The FASEB Journal • Research Communication

Retinal functional alterations in mice lacking intermediatefilament proteins glial fibrillary acidic proteinand vimentin

Kirsten A. Wunderlich,*,†,‡,1 Naoyuki Tanimoto,§,1 Antje Grosche,{ Eberhart Zrenner,‡

Milos Pekny,k,#,** Andreas Reichenbach,†† Mathias W. Seeliger,§ Thomas Pannicke,††

and Maria-Thereza Perez*,‡‡,2

*Department of Clinical Sciences, Division of Ophthalmology, and ‡‡NanoLund, Nanometer StructureConsortium, Lund University, Lund, Sweden; †Graduate School of Cellular and Molecular Neuroscience,‡Center for Integrative Neuroscience (CIN), and §Division of Ocular Neurodegeneration, Institute forOphthalmic Research, University of Tubingen, Tubingen, Germany; {Institute of Human Genetics,University of Regensburg, Regensburg, Germany; kCenter for Brain Repair and Rehabilitation,Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology,Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; #Florey Institute of Neuroscienceand Mental Health, Parkville, Victoria, Australia; **Hunter Medical Research Institute, University ofNewcastle, Newcastle, New South Wales, Australia; and ††Paul Flechsig Institute of Brain Research,University of Leipzig, Leipzig, Germany

ABSTRACT Vimentin (Vim) and glial fibrillary acidicprotein (GFAP) are important components of the inter-mediate filament (IF) (or nanofilament) system of astro-glial cells. We conducted full-field electroretinogram(ERG) recordings and found that whereas photoreceptor re-sponses (a-wave) were normal in uninjured GFAP2/2Vim2/2

mice, b-wave amplitudes were increased. Moreover, wefound that Kir (inward rectifier K+) channel protein ex-pression was reduced in the retinas of GFAP2/2Vim2/2

mice and that Kir-mediated current amplitudes werelower in Muller glial cells isolated from these mice.Studies have shown that the IF system, in addition, isinvolved in the retinal response to injury and that atten-uated Muller cell reactivity and reduced photoreceptorcell loss are observed in IF-deficient mice after experi-mental retinal detachment. We investigated whether thelack of IF proteins would affect cell survival in a retinalischemia–reperfusion model. We found that although celllosswas induced inbothgenotypes, thenumberof survivingcells in the inner retina was lower in IF-deficient mice. Ourfindings thus show that the inability to produce GFAP andVim affects normal retinal physiology and that the effect ofIF deficiency on retinal cell survival differs, depending ontheunderlying pathologic condition.—Wunderlich, K. A.,Tanimoto, N., Grosche, A., Zrenner E., Pekny, M.,Reichenbach, A., Seeliger,M.W., Pannicke, T., Perez,M.-T.Retinal functional alterations in mice lacking intermediate

filament proteins glial fibrillary acidic protein andvimentin. FASEB J. 29, 000–000 (2015). www.fasebj.org

Key Words: GFAP • Muller glia • electroretinogram • Kirchannels • retinal ischemia

Intermediate filament (IF) proteins, also known as nano-filament proteins, constitute a diverse family of proteinsencoded by at least 70 genes in the human genome (1).IFs are intermediate in size (;10 nm on a cross-section),between actin filaments (6–7 nm) and microtubules(23–25 nm). Expression of IF proteins is tissue- and celltype–specific and for some of themalso age and activationstage dependent (1–3).

In the adult healthy retina, the IF protein vimentin(Vim) is expressed by Muller glial cells (4). These cellshave their soma in the middle of the retina and extendradial processes across the retina toward the vitreousside of the retina, where they terminate in funnel-shaped end feet at the inner limitingmembrane (ILM),and toward the subretinal space, where they contactphotoreceptors and each other at the level of the outerlimiting membrane (OLM) and terminate as apicalmicrovilli that protrude into the subretinal space.Throughout the retina, Muller cells also extend sidebranches that contact neuronal cells and blood vessels(5–7). As the main glial cell type in the retina, Muller

Abbreviations: AQP4, aquaporin-4; Brn, brain specifichomeobox/POU domain protein; cd, candela; CRALBP, cellularretinaldehyde-binding protein; DA, dark-adapted; dKO, doubleknockout; ERG, electroretinogram; GAPDH, glyceraldehyde3-phosphate dehydrogenase; GCL, ganglion cell layer; GFAP, glialfibrillary acidic protein; GLAST, glutamate-aspartate transporter;

(continued on next page)

1 These authors contributed equally to this work.2 Correspondence: Department of Clinical Sciences Lund,

Division of Ophthalmology, Lund University, BMC B11,Klinikgatan 26, SE-221 84 Lund, Sweden. E-mail: maria_thereza.perez@med.lu.sedoi: 10.1096/fj.15-272963This article includes supplemental data. Please visit http://

www.fasebj.org to obtain this information.

0892-6638/15/0029-0001 © FASEB 1

The FASEB Journal article fj.15-272963. Published online August 6, 2015.

cells exert a range of important functions necessary tomaintain ion, pH, fluid, and neurotransmitter homeo-stasis, neuronal survival, and the organization of theblood–retina barrier [reviewed in (5–7)]. They alsoparticipate in the visual cycle by contributing to theregeneration of cone photopigment (8) and act, be-cause of their geometry, as optical fibers that facilitateimage transfer through the retina withminimal loss anddistortion (9).

Muller cells are activated under all pathologic con-ditions that affect the retina, such as retinal detach-ment, phototoxic insult, uveitis, diabetic retinopathy,inherited retinal degeneration, stick injury, and retinalischemia (10–19). Under these conditions, Muller cellscan proliferate, migrate, change shape, and up-regulatethe synthesis of a large number of molecules, suchas cytokines and trophic factors, some of which havea neuroprotective effect when applied exogenously(10–20). The activation of Muller cells, however, notonly has a positive effect, but also limits the outcome ofseveral therapeutic approaches, preventing, for ex-ample, the integration of transplanted cells with hostretinal cells and even the efficacy of gene delivery(21–24).

Similar to what occurs in brain astrocytes (25), theexpression of Vim and another IF protein, glial fibrillaryacidic protein (GFAP), is increased inMuller cells in theprocess of activation (4–7). Important insight into therole that these IF proteins play under pathologic con-ditions has been gained from studies of mice devoid ofGFAP and Vim (GFAP2/2Vim2/2). The retinas of thesemice show attenuated Muller cell reactivity and exhibitbetter neural integration of retinal transplants and re-duced monocyte infiltration and photoreceptor celldeath after experimental retinal detachment, com-pared to wild-type (WT) mice (26–30). Considering theimportant roles of Muller cells in regulating retinalhomeostasis, it is important to examine in detailwhether and how the lack of the IF proteins affects thefunction of anunchallenged retina. For this purpose, weperformed an analysis ofGFAP2/2Vim2/2mouse retinalresponses to light stimuli by electroretinography, alongwith a screening of cell type–specificmarkers and patch-clamp recordings of membrane currents of isolatedMuller cells. Moreover, given the neuroprotectiveeffects observed in GFAP2/2Vim2/2 mice after retinaldetachment (28), we sought to determine to what ex-tent GFAP and Vim deficiencies support neuronal sur-vival in a model of transient retinal ischemia.

MATERIALS AND METHODS

Animals

Mice of various ages (specified below) on a mixed C57BL/6J,129/Sv, 129/Ola genetic background with null mutations inthe GFAP and Vim loci (31–34) and age-matched controlswere examined: 1) WT (GFAP+/+Vim+/+); 2) single-knockout:GFAP+/+Vim2/2 (Vim2/2) andGFAP2/2Vim+/+ (GFAP2/2); and3)double-knockout (dKO; GFAP2/2Vim2/2) mice. The lack of GFAPand Vim expression was verified by PCR genotyping and was sub-sequently confirmed by immunohistochemistry and Western blotanalysis (not shown). The animals were kept on a 12 h light–dark cycle with free access to food and water. They were han-dled according to the guidelines set by the European CouncilDirective 2010/63/EUand theARVOStatement for theUse ofAnimals in Ophthalmic and Vision Research, and all experi-ments were approved by the local animal experimentationethics committees.

Electroretinographic analysis

Full-field electroretinograms (ERGs) were recorded binocularlyfrom WT (n = 4), Vim2/2 (n = 6), GFAP2/2 (n = 6), and GFAP2/2

Vim2/2 (n = 6) mice at the age of 8 wk (35, 36). Mice were dark-adapted (DA) overnight before the experiments and anesthetizedwith a subcutaneous injection of a mixture of ketamine, xylazine,and physiologic saline. Ketamine and xylazine were given at 66.7and11.7mg/kgbodyweight, respectively.Thepupilsweredilatedandsingle-flash ERG responses were obtained under DA and light-adapted (LA) conditions [with a background illumination of30 candela (cd)/m2 starting 10 min before recording]. Singlewhite-flash stimuli ranged from24 to 1.5 log cd s/m2 under DAconditions and from 22 to 1.5 log cd s/m2 under LA conditions,divided into 10 and 8 steps, respectively. Ten responses were aver-agedwith interstimulus intervals of 5 s (for24 to20.5 log cd s/m2)or 17 s (for 0–1.5 log cd s/m2). Responses to trains of flashes(flicker) of a fixed intensity [0.5 log cd s/m2; the InternationalSociety for Clinical Electrophysiology of Vision standard flash in-tensity (37)] with various frequencies (0.5, 1, 2, 3, 5, 7, 10, 12, 15, 18,20, and30Hz)wereobtainedwithout anybackground illumination(0 cd/m2). Flicker responses were averaged either 20 (at 0.5–3Hz)or 30 (at 5 Hz and above) times. Band-pass filter frequencies were0.3 and 300 Hz for all ERG recordings.

Retinal ischemia

Transient retinal ischemiawas induced by the high intraocularpressure (HIOP) method (38). Anesthesia was induced withketamine [100 mg/kg body weight, i.p.], xylazine (5 mg/kg,i.p.), and atropine sulfate (100 mg/kg, i.p.). Ischemia was in-duced in 1 eye inWT (n = 9) andGFAP2/2Vim2/2 (n = 6) mice(16–33 wk old), whereas the other eye remained untreatedand served as an internal control. The anterior chamber of thetest eye was cannulated from the pars plana with a 30-gaugeinfusion needle, connected to a saline bottle. The IOP wasincreased to 160 mm Hg for 90 min by elevating the bottle.After the needle was removed, the animals survived for 7 d andwere subsequently euthanized with carbon dioxide. As we haveshown before in WT mice, reduction of inward currentamplitudes (whole-cell currents recorded from freshly iso-lated Muller cells, see below) and of retinal thickness are ob-served in eyes 7 d after induction of transient ischemia byHIOP (38). In the present study, further analysis was thereforeperformed only in eyes where morphologic retinal damageand a decrease of inward currents were noted.

(continued from previous page)GS, glutamine synthetase; H&E, hematoxylin and eosin; HEPES,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HIOP, highintraocular pressure; IF, intermediate filament; ILM, inner limit-ing membrane; INL, inner nuclear layer; IPL, inner plexiformlayer; Kir, inwardly rectifying potassium; LA, light-adapted; NeuN,neuronal nuclei specific protein; OLM, outer limiting membrane;ONL, outer nuclear layer; OPL, outer plexiform layer; PBS-TB,PBS containing Triton X-100 (0.25%) and bovine serum albumin(1%); PIII, peak three; PFA, paraformaldehyde; PN, postnatal;PNA, peanut agglutinin; RPE, retinal pigment epithelium; Sox-9,sex-determining region Y-box 9; Vim, vimentin; WT, wild-type

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Morphologic analysis

WT and dKO mice [postnatal (PN) d 0–270] were euthanizedwith carbon dioxide, and the eyes (4–6 eyes per age) were quicklyenucleated and immersed for 24 h in Bouin’s solution (Sigma-Aldrich, St. Louis, MO, USA) or 4% paraformaldehyde (PFA) in0.1M Sorensen’s buffer (28mMNaH2PO4 and 72mMNaHPO4;pH 7.2). After they were rinsed and dehydrated, the eyes wereembedded in paraffin and sectioned (4–5 mm). Sections werecollectedonglass slides andcounterstainedwithhematoxylin andeosin (H&E).

Immunohistochemistry

Mice (PN0–270) were killed with carbon dioxide, and the eyes(4–6 eyes per age) were quickly enucleated, fixed in 4% PFA inSorensen’s buffer for 2 h at 4°C, rinsed, cryoprotected, embed-ded, and frozen, as described in detail (16). For immunohisto-chemical analysis, 12 mm cryostat sections were permeabilizedand blocked with phosphate-buffered saline (PBS) containingTritonX-100 (0.25%) andbovine serumalbumin (1%) (PBS-TB)and the corresponding normal serum (5%), followed by in-cubation with the primary antiserum, diluted in PBS-TB con-taining 2% normal serum. Lectin labeling was performed byincubating sections in lectin buffer [300 mM NaCl and 100 mMCaCl2 in 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (HEPES; pH 7.5)] containing fluorescein-conjugatedpeanut agglutinin (PNA). For the analysis of control and ische-mic retinas, they were fixed for 1 h in 4% PFA, rinsed, and em-bedded in PBS containing 3% agarose (w/v), and 70 mm thicksections were cut by using a vibrating microtome. Subsequently,the tissue was permeabilized and blocked with PBS containingTritonX-100 (0.3%),DMSO(1.0%), and 5%normal serum.Thesections were subsequently incubated with different antibodies,as described above. Theprimary antibodies and lectin used, theirdilutions, and their suppliers are listed inSupplementalTable S1.

After incubation with primary antibodies and washes withPBS, the retinal sections were incubated with secondary anti-bodies (Supplemental Table S1). Cell nuclei were labeled withTO-PRO-3 (1:1000; Life Technologies, Carlsbad, CA, USA).Immunostained sections were mounted with an antifademedium (Vectashield; Vector Laboratories, Burlingame, CA,USA) and visualized with a fluorescence microscope (Axio-phot; Carl Zeiss Meditec, AG, Oberkochen, Germany) or witha laser scanning microscope (LSM 510 Meta; Carl Zeiss Medi-tec). Images were taken with a digital camera and accompany-ing software (Axiovision 4.2; Carl ZeissMeditec) using the sameillumination and acquisition settings for all sections processedwith the same antibody. Negative controls were included inwhich the primary antibody was omitted (not shown).

For quantification of the cells in control and postischemicretinas, cell nuclei (TO-PRO-3 positive) and immunolabeled cellswere counted in the nuclear layers in retinal sections ina 200 mm wide area next to the optic nerve head (optical slicethickness, 1.5 mm) (38).

Western blot analysis

Western blot analysis was performed to verify the specificity of theantibodies used and compare the expression levels of certainproteins between WT and dKOmice of the same age. Mice werekilled, and the retinas were quickly isolated through a cornealincision.The specimenswerepooled for each timepoint (1 retinafrom each animal at PN7, -14, -21, -28, and -60), and each pooledsample was analyzed for each protein. The tissue in each sam-ple was homogenized, and retinal proteins (10 or 20 mg) were

separated on 10–15% SDS-polyacrylamide gels and semidryblotted onto PVDF transfer membranes (Immobilon; Millipore,Billerica, MA, USA). Membranes were blocked with 5% nonfatdry milk and subsequently incubated with primary antibodies(Supplemental Table S1), rinsed, incubated with peroxidase-conjugated secondary antibodies, and washed. The reaction wasvisualized by ECL, with an ECL Western Blot detection kit(Amersham Biosciences, Uppsala, Sweden). Bands correspond-ing to the inwardly rectifying potassium (Kir)2.1 and Kir4.1channels were analyzed by using ImageJ 1.48p software (ImageJ;National Institutes of Health, Bethesda, MD, USA). The amountof a-tubulin or glyceraldehyde 3-phosphate dehydrogenase(GAPDH) expression in each respective sample was used as aninternal loading control. The relative amounts of Kir2.1 andKir4.1 expressed in adult WT and dKO retinas were calculatedafter setting theamountofa-tubulin (orGAPDH) in each sampleto 100% [Kir2.1: 9 (WT) and 7 (dKO) retinas in 4 pools each;4 gels; Kir4.1: 4 (WT), 5 (dKO) retinas in 2 pools each; 4 gels].

As a technical note, an antibody against Kir2.1 obtained fromAlomone Labs (APC-026; Jerusalem, Israel) exhibited a singleband by Western blot that was.10 kDa larger than indicated inthe data sheet. The staining obtained also did not correspond tothat described by others using the same product and dilution(39). We therefore used an antibody provided by Sigma-Aldrich(Supplemental Table S1).

Whole-cell patch-clamp records of isolated Muller cells

Retinal tissue frommice subjected toHIOP-induced ischemiawasincubated in Ca2+/Mg2+-free PBS (pH 7.4) containing papain(0.2 mg/ml; Roche, Mannheim, Germany) for 30 min at 37°C,followed by several washing steps with PBS. After short (2 min)incubation inPBS supplementedwithDNase I (200U/ml; Sigma-Aldrich), the tissue pieces were triturated by a 1 ml pipette tip,to obtain isolated retinal cells. The cells were stored at 4°C inserum-free minimum essential medium (Sigma-Aldrich) untiluse within 4 h after cell isolation. Muller cells were identified inthe retinal cell suspensions according to their characteristic cellmorphology.

The whole-cell currents of freshly isolated Muller cells wererecorded at room temperature with theAxopatch 200A amplifier(Axon Instruments, Foster City, CA, USA) and the ISO-2 com-puter program (MFK, Niedernhausen, Germany). The signalswere low-pass filtered at 1 or 6 kHz (8-pole Bessel filter) anddigitized at 5or 30 kHz, respectively, usinga 12-bit A/Dconverter.Patch pipettes were pulled from borosilicate glass (ScienceProducts,Hofheim,Germany)andhad resistancesbetween4and6 MV when filled with a solution containing (in millimolar)10 NaCl, 130 KCl, 1 CaCl2, 2 MgCl2, 10 EGTA, and 10 HEPES,adjusted to pH 7.1 with Tris. The recording chamber was contin-uously perfused with extracellular solution containing (in milli-molar) 135NaCl, 3 KCl, 2 CaCl2, 1MgCl2, 1 Na2HPO4, 10HEPES,and11glucose, adjusted topH7.4withTris. Toevokepotassiumcurrents, we applied de- and hyperpolarizing voltage stepsof 250 ms duration in increments of 10 mV from a holdingpotentialof280mV.Theamplitudeof thesteady-stateKir currents(inward current) wasmeasured at the end of the 250ms voltagestep from 280 to 2140 mV. For the calculation of the relativeinward currents, the mean current amplitude recorded fromcells of the control eye was set to 100% for eachmouse, and therelative value for each cell from the contralateral postischemiceye was calculated. The membrane capacitance of the cell wasmeasured by the integral of the uncompensated capacitive ar-tifact (filtered at 6 kHz) evoked by a 10 mV voltage step in thepresence of extracellular BaCl2 (1mM). Current densities werecalculated by dividing inward current amplitudes evokedby 60mVhyperpolarization by the membrane capacitance. The restingmembrane potential was measured in the current-clamp mode.

IFs IN RETINAL CELL FUNCTION AND SURVIVAL 3

Statistics

Data are expressed asmeans6 SEM or SD, unless stated otherwise.Statistical analysiswasperformedwithPrism(GraphpadSoftware,SanDiego,CA,USA), andsignificance(P,0.05)wasdeterminedby the nonparametric Mann-Whitney U test (quantification ofcells and patch-clamp data) or a 2-tailed paired Student’s t test(Kir2.1 and Kir4.1 protein levels).

RESULTS

GFAP and Vim deficiency affects retinal responses tolight stimuli

Toexaminewhether the lackofGFAP, Vim, or both causesalterations in the in vivo retinal function, we performedERG recordings in WT, GFAP2/2, Vim2/2, and GFAP2/2

Vim2/2mice. Full-field ERG is amass response of transientelectrical activity of the entire retina to light stimulation,but the functionality of certain retinal cell types can beassessed by varying stimulus intensity, stimulus frequency,and static background light.

Figure 1A shows representative single-flash ERG tracesfrom each genotype recorded under DA conditions. Theinitial negative-going response at intensities brighter than22 log cd s/m2 corresponds to the a-wave, which is initi-ated by the activity of rod photoreceptor cells inmice. Thewhole activation phase of the rod outer segments is visible,when it saturates at high stimulus intensities, such as 1.0and 1.5 log cd s/m2, as the negative response reaches itstroughbefore theonset of the followingpositive deflection(b-wave). A comparison of all 4 mouse lines showed nodifference in the DA single-flash a-wave up to the highestintensity of the protocol (Fig. 1A), indicating that the lackofGFAP,Vim,or bothhadnomeasurable effect on the rodphotoreceptor responses.

The single-flash ERG b-wave is evoked by both the rodand the cone systems, depending on the ERG paradigm:responses at stimulus intensities up to22 log cd s/m2 un-der DA conditions are generated exclusively by the rodsystem (scotopic conditions); at stimulus intensitiesbrighter than22 log cd s/m2underDAconditions by bothsystems (mesopic conditions); and at any stimulus intensityunder LA conditions, usually by the cone system (photopicconditions) (35). Under all 3 recording conditions, single-knockout mice (i.e., GFAP2/2 and Vim2/2), showed re-sponses comparable to those in WT mice (Fig. 1A, B, D),indicating that the output signals from rod and conephotoreceptors, as well as the responsiveness of both typesof depolarizing bipolar cells, were not affected by a lack ofVim or GFAP.

In contrast, GFAP2/2Vim2/2 mice showed some b-wave changes in mesopic conditions of the DA single-flash ERG (Fig. 1A, C, D). Whereas the a-wave and theascending edge of the b-wave were unchanged (Fig. 1C),the top of the GFAP2/2Vim2/2 b-wave was enhanced(i.e., larger b-wave amplitudes; Fig. 1C, D), and this re-sponse was accompanied by an alteration of the trailingedge of the b-wave. In WT, GFAP2/2, and Vim2/2 mice,the shape of the trailing edge was concave, whereasin the GFAP2/2Vim2/2 mice this concavity becameless prominent, particularly at high stimulus intensities

(Fig. 1A, C). As a result, the light-evoked responses ofGFAP2/2Vim2/2mice returned to baselinemuch slowerthan those in WT, GFAP2/2, and Vim2/2 mice.

In addition, we examined the ability of WT andGFAP2/2Vim2/2mice torespondto trainsofflashes(flicker)by performing a DA flicker ERG frequency series at a fixedmesopic intensity [0.5 log cd s/m2; the International So-ciety forClinical Electrophysiology of Vision standardflashintensity (37)], which provides an overview of the func-tionality of both photoreceptor systems without using anybackground light (35, 36). In this protocol, the responsesbelow5Hzaredominatedby the rod systemandareusuallyentirely driven by the cone system at 5 Hz and higher fre-quencies. The phenotype of GFAP2/2Vim2/2 mice de-tected at 0.5 Hz in the flicker ERG was similar to thatobserved in the DA single-flash ERG [i.e., the negative-going response (a-wave analog) and the onset and theinitial part of the positive-going response (b-wave analog)were normal in GFAP2/2Vim2/2 mice], whereas the topand trailing edges of the positive-going responses wereenhanced (Fig. 1E–G); as a consequence, the responses inGFAP2/2Vim2/2 mice returned to baseline slower thanthose inWTmice.With increasing stimulus frequency, theresponse amplitude became smaller in WT mice becausethe intervals offlicker stimuli were shorter. This amplitudedecline was much more prominent in GFAP2/2Vim2/2

mice; therefore,flicker responses inGFAP2/2Vim2/2micebecame smaller than those in WTmice at 5 Hz and above,and the GFAP2/2Vim2/2 retina could not resolve theflickering stimulus at 30 Hz. We found notable amplitudereductions in the frequency range usually attributable tothe cone system (35). Because we found significant alter-ations of the ERG only in GFAP2/2Vim2/2 mice, furtherexperiments concentrated on this strain.

GFAP and Vim deficiency does not alter distributionof neuronal markers

The overall morphology of retinas from WT andGFAP2/2Vim2/2 (dKO) mice was comparable at all agesexamined, except that in the latter, as we described else-where (27), the ILM was often separated from the retinalsurface (Fig. 2A, B; representative morphologies at PN60).To evaluate whether the differences in ERG recordings be-tween WT and GFAP2/2Vim2/2 mice were accompaniedby alterations in specific retinal cell subpopulations, weperformed an immunohistochemical analysis with well-established retinal neuronal markers (Fig. 2C–L and Sup-plemental Fig. S1 show representative examples of themarkers’ labeling pattern at PN60). The distribution ofmost markers agreed with that described previously (ref-erences areprovided inSupplementalTableS1).Ganglioncells were labeled with the transcription factor Brn3a (Fig.2C, D) and a neuron-specific nuclear protein (NeuN)(Fig. 2E, F) in all specimens analyzed. NeuN also labeleda fewcells in the innerpart of the innernuclear layer (INL)(Fig. 2E, F). The calcium-binding proteins parvalbuminandcalbindinweredetected incell bodieswith thepositionof amacrine cells and, in addition, weakly in a few cells inthe ganglion cell layer (GCL) (Fig. 2G–J). Several hori-zontal cells were also positive for calbindin in both WTand dKO mice (Fig. 2I, J). Staining for recoverin, a small

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Ca2+-binding protein, resulted in labeling of a small sub-population of cells located in the outer half of the INL,which are likely to correspond to cone bipolar cells (Fig.2K, L). Immunoreactivity was also detected in the innerplexiform layer (IPL), next to the INL. In addition, stronglabeling of photoreceptors was observed, including theirinner and outer segments, cell bodies, and terminals in the

outer plexiform layer (OPL) (Fig. 2K, L). Again, no dis-cernible differences in marker distribution were notedbetween the 2 genotypes.

Immunolabeling with other markers associated withphotoreceptors showed also the same pattern in both WTand GFAP2/2Vim2/2 mice. Rod outer segments weredensely immunopositive for the rod-specific visualpigment

Figure 1. Functional characterization of WT, Vim2/2, GFAP2/,2 and GFAP2/2Vim2/2 mice in vivo by electroretinography. A andB) Representative single-flash ERG recordings from WT, Vim2/2, GFAP2/2, and GFAP2/2Vim2/2 mice (age 8 wk) for increasingstimulus intensity under dark-adapted (DA) (A) and light-adapted (LA) (B) conditions. C) Overlay of the response traces of WT(black) and GFAP2/2Vim2/2 (red) mice [left: from (A), right: from (B)]. D) Box-and-whisker plot of DA and LA single-flashb-wave amplitudes in WT (black), Vim2/2 (blue), GFAP2/2 (green), and GFAP2/2Vim2/2 (red) mice. The top and the trailing edgeof the GFAP2/2Vim2/2 b-wave was increased at middle and high stimulus intensities under DA conditions, resulting in a delayedreturn of the light-evoked responses to baseline. E) Representative flicker ERG frequency series from WT and GFAP2/2Vim2/2 miceat a flash intensity of 0.5 log cd s/m2 under DA conditions. F) Overlay of the response traces of WT (black) and GFAP2/2Vim2/2

(red) mice from (E). G) Top: box-and-whisker plot of flicker ERG response amplitudes in WT (black) and GFAP2/2Vim2/2 (red)mice. Bottom: difference in median amplitudes (GFAP2/2Vim2/2 median amplitude 2 WT median amplitude, for each stimulusfrequency). Although the GFAP2/2Vim2/2

flicker responses were larger than the WT responses at low stimulus frequencies (blueshading), they decreased much faster with increasing stimulus frequency and became smaller than the WT responses at 5 Hz andabove (red shading). In all quantitative plots (D, G), boxes indicate the 25th and 75th percentile range, whiskers indicate the 5th and95th percentiles, and the asterisks indicate the median of the data.

IFs IN RETINAL CELL FUNCTION AND SURVIVAL 5

rhodopsin, whereas the cell bodies showed weak orno labeling, as expected for healthy, differentiated rodphotoreceptors (Supplemental Fig. S1A, B). Fluorophore-conjugated PNA, a carbohydrate-specific lectin that selec-tively binds to the surface of cones, was detected in thesynaptic pedicles at the level of theOPL and the inner andthe outer segments of the cones (Supplemental Fig. S1C,D). There were no consistent differences between retinasof WT and knockout mice with the same variation inlabeling pattern and intensity in both genotypes and inindividual sections of the same genotype. The distributionof calretinin and PKCa was also analyzed and is describedbelow in connection with the results obtained afterischemia.

GFAP and Vim deficiency reduces retinal cell survival

Sections from control (unchallenged) and postischemicWTandGFAP2/2Vim2/2retinaswere stainedwith TO-PRO-3

and 3 other cell markers, to assess alterations in retinalstructure (Fig. 3) anddetermine thenumberof cell nucleiin the different retinal layers (Fig. 4). The number of cellnuclei in the INL and in the outer nuclear layer (ONL)was not significantly different between control retinasof WT and GFAP2/2Vim2/2 mice (Fig. 4A). However, inthe GCL, the number of cells was significantly lowerin theGFAP2/2Vim2/2mice (Fig. 4A).Use of an antibodyagainst glutamine synthetase (GS; converts glutamate toglutamine in Muller glial cells) revealed changes in theinner retina of the dKO mice. Although, in WT retinas,the Muller cell end feet formed the retinal surface at theborder to the vitreous (Fig. 3A), this border was oftendisrupted inGFAP2/2Vim2/2mice (Fig. 3G), confirmingthe observations made with H&E (Fig. 2A, B). Immunos-taining against the cell type–specific markers calretinin(acalcium-bindingproteinexpressedbya subpopulationofamacrine and ganglion cells) and PKCa (expressed by rodbipolar cells) was also examined and quantified. In WTretinas, calretinin-positive cells were observed in the INL, as

Figure 2. Inner retinal structure and localization of neuronal markers at PN60 in WT and GFAP2/2Vim2/2 dKO mice. A and B)Retinas stained with H&E. B) Separation and disruption (asterisk) of the ILM (arrows) in dKO mice. (C–L)Immunohistochemical staining revealed no obvious differences in the distribution of specific neuronal cell markers in retinasof WT and dKO mice. C and D) Brn3a staining in the nuclei of ganglion cells in the GCL. E and F) NeuN staining in ganglioncells, a few amacrine cells in the INL, and displaced amacrine cells in the GCL. G and H) Parvalbumin (Parvalb) staining in someamacrine cells. I and J ) Calbindin (Calb) staining in horizontal cells in the INL and some amacrine cells. K and L) Recoverin(Recov) staining in photoreceptors and cone bipolar cells. Scale bars, 25 mm.

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well as in the GCL (Fig. 3A, B). In agreement with the datafrom TO-PRO-3 staining, the number of calretinin-positivecells in the GCL was lower in the unchallenged GFAP2/2

Vim2/2mice compared with the number in the WT mice,whereas no difference was found in the INL (Figs. 3G, Hand 4B). PKCa labeling was observed in the cell bodies inthe INL and in processes extending toward the OPL andIPL, with no apparent differences between the 2 geno-types in distribution (Fig. 3C, I), the number of cells (Fig.4B), or the levels of expression (Supplemental Fig. S2).

At 7 d after ischemia, the retinal thickness was decreasedinboth strains (Fig. 3D, J). InWTretinas, thenumberof cellsin the GCL and INL was reduced to ;70%, whereas thenumber of photoreceptor nuclei in the ONL was reducedto ;10% (Fig. 4). In retinas from GFAP2/2Vim2/2 mice,a similar percentage of photoreceptors survived, whereasthe number of surviving cells in the GCL and INL was sig-nificantly lower than in WT mice (Fig. 4A–C). Immuno-histochemical staining demonstrated that GS labeling wasreduced inGFAP2/2Vim2/2mice (Fig. 3D, J) and that fewercalretinin-positive cells in theGCL and fewer PKCa-positive

cells in the INL survived in the GFAP- and Vim-deficientmice than in the WT (Figs. 3E, F, K, L and 4B, C).

Muller cells from GFAP2/2Vim2/2 mice displayaltered Kir channel distribution

Since alterations in GS were noted between unchallengedWT andGFAP2/2Vim2/2mice, we screened for a few otherproteins that can be used to identify glial Muller cells or areinvolved in some of themain functions of these cells (Figs. 5and 6 show representative examples of their distribution atPN60, unless otherwise specified). Antibodies against thehigh-mobility group (HMG)-box transcription factor, sex-determining region Y-box 9 (Sox9), recognized the proteinin the retinal pigment epithelium (RPE; not shown) and inthe nuclei of Muller cells, with no obvious differences be-tween the 2 genotypes (Fig. 5A, B). Cellular retinaldehydebinding protein (CRALBP), which participates in the pro-cessingof visual retinoids, was expressed inWTretinas in theRPE(not shown) and in theMuller cells, from their end feet

Figure 3. Localization of retinal cell markers in control retinas (left) and postischemic retinas (7 d after 90 min transientischemia) of WT (GFAP+/+Vim+/+, WT) and GFAP2/2Vim2/2 (dKO) mice. A, D, G, and J ) Merged immunostaining imagesshowing TO-PRO-3 staining in cell nuclei (blue); GS expression, mainly in Muller cell end feet next to the ILM and along theOLM (green); calretinin (Calr) expression in amacrine cells in the INL, and displaced amacrine cells and ganglion cells in theGCL and in processes in the IPL (red). Postischemic retinas showed an overall thinning, a reduction of GS staining, and a loss ofcalretinin-positive cells (D, J ), changes that were all more prominent in dKO mice (J ). D and J, insets) nuclear staining by TO-PRO-3 only. Although the OPL is strongly reduced, photoreceptor nuclei can still be discriminated because of their brighterstaining. B, E, H, and K) Single staining corresponding to calretinin in the merged images. C, F, I, and L) Single staining showingexpression of PKCa in bipolar cell bodies in the INL and their processes in the OPL and IPL. As with the other markers, PKCastaining was reduced after ischemia in both genotypes, but the effect was more pronounced in the dKO mice. PKCa staining wasapplied to the same section as in the merged image, but is shown separately for more clarity. G and J ) Disruption of the ILM indKO mice (asterisks). Scale bar, 20 mm.

IFs IN RETINAL CELL FUNCTION AND SURVIVAL 7

at the ILMto theOLM, including their cell bodies in the INL(Fig. 5C, D). Immunolabeling corresponding to the gluta-mate aspartate transporter (GLAST) and to the waterchannelaquaporin(AQP)-4 resembled thatofCRALBPandwas observed in all retinal layers in association with Mullercell processes and alsowith blood vessels in the case ofAQP4(Fig. 5E–H). The distribution of the 3markers was similar inWTandGFAP2/2Vim2/2 retinas,but stainingof the ILMwasoftenweaker or absent in the latter (Fig. 5D, F).Westernblotanalysis showed that the expression of these glial proteinsincreased steadily with age in both genotypes and that theirlevels were comparable (Supplemental Fig. S2).

We also examined the distribution of the Kir4.1 andKir2.1 channels. InWT retinas, Kir4.1 was observed at thelevel of the OLM and ILM in the distal and proximalprocesses of Muller cells and in association with retinalblood vessels (Fig. 6A, C). In contrast, in GFAP2/2Vim2/2

mice, labeling of the Muller cell end feet, of the ILM, andof theprocesses traversing the IPLwas substantiallyweakerthan in WT mice (Fig. 6B, D). A reduction in Kir4.1 ex-pression was observed by Western blot analysis (Fig. 6),which showed 22% lower levels in adult GFAP2/2Vim2/2

mice compared to that in WT (P, 0.005). The alterationof Kir4.1 immunoreactivity was not observed in GFAPsingle-knockout mice (Fig. 6E), but was evident in Vimsingle knockouts (Fig. 6F). The distribution of Kir2.1appeared similar in WT and GFAP2/2Vim2/2 retinas andwas observed mainly in the proximal radial processes ofMuller cells and in cell bodies in the INL and GCL (Fig.6G, H). In contrast, Western blot analysis revealed a 39%reduction in the levels of Kir2.1 (Fig. 6) in adult GFAP2/2

Vim2/2mouse retinas compared toWT levels (P, 0.005).

Muller cells from GFAP2/2Vim2/2 mice displaydecreased K+ currents

Given that the expression of Kir channels was altered inGFAP2/2Vim2/2 retinas, we recordedmembrane currents

in freshly isolated Muller cells in the voltage-clamp mode.As shown in Fig. 7, large inward currents were evoked byhyperpolarizing steps, whereas depolarizing steps evokedoutward currents of slightly smaller amplitude. Althoughthe current pattern was similar in both genotypes, the in-ward current amplitudes in Muller cells from GFAP2/2

Vim2/2mice were significantly smaller than inWT. Detailsandmeanvalues aregiven inTable 1. Inwardcurrentswereblocked by Ba2+ ions (0.1 and 1 mM), indicating that thecurrents were mediated by inwardly rectifying (Kir) chan-nels (data not shown).

After transient retinal ischemia, significantly decreasedcurrent amplitudes (Fig. 7) were recorded in cells fromboth strains. There was no significant difference betweenthe absolute inward current amplitudes recorded frompostischemic cells of both genotypes. When the currentamplitudes recorded in cells from control eyes were set to100%, the relative values recorded in cells from ischemiceyeswere similar inWTandGFAP2/2Vim2/2mice (Table1).The membrane capacitance in cells from GFAP2/2Vim2/2

mice was also smaller than in those from WT mice andincreased after ischemia. Moreover, the membrane po-tential was less negative in ischemic cells from bothgenotypes.

DISCUSSION

Mature Muller glial cells respond to structural and meta-bolic disruptions of normal neuron–glia interactions witha massive and relatively fast up-regulation of GFAP andVim; increased synthesis of cytokines, growth factors, andseveral other proteins; and increased stiffness in a processknown as reactive gliosis (10–20). Earlier studies showedthat the lack of GFAP and Vim attenuates the responses ofMuller cells to stress (26–30) and that cell survival is im-proved in a model of retinal detachment (28). In thepresent study, in an unchallenged retina, the inability to

Figure 4. Quantification of cells in control retinasand postischemic retinas (7 d after 90 min oftransient ischemia) of WT and GFAP2/2Vim2/2

mice. Cells were counted in the GCL, INL, andONL. A) The number of TO-PRO-3-stained cