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(Investigative Ophthalmology and Visual Science. 2001;42:1072-1079.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Electrophysiology of Rabbit Müller (Glial) Cells in Experimental Retinal Detachment and PVR

Mike Francke1,2, Frank Faude2, Thomas Pannicke1, Andreas Bringmann1, Peggy Eckstein1,2, Winfried Reichelt1, Peter Wiedemann and Andreas Reichenbach1

1 From the Paul Flechsig Institute for Brain Research and the 2 Department of Ophthalmology, Eye Hospital, University of Leipzig, Germany.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the electrophysiological properties of Müller (glial) cells from experimentally detached rabbit retinas.

METHODS. A stable local retinal detachment was induced by subretinal injection of a sodium hyaluronate solution. Müller cells were acutely dissociated and studied by the whole-cell voltage-clamp technique.

RESULTS. The cell membranes of Müller cells from normal retinas were dominated by a large inwardly rectifying potassium ion (K+) conductance that caused a low-input resistance (<100 M{Omega}) and a high resting membrane potential (-82 ± 6 mV). During the first week after detachment, the Müller cells became reactive as shown by glial fibrillary acidic protein (GFAP) immunoreactivity, and their inward currents were markedly reduced, accompanied by an increased input resistance (>200 M{Omega}). After 3 weeks of detachment, the input resistance increased further (>300 M{Omega}), and some cells displayed significantly depolarized membrane potentials (mean -69 ± 18 mV). When PVR developed (in 20% of the cases) the inward K+ currents were virtually completely eliminated. The input resistance increased dramatically (>1000 M{Omega}), and almost all cells displayed strongly depolarized membrane potentials (-44 ± 16 mV).

CONCLUSIONS. Reactive Müller cells are characterized by a severe reduction of their K+ inward conductance, accompanied by depolarized membrane potentials. These changes must impair physiological glial functions, such as neurotransmitter recycling and K+ ion clearance. Furthermore, the open probability of certain types of voltage-dependent ion channels (e.g., Ca2+-dependent K+ maxi channels) increases that may be a precondition for Müller cell proliferation, particularly in PVR when a dramatic downregulation of both inward current density and resting membrane potential occurs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Normal mature Müller cells are responsible for a wealth of glial–neuronal interactions.1 Among these are the clearance of excess extracellular potassium (K+) ions by a mechanism called spatial buffering2 3 or potassium siphoning,4 the uptake of neurotransmitters such as {gamma}-aminobutyric acid (GABA) and glutamate (for recent review, see Reference 1 ), and the supply of the free radical scavenger glutathione, the synthesis of which depends on glial glutamate uptake.5 All these functions, as well as many others, critically depend on both a high activity of inwardly rectifying potassium channels providing the pathways for spatial buffering currents6 and a high (i.e., relatively hyperpolarized) resting membrane potential used as the driving force for most (e.g., neurotransmitter) uptake processes.1 In contrast to this normal situation, Müller cells are known to proliferate after retinal detachment or other neurodegenerative alterations,7 8 9 10 particularly in a condition referred to as proliferative vitreoretinopathy (PVR), a frequent complication of retinal detachment.11 12 Generally, in various cell types studied so far, hyperpolarized resting membrane potentials seem to be incompatible with cell proliferation, in that proliferating cells display depolarized membrane potentials (Em) of less than approximately -40 mV.13 14 15 Human Müller cells isolated from diseased retinas were found to display depolarized resting membrane potentials as well as an altered pattern of K+ channels,16 17 18 19 but nothing is known about the time course of these changes. Thus, we decided to use a rabbit animal model of experimental retinal detachment to study the electrophysiological changes of Müller cells.

To enhance the comprehensibility of the observations on reactive cells, a brief list of the relevant features of normal rabbit Müller cells is given. In the healthy mature rabbit retina, Müller cells are devoid of the intermediate filament protein glial fibrillary acidic protein (GFAP).20 21 Their membrane conductance is dominated by K+ channels. At least four distinct types of these channels are found: (1) inwardly rectifying K+ (Kir) channels with rather weak rectification, allowing also for outward currents in the physiological range of Em; (2) A-type transient outwardly rectifying channels; (3) delayed rectifying channels, both of which mediate solely outward currents (when the membrane is strongly depolarized); and (4) Ca2+-dependent K+ channels of big conductance (BK channels), which may provide both inward and outward currents at sufficient depolarization and internal Ca2+ concentrations.6 22 23 The high K+ conductance (mainly mediated by Kir) is mirrored by a low-input resistance (Rin) of less than 100 M{Omega}22 24 ; this "leakiness" is considered to facilitate K+ siphoning.1 The resting membrane potential of the cells is close to -80 mV (i.e., relatively hyperpolarized), largely because of the activity of the Kir channels, in that the Müller cells in other mammalian species are shown to depolarize when these channels are genetically eliminated25 or specifically blocked by Ba2+ ions.26 Finally, rabbit Müller cells possess an electrogenic glutamate uptake carrier that causes measurable inward currents in response to glutamate application, the efficacy of which depends on Em.27


