the vitreous rather than within the retinal tissue.¹⁰⁶

Müller cells sense mechanical deformations of the retina by calcium-dependent mechanisms. Stretching of the retinal tissue induces calcium responses, activation of extracellular signal-regulated kinases (ERK1/2), and upregulation of the transcription factor c-Fos and of bFGF in Müller cells.¹⁰⁷ This may have implications also for postnatal eye growth. Because bFGF counteracts the development of form deprivation-induced axial myopia,¹⁰⁸ Müller cell-derived bFGF might prevent retinal overstretching and the development of myopia during ocular growth.¹⁰⁷

Müller cells have stretch-activated calcium-permeable cation channels.¹⁰⁹ Activation of the channels results in an increased activity of calcium-activated BK channels.¹⁰⁹ The potassium efflux through BK channels is associated with an efflux of cell water and results in decreased Müller cell volume.¹⁰⁹ Stretch-induced Müller cell responses may be implicated in epiretinal membrane formation. Vitreous fibers, which adhere to Müller cell endfeet at sites of vitreoretinal attachment after partial detachment of the vitreous from the retina, exert tractional forces on to the cells; this activates Müller cells and results in cell hypertrophy and proliferation, as well as in vascular leakage.¹¹⁰ Mechanically stressed Müller cells release growth factors (e.g., bFGF) and ATP.^107,^111,¹¹² Calcium influx (through stretch-activated and/or voltage-gated channels) and activation of BK channels are required for the growth factor- and ATP-induced proliferation of Müller cells.^1,^113,¹¹⁴

Regulation of neuronal activity by release of gliotransmitters

In addition to the uptake of neurotransmitters, Müller cells regulate the synaptic transmission by the release of “gliotransmitters,” in particular of glutamate and of the purines, ATP and adenosine. Gliotransmitters provide excitatory (glutamate, ATP) and inhibitory effects (adenosine) on neighboring neurons.^98,¹¹⁵

Retinal glial cells release glutamate in response to electrical and mechanical stimuli, and after activation of receptors which are coupled to intracellular calcium responses, including VEGF receptor-2 and P2Y receptors.^1,¹¹⁶ Glia-derived glutamate activates inhibitory amacrine cells which results in a depression of the light-evoked activity in a population of ganglion cell layer neurons.^116,¹¹⁷ Glutamate released from Müller cells by calcium-dependent exocytosis directly activates ganglion cell layer neurons.¹ Glia-derived glutamate (acting at ionotropic glutamate receptors) and ATP (acting at ionotropic purinergic P2X₇ receptors) might contribute to the swelling and degeneration of retinal ganglion cells, e.g., in glaucoma.^1,^118,¹¹⁹ In addition, Müller cell-derived glutathione and polyamines, which increase the currents of ionotropic glutamate receptors, might contribute to glutamate-induced neuronal degeneration.^120,¹²¹

Receptor agonists such as glutamate as well as electrical and mechanical stimuli trigger calcium waves in the glial network of the rat retina.^111,¹¹⁶ The propagation of the waves among Müller cells depends upon the release of ATP and autocrine/paracrine activation of P2Y receptors.^111,¹¹⁶ The glial calcium waves are associated with both enhancement and depression of light-evoked activity of distinct ganglion cells.¹¹⁷ Glial ATP released into the inner plexiform layer is converted extracellularly to adenosine that activates A₁ receptors in a population of ganglion cells, resulting in reduced spontaneous activity.¹¹² Müller cell-derived adenosine, which is also released via nucleoside transporters,¹ may act as a negative-feedback regulator of neurotransmission; this adapts the level of neuronal activity to the capacity of Müller cells to maintain retinal homeostasis.

Müller cells are involved in setting the sensitivity of ganglion and amacrine cells by the release of d-serine, which potentiates N-methyl-d-aspartate receptor currents.¹²² They are implicated in providing the direction selectivity of direction-selective ganglion cells by the secretion of acyl coenzyme A-binding protein.¹²³ Müller cells also produce nitric oxide; the production of nitric oxide is strongly enhanced during dark adaptation.¹²⁴ Nitric oxide diffuses out of the cells and has multiple effects on neighboring neurons, including a potentiation of phototransduction, closure of gap junctions and N-methyl-d-aspartate receptor channels, and an increase of calcium channel currents.¹ Under ischemic-hypoxic conditions, glial cells are a major source of retinal nitric oxide. Nitric oxide dilates blood vessels, prevents platelet aggregation, and protects neurons from apoptosis by closure of N-methyl-d-aspartate receptor channels.¹²⁵ However, production of excess nitric oxide and subsequnt formation of free nitrogen radicals and protein nitrosylation have toxic effects on surrounding neurons; these effects are implicated in the pathogenesis of diabetic retinopathy, for example.¹²⁶^–¹²⁸

Ionotropic receptors of Müller cells

Müller cells can sense neuronal activity,¹²⁹ primarily by membrane depolarization upon activation of receptor channels. Human Müller cells express GABA_A and P2X₇ receptors (Fig. 17.7D),^37,¹³⁰ which are ligand-gated chloride and cation channels, respectively. GABA evokes two types of inward currents in Müller cells, a fast transient GABA_A receptor current and a sustained current mediated by electrogenic GABA transporters (Fig. 17.7F).³⁷ Both currents depolarize the Müller cells. Zinc ions released from photoreceptors²⁹ increase the GABA_A receptor currents.³⁷ GABA_A receptors may have different functional roles; because the receptor channels are also permeable for bicarbonate, they may be implicated in the regulation of the extracellular pH. The chloride efflux through the receptor channels may also stimulate GABA uptake by the cells (which is driven by a cotransport of sodium and chloride) and may compensate the decrease in the extracellular chloride level caused by chloride influx into activated neurons.

P2X₇ receptor channels are calcium-permeable cation channels. Opening of the channels by extracellular ATP results in a calcium influx into the cells, calcium release from intracellular stores, and activation of BK channels (Fig. 17.7B, C).^1,¹³⁰ Moreover, the opening of P2X₇ channels causes cellular depolarization which decreases the efficiency of the electrogenic neurotransmitter uptake.¹³⁰ However, the depolarization is counterregulated by hyperpolarization due to a potassium flux through BK channels. The BK channel-mediated hyperpolarization increases the driving force for the calcium influx. P2X₇ receptor currents are increased when the extracellular fluid contains low concentrations of divalent cations,¹³⁰ suggesting that the light-induced decrease in extracellular calcium will facilitate the gating of P2X₇ receptors in Müller cells.

Müller cell gliosis

In response to virtually every pathological stimulus, Müller cells become activated.¹ Reactive gliosis is thought to represent a cellular attempt to protect the tissue from further damage, to promote tissue repair, and to limit tissue remodeling. It includes morphological, biochemical, and physiological changes; these responses vary with the type and severity of the insult. An upregulation of the intermediate filaments, vimentin and GFAP in glial cells (Fig. 17.12) is a very sensitive and early retinal stress indicator.^1,¹³¹

In fact, Müller cell gliosis has both cytoprotective and cytotoxic effects on retinal neurons.¹ Particularly under conditions of massive (proliferative) gliosis, the regular glial–neuronal interactions are disrupted and the retina degenerates (Fig. 17.14). An inverse relation between Müller cell proliferation and the expression of neuron glia symbiosis-mediating proteins has been observed. However, also in the case of “conservative” gliosis (associated with short-term, or low-level, Müller cell proliferation), a dysfunction of Müller cells may contribute to neurodegeneration. After retinal detachment, for example, the downregulation of CRALBP and of glutamine synthetase^132,¹³³ disrupts glia–neuronal interactions involved in photopigment and neurotransmitter recycling. The downregulation of carbonic anhydrase^132,¹³³ and Kir channels (Fig. 17.8A, B)¹ results in disturbances of the retinal acid–base, ion, and water homeostasis. Because the electrogenic uptake carriers are voltage-dependent (Fig. 17.6A), membrane depolarization reduces the efficiency of glial neurotransmitter recycling which, together with an increase in extracellular potassium, will aggravate neurotoxicity. Because the potassium currents of Müller cells are also a major driving force for the water transport through the cells,¹ downregulation of functional Kir channels also impairs retinal water homeostasis, contributing to the development of edema.¹³⁴

Fig. 17.14 Müller cell reactivity in proliferative vitreoretinopathy (PVR). (A) Ophthalmoscopic image of an experimentally induced PVR in the rabbit eye. Note the large, folded cellular masses on the vitreal surface of the retina (arrows). (B) Schematic drawing of increasing degrees of Müller cell reactivity (from left to right). Müller cells re-enter the proliferation cycle, migrate out of the neural retina, and participate in the formation of periretinal fibrocellular membranes. (C) Transmission electron micrograph of a reactive Müller cell (MC) of the rabbit retina which is migrating through a hole in the inner limiting membrane (arrowheads) into the vitreous body (asterisk). The nucleus (N) of the Müller cell is translocated to the innermost retinal layer.

The involvement of reactive Müller cells in immune and inflammatory responses may also exert detrimental effects. After retinal injuries such as retinal detachment, Müller cells upregulate inflammatory factors, including monocyte chemoattractant protein (MCP)-1, which recruit phagocytotic monocytes/macrophages and microglial cells to the injured area.^135,¹³⁶ Blood-derived monocytes/macrophages and neutrophils release oxygen free radicals and cytotoxic cytokines which play critical roles in the photoreceptor apoptosis after retinal detachment¹³⁷ and in neuronal degeneration after ischemia-reperfusion.¹³⁸ Mice deficient in vimentin and GFAP display an attenuation of the detachment-induced glial cell responses (activation of ERK1/2 and c-Fos, induction of MCP-1) and, as a consequence, a decrease in monocyte infiltration and photoreceptor apoptosis was observed.¹³⁹

In contrast, reactive Müller cells might protect photoreceptors and neurons from cell death by various mechanisms, including the secretion of neurotrophic and growth factors, in particular bFGF.^1,^46,¹⁴⁰ Various cytokines might reduce (e.g., nerve growth factor) or increase (e.g., GDNF) the production of bFGF by Müller cells.^46,^141,¹⁴² GDNF induces phosphorylation of ERK1/2 in Müller cells, resulting in a transcriptional upregulation of bFGF that in turn supports photoreceptor survival.^141,¹⁴² GDNF, which is produced by photoreceptor and glial cells,^141,¹⁴³ also protects photoreceptors and ganglion cells from apoptosis through upregulation of the glial glutamate transporter GLAST.^144,¹⁴⁵ Retinal preconditioning with bright light or mechanical stress protects photoreceptors from apoptosis because these stimuli induce an upregulation of bFGF and ciliary neurotrophic factor in Müller cells.^45,¹⁴⁰ Likewise, argon laser photocoagulation slows photoreceptor degeneration by induction of bFGF in Müller cells.¹⁴⁶

Müller stem cells

As a precondition for proliferation and migration, Müller cells reactivate distinct cellular programs normally used in ontogenetic development. In the injured retina, a population of Müller cells dedifferentiates to cells with properties similar to pluripotent retinal progenitor/stem cells, and may even express distinct neuronal and photoreceptor markers.^147,¹⁴⁸ The decrease in Kir currents (Fig. 17.10A) and the increase in voltage-gated sodium currents (Fig. 17.10B) observed in Müller cells from patients with proliferative retinopathies³⁷ may reflect such a transdifferentiation of the cells. However, the neuron-regenerating capability of mammalian Müller cells in situ is very restricted. Attempts to facilitate the neurogenic program of Müller cells, e.g., by transdifferentiation of cultured Müller cells, are ongoing.^148,¹⁴⁹ The tedious progress of this approach underlines the general issue that a better understanding of gliotic mechanisms is essential for the development of efficient therapeutic strategies which increase the protective and regenerative, and decrease the destructive, roles of reactive Müller cells.