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental Retinal Detachment
All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The surgical procedure was modified from earlier reports.28 29 30 Briefly, adult pigmented rabbits (both sexes; 2–3 kg) were anesthetized by an intramuscular application of a mixture of ketamine hydrochloride (1 ml/kg) and xylazine hydrochloride (0.1–0.2 ml/kg), the pupil of the right eye was dilated with topical application of 1% tropicamide and 5% phenylephrine hydrochloride, and the eye was protruded and immobilized. After pars plana sclerotomy, a circumscript vitrectomy was performed in the area of the future detachment (i.e., the ventronasal quadrant, just below the medullary rays). Thin glass micropipettes attached to 250-µl glass syringes (Hamilton, Reno, NV) were used, first to raise a subretinal bleb and then to create a local retinal detachment by injection of a solution of 0.25% sodium hyaluronate (Healon; Pharmacia & Opion, Dübendorf, Switzerland). The sclerotomies and the overlying conjunctiva were then closed. At the end of the scheduled survival time (described later), the rabbits were anesthetized as described and subjected to video ophthalmoscopy to document the state of the retinal detachment. Of 24 rabbits used for this study, 20 (5, 7, 3, and 5 animals with survival times of 2 days, 1 week, 2 weeks, and 3 weeks, respectively) maintained the experimental retinal detachment (diameter: 5–10 mm, i.e., approximately 4%–16% of the total retinal area) over their entire survival time. The remaining four animals with a survival time of 3 weeks showed massive (macroscopically visible) PVR with extensive preretinal membranes and secondarily enlarged areas of retinal detachment. After the ophthalmoscopy, the anesthetized animals were killed by an intravenous application of T61 (embutramid mebezonium iodide, 3 ml; Hoechst, Unterschleissheim, Germany), and both the treated and the control eyes were excised.

Isolation of Müller Cells
The electrophysiological experiments were performed on acutely isolated, noncultured Müller cells (Fig. 1G ). The isolation was performed as described previously.31 Briefly, retinal pieces approximately 5 mm in diameter were dissected from the detached bleb and the nondetached retina (1–6 mm distant from the margin of the bleb). These pieces were roughly halved. One part was used for immunocytochemistry, and the other was enzymatically digested in Ca2+-Mg2+–free phosphate-buffered saline (PBS) using Nagarse (0.25 mg/ml; Serva, Heidelberg, Germany) or papain (0.5 mg/ml; Boehringer–Mannheim, Germany) at 35°C for 30 minutes. The tissue was then triturated using a 1-ml pipette tip until the Müller cells separated. During the first mechanical trituration, DNase I (200 units; Sigma, Deisenhofen, Germany) was added to the solution to prevent the Müller cells from sticking together in DNA released from destroyed cells. Suspensions of freshly dissociated Müller cells were harvested on ice in control extracellular solution until use (generally, up to 8 hours).



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  		Figure 1. Immunocytochemistry of Müller cells in retinal detachment; labeling of immunoreactivity for GFAP (A, B, and C: immunofluorescence; D, E, and F: DAB reaction). As early as 2 days after detachment, in the detached retina (A) GFAP immunoreactivity was found in many Müller cells. GFAP-immunopositive cells were found even in the neighboring nondetached retina (B), and faint GFAP immunoreactivity occurred in the far distant retinal periphery (C). There was no obvious GFAP immunoreactivity in the control retina (not shown). One week after detachment surgery, the retinal GFAP immunoreactivity increased dramatically and was present throughout the entire length of virtually all Müller cells in the detached (D) and in the neighboring nondetached (E) retina. GFAP-immunolabeled Müller cells were also found in the retinal periphery (F). (G) Dissociated unlabeled Müller cell from a control retina, with the attached micropipette. NFL, nerve fiber layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRS, photoreceptor segments.

 
Immunohistochemistry
Retinal cryosections for immunocytochemistry were prepared as follows. Retinal pieces were fixed for 4 hours in 4% paraformaldehyde (PFA) in PBS (pH 7.4) or in a 1:9 formalin (37%)-alcohol (96%) mixture. Then the tissue was stored in 30% sucrose (in PBS) with 0.01% sodium-azide up to 24 hours. Ten-micrometer-thick sections were cut on a microtome cryostat (Microm–Laborgeräte, Walldorf, Germany). The sections were placed on slides that had been coated with 1% gelatin and 0.1% chromium potassium sulfate (Sigma, St. Louis, USA) in distilled water, to promote adhesion of the sections to the glass surface. Some PFA-fixed material was processed for paraffin embedding, and 10-µm-thick sections were cut and processed for immunocytochemistry.

All antibodies were diluted in PBS containing 1% dimethylsulfoxide and 0.3% Triton X-100. Retinal cryostat sections were blocked with 10% normal goat serum (Dianova, Hamburg, Germany) for 60 minutes and incubated with the primary antibodies overnight at 4°C. Rabbit anti-cow GFAP antiserum (Dako, Glostrup, Denmark) was used at a 1:200 dilution. The secondary antibodies were added for 90 minutes at room temperature. Cy3-conjugated goat anti-rabbit Ig (Dianova) was used at a 1:150 dilution. Finally, the sections were mounted (Entellan; Merck, Darmstadt, Germany) and viewed by a fluorescence microscope (Axiophot photomicroscope; Carl Zeiss, Oberkochen, Germany). The same rabbit anti-cow GFAP antiserum was used at a dilution of 1:200 for the paraffin sections. A kit (Vectastain Elite ABC Kit; Vector, Burlingame, CA) was used for staining. Diaminobenzidine (DAB) nickel was used as the chromogen. The paraffin sections were counterstained with Mayer’s hemalum and viewed by a photomicroscope (Axiophot; Carl Zeiss).

Electrophysiological Experiments
For whole-cell voltage-clamp experiments, we used patch-clamp amplifiers (EPC 7; List, Darmstadt, Germany; RK-400; Biological, Claix, France; Axopatch 200A; Axon Instruments, Foster City, CA) and a discontinuous single-electrode voltage-clamp amplifier (SEL-1L; NPI, Tamm, Germany). Current signals were low-pass filtered at 1 to 3 kHz with eight-pole Bessel filters (of the amplifier or from Frequency Devices, Haverhill, MA), digitized online with a 12-bit analog–digital converter and saved at 5 to 40 kHz with the patch-clamp software (Tida 5; Batelle, Frankfurt am Main, Germany; ISO-2; MFK-Computer, Niedernhausen, Germany) on IBM-compatible microcomputers. The data were analyzed with this software, and the patch-clamp software boards also delivered the voltage command pulses.

Suction electrodes were made from borosilicate glass (GB150-8P; Science Products, Frankfurt am Main, Germany) and had resistances from 3 to 8 M{Omega} when filled with intracellular solution containing (in millimolar) 10 NaCl, 130 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, 10 EGTA, adjusted to pH 7.1 by Tris-base. The patch pipettes were sealed to the Müller cell membrane (mainly at their somata; Fig. 1G ). The cells were continuously perfused with extracellular solution at room temperature by an application system that allowed the addition of test substances to the bath solution. The control extracellular solution contained (in millimolar): 110 NaCl, 3 KCl, 1 Na2HPO4, 2 CaCl2, 1 MgCl2, 10 HEPES-Tris, 11 glucose, and 25 NaHCO3. The pH was adjusted to 7.4 by gassing the solution permanently with a 5% CO2-95% O2 mixture. For all measurements (with the exception of capacitance calculation), capacitance compensation and series resistance compensation were used to minimize voltage errors.

The membrane potentials of Müller cells were determined by reading the zero current potentials from the steady state current/voltage (I/V) curves recorded in control solution. The Rin was calculated from the steady state currents evoked by a hyperpolarizing 10-mV step from a holding potential of -80 mV in control extra- and intracellular solutions, according to Ohm’s law. For estimation of the membrane capacitance (Cm), we applied 10-mV depolarizing and hyperpolarizing voltage steps from a holding potential of -80 mV. To block all the voltage-gated K+ currents at these potentials, barium chloride (1 mM) was added to the extracellular solution. Thus, the membrane resistance at these potentials was limited by leak currents and the seal resistance. The capacitive artifact was then integrated by means of the patch-clamp software on IBM-compatible microcomputers. The density of inward currents (CDin, given in picoamps [pA] per picofarad [pF]) was calculated by dividing the current amplitude at a 10-mV hyperpolarizing voltage step by the membrane capacitance. Because Cm is roughly proportional to the surface area of the cells (approximately 1 µF/cm2), this procedure normalized the currents with regard to differences in cell size, and the CDin values provide an idea of the density of current-mediating channels within the cell membrane.

To determine the current density of the glutamate uptake current (CDglut) evoked by an application of 100 µM L-glutamate (Sigma) to the bath solution for 10 to 20 seconds, the evoked inward current was divided by the membrane capacitance. The glutamate uptake current of all Müller cells (from both control and surgically altered eyes) was measured at a holding potential of -80 mV.

All data are expressed as means ± SD, with the exception of Figure 3 , in which means ± SEM are shown. The number (n) of cells used for each experiment is given in parentheses. Significant differences between data were evaluated by use of the Student’s t-test (SigmaPlot; Jandel, San Rafael, CA).



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  		Figure 3. Time-dependent changes of averaged membrane parameters of Müller cells during retinal detachment, compared with that of control cells and cells isolated from retinas with proliferative vitreoretinopathy (PVR). The hatched bars represent the mean values of parameters of Müller cells obtained from the attached area of the surgically altered eye. (A) Membrane potentials; (B) input resistances (Rin); and (C) inward current densities (CDin). Within (or above) each bar, the mean values are shown, followed by the number (n) of cells studied in parentheses. For the control conditions, n is shown in the order of the experimental groups. For example, in (A) 23 control cells were studied from the left, non–surgically altered eyes 2 days after detachment, 37 cells from the left eyes after 1 week, and so forth. The bars over the columns give the positive SEM. *Probability result of Student’s t-test.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Müller Cell Reactivity
According to earlier reports,21 32 33 34 GFAP immunoreactivity of Müller cells was used as an indicator of reactive changes. As early as 2 days of detachment, many Müller cells in the detached area displayed strong GFAP immunoreactivity almost throughout their length (Fig. 1A) . At this time, faint GFAP immunoreactivity was also expressed by some Müller cells in the nondetached areas of the same retinas (Figs. 1B 1C) . In the retina detached for 1 week, all Müller cells showed strong GFAP immunolabel throughout their length (Fig. 1D) . A similar strong GFAP immunoreactivity was also observed in Müller cells of the neighboring nondetached retinal areas (Fig. 1E) . Even in far distant peripheral retinal regions, faint GFAP immunoreactivity appeared (Fig. 1F) . After 3 weeks, virtually all Müller cells were strongly GFAP immunopositive throughout the entire retina, whether or not PVR occurred (data not shown). Thus, Müller cell reactivity developed early in the detached retinal tissue and rapidly spread into the nondetached retina.

Electrophysiology
Müller Cells from Normal Retinas.
Living isolated cells displayed a well-maintained morphology including the presence of fine side branches (Fig. 1G) . Typical potassium currents of a Müller cell from a healthy rabbit retina are shown in Figure 2A . The current pattern comprises outwardly (upward in all figures) as well as inwardly (downward) directed potassium currents. The inward currents are mediated by Kir channels.22 24 The outward currents are mediated by delayed rectifier, A-type,22 and Ca2+-activated K+ BK channels,35 36 which were not further distinguished in the standard experiments. Normal rabbit Müller cells had a Cm of 84 ± 32 pF (n = 133) and an Em of -82 ± 6 mV (n = 152). They had an Rin of less than 100 M{Omega} (60 ± 51 M{Omega}, n = 156) for inward currents elicited by a 10-mV hyperpolarizing step from a holding potential of -80 mV. These values are typical for Müller cells in many mammalian species.24



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  		Figure 2. Comparison of voltage-clamp recordings from rabbit Müller cells isolated from control retina (A, D), from a retina 3 weeks after retinal detachment without PVR (B, E), and from a retina with proliferative retinopathy (also 3 weeks after detachment; C, F). (A, B, and C) Typical current responses of Müller cells from the three experimental groups. The control Müller cell (A) clearly exhibited both outward and inward K+ currents, whereas the inward currents were strongly reduced after retinal detachment (B) and even completely disappeared in the retina with PVR (C). The outward current amplitude was only slightly diminished. The voltage step paradigm for (A), (B), and (C) was as follows: holding potential -80 mV, voltage range -170 mV to +110 mV, and increment 10 mV. Every second trace is shown. (D, E, and F) Steady state I/V curves were evaluated from the three experiments.

 
Müller Cells in Retinal Detachment.
When Müller cells were isolated from detached retinas without PVR, a gradual loss of the potassium inward currents became evident. As early as 2 days after surgical detachment (the shortest interval studied) the mean CDin decreased significantly from approximately 3 pA/pF to 2 pA/pF (Fig. 3C ). Significant changes of all studied parameters were observed after 1 week (Em = -73 ± 15 mV, n = 32; Rin = 212 ± 310 M{Omega}, n = 32; CDin = 1.0 ± 0.5 pA/pF, n = 25) and altered further until 2 (data not shown) and 3 weeks after detachment (Figs. 3 4) . Three weeks after detachment, the inward currents were dramatically reduced, whereas the outward currents were almost unchanged (Fig. 2B) . The resting membrane potential of many, but not all cells was significantly reduced; a mean of -69 ± 18 mV (n = 42) was measured after 3 weeks of detachment, and the Rin values increased to more than 300 M{Omega}. It is noteworthy that similar but less pronounced changes were observed also in Müller cells isolated from the adjacent attached retina (Figs. 3A 3B 3C) , without any apparent delay.



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  		Figure 4. Time-dependent changes of membrane potentials and inward current densities of individual Müller cells during retinal detachment, compared with control cells and cells isolated from retinas with PVR. Each point represents the data of one cell. Data of Müller cells from detached retinas without PVR are given both as a summary (left middle) and separately for each of the three stages (middle column: detached retinal area; right column: nondetached distant retina). Left column (control and detached cells): These diagrams include the cells of rabbits with a survival time of 2 weeks (not shown in Fig. 3 ).

 
When Müller cells were isolated from the four 3-week detached retinas (underlying the preretinal membranes) in eyes in which massive PVR had developed, their membrane properties were found to be changed even more dramatically. The inward currents were virtually completely lost (Fig. 2C) . The membrane potential of all these cells was reduced; a mean of -44 ± 16 mV (n = 40) was measured (Fig. 3A ; cf. Fig. 2F ). This was accompanied by a marked increase in the Rin (1177 ± 1048 M{Omega}, n = 41; Fig. 3B ) and a dramatic reduction in CDin (0.2 ± 0.2 pA/pF, n = 34; Fig. 3C ).

Thus, Müller cells from detached retinas experienced a gradual reduction of both mean Em and mean CDin (Figs. 3A 3C) , which was even more pronounced in the presence of PVR. Because CDin is a measure of the number of (open) inwardly rectifying K+ channels and as these channels are thought to be responsible for the high Em of normal Müller cells,1 it seems reasonable to suppose a close relationship between the two parameters. Figure 4 shows that, when individual cells were studied, this relationship was less linear than may have been assumed. In extreme cases, such individual cells had a low CDin of less than 0.5 pA/pF, but a high Em of -80 mV, whereas others displayed a moderately high CDin level of 2 pA/pF but a lowered Em close to -50 mV (Fig. 4) . There was, however, a general tendency: Normal Müller cells were characterized by high membrane potentials and by high CDin levels (1–9 pA/pF; Fig. 4 , control). At the other end of the scale, there were Müller cells from retinas with PVR that displayed low CDin levels (<1 pA/pF) and, with a few exceptions, greatly reduced membrane potentials (most cells less than -60 mV, some less than -20 mV; Fig. 4 , PVR). In cells from detached retinas without PVR, a less clear picture was found (Fig. 4 , retinal detachment). This pattern is better understood if the time course of the changes is given in detail (Fig. 4 , middle vertical column). After two days of detachment, the pattern was closely similar to that of the controls but after 1 week, there were no longer any cells with CDin levels above 2 pA/pF, and several cells displayed significantly reduced membrane potentials. Similar but less severe changes were observed in the neighboring nondetached retina (Fig. 4 , right vertical column).

Glutamate Uptake Currents
To test whether the depolarization of Em may impair the physiological function(s) of Müller cells, the currents through the electrogenic glutamate uptake carriers were measured in Müller cells from control and detached retinas (Fig. 5) . All cells displayed such currents with virtually identical current densities (CDglut); if measured at a holding potential of -80 mV, CDglut was 0.50 ± 0.20 pA/pF in control cells (n = 28, six animals) and 0.52 ± 0.17 pA/pF in cells from detached retinas (n = 28, seven animals). There were no significant differences in CDglut between Müller cells from detached (n = 28) and attached retinal areas (n = 9) or between cells after different periods of retinal detachment, or in comparison with cells from retinas with PVR (Table 1) . The known strong voltage dependence of the glutamate uptake current27 is exemplified in Figure 5 : Lowering the holding potential from -80 to -60 mV caused a reduction of the current amplitude by almost 40%. There was no obvious compensatory increase of the current densities in the reactive cells, although their resting membrane potentials were depolarized (Fig. 3A) .



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  		Figure 5. The current evoked by application of glutamate (100 µM) recorded from a Müller cell of a retinal region detached for 1 week. When the holding potential was changed from -80 mV (left trace) to -60 mV (right trace), the evoked current was reduced by 37.7%.

 

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  		Table 1. Summary of the Key Parameters of Müller Cells from Control and Detached Retinae

 
A short summary of the key parameters of Müller cells from control and detached retinas is given in Table 1 .


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Müller Cell Reactivity: A Triad of GFAP Upregulation, Loss of K+ Inward Conductance, and Membrane Depolarization
Retinal detachment is known to initiate a series of degenerative and reactive events in retinal neurons and glial cells, respectively.28 29 30 37 We confirm that Müller cell reactivity (visualized by GFAP immunocytochemistry32 ) is an early event after detachment surgery (Fig. 1) . Further, we show that Müller cells become reactive even in nondetached retinal regions. A similar spread of Müller cell reactivity has been observed when multiple laser lesions were applied to the rabbit retina.21 Which pathway mediates this spread of reactivity remains to be elucidated. In the present study, the reactive cellular response was accompanied by changes of the electrophysiological properties of the Müller cells. As in the case of GFAP expression, the alterations were observed at day 2 after detachment, and large, widespread changes were found after 3 weeks (Fig. 3) . It is noteworthy that in Müller cells from eyes with PVR the electrophysiological alterations were extremely dramatic. Although it remains unclear how these reactive changes are regulated, it can be safely stated that reactive rabbit (and human16 17 ) Müller cells are characterized by a loss of (at least, functionally active) inwardly rectifying K+ channels.

Influence on Retinal Function
The observed loss of inwardly rectifying K+ channels should have functional consequences. Indeed, it has recently been shown that in knockout mice without Kir4.1 (an important type of glial inwardly rectifying K+ channels), Müller cells displayed dramatically enhanced Rin and depolarized Em, accompanied by functional alterations of retinal signal processing, as measured electroretinographically.25 One of the functions of normal Müller cells is the clearance of excess extracellular K+ ions through inwardly rectifying K+ channels6 by a mechanism called spatial buffering2 3 or potassium siphoning.4 It is obvious that this mechanism must be severely impaired when the current density through these channels is reduced, and the input resistance is increased in the reactive Müller cells (e.g., Figs. 3B 3C ). This functional deficit was not improved when high extracellular K+ was applied (data not shown). Other important functions of normal Müller cells are the uptake of neurotransmitters such as GABA and glutamate.1 These latter functions must critically depend on a high resting membrane potential used as the driving force for most uptake processes.1 Indeed, the glutamate uptake current is almost halved when the membrane potential is depolarized from -80 to -60 mV (Fig. 5 ; cf. also Reference 29 ). Because the resting membrane potentials of the reactive Müller cells are depolarized (Figs. 3A 4) , and no compensatory upregulation of glutamate uptake carriers occurs, transmitter clearance and recycling, as well as glutathione synthesis,5 must be impaired in the case of Müller cell reactivity.

Because similar changes in membrane physiology were observed on reactive human Müller cells in various retinal diseases,16 17 the surprising conclusion is that reactive Müller cells are less well suited than normal ones to perform crucial glioneuronal interactions. In contrast, the neuronal degeneration in retinal detachment and other retinal diseases is thought to be accompanied by an increased release of K+ ions and excitotoxic neurotransmitters into the extracellular clefts38 39 and by an increased production of cytotoxic free radicals.40 Thus, the reactive changes in Müller cell properties are certainly no adaptation to the enhanced requirements but rather accelerate the underlying neuronal degeneration. However, there may be a benefit for the Müller cells themselves: a reduction of their energetic requirements. Reduced K+ leak currents can be balanced by reduced energy-consuming Na+K+ pump currents.41 Furthermore, if the uptake of glutamate is reduced, less energy is required for the (adenosine triphosphate [ATP]–dependent) glutamine synthetase reaction, and so on. Thus, the membrane physiology of reactive Müller cells is compatible with the idea that such cells are in an ambivalent waiting position with reduced energy waste, able either to recover their normal functional state or to undergo further transdifferentiation.

Changes Facilitate Proliferation
In reactive Müller cells the dominance of the inward rectifier–mediated (hyperpolarizing) conductance over other (depolarizing) membrane conductances is reduced. This makes the cells more susceptible to depolarizing forces, whether in the form of a further blockade of K+ currents or an activation of Na+, Ca2+, or nonspecific cation channels by intra- or extracellular signals. Such depolarizations of the membrane cause a (further) activation of Ca2+ channels42 43 and thus the occurrence of substantial Ca2+ influxes. Furthermore, membrane depolarization and/or elevated intracellular Ca2+ facilitates the activation of BK channels.19 35 Among the conditions known to be permissive and/or necessary for cell proliferation, membrane depolarization toward -40 mV or less13 14 15 and elevated activity of BK channels23 44 45 were found in reactive rabbit (depolarization: Figs. 3A 4 ; BK channels: A. Bringmann, unpublished data, 2000) and human16 17 18 19 Müller cells, particular in cases of PVR.

PVR is characterized by cellular proliferation on both surfaces of the detached neuroretina. Proliferating dedifferentiated Müller cells are well-established constituents of these membranes.11 37 There are many reports of Müller cell proliferation after retinal detachment,7 8 9 often accompanied by a migration of some Müller cells into the vitreous cavity or subretinal space.30 37 46 Furthermore, there is a striking similarity between the current patterns of cells from retinas with PVR (present study) and neonatal rabbit Müller cells and/or their immediate progenitors, which have a mean CDin of 0.2 pA/pF, a mean Em of -40 mV, and a high open probability of BK channels.23 It is noteworthy in this context that up to 30% of the latter cells may be mitotically active.47 Thus, an almost complete absence or inactivation of inwardly rectifying K+ channels and a strongly depolarized membrane potential, together with elevated BK channel activity, may be involved in Müller cell proliferation and PVR.

Clinical Implications
What are the possible conclusions of these considerations, in regard to therapeutical intervention of retinal detachment and PVR? The best way would certainly be to interfere with the transition between the normal and the (potentially proliferative) reactive Müller cell phenotype—for example, by blocking the (still unknown) signals triggering this transition. A less difficult way may be to prevent the activation of noninwardly rectifying (e.g., BK type K+ or voltage-activated Ca2+) channels required for proliferation. This might be done by specific blockers of these channels that could be applied during retinal surgery or even in a noninvasive way.48 The main conclusion from our data is that fatal glial reactions may occur very rapidly and that any therapeutic measures should be performed as early as possible.


   Acknowledgements
 
The authors thank Grit Müller for reliable technical assistance in operating the animals and an anonymous reviewer of Investigative Ophthalmology & Visual Science for a thorough reading of an earlier version of the manuscript and helpful comments.


   Footnotes
 
Supported by the Bundesministerium für Bildung und Forschung; by Grant 01KS9504, (Project C5) of the Interdisciplinary Center for Clinical Research at the University of Leipzig; and by Grant Re. 849/8-1 from the Deutsche Forschungsgemeinschaft (and Graduiertenkolleg "Intercell," GRK 250/1-96).

Submitted for publication August 7, 2000; revised October 31, 2000; accepted December 20, 2000.

Commercial relationships policy: N.

Corresponding author: Andreas Reichenbach, Paul Flechsig Institute for Brain Research, University of Leipzig, Jahnallee 59, Leipzig D-04109, Germany. reia{at}server3.medizin.uni-leipzig.de


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Newman, EA, Reichenbach, A. (1996) The Muller cell: a functional element of the retina Trends Neurosci 19,307-312[Medline][Order article via Infotrieve]
  2. Orkand, RK, Nicholls, JG, Kuffler, SW (1966) Effect of nerve impulses on the membrane potential of glial cells in the central nervous system of amphibia J Neurophysiol 29,788-806[Free Full Text]
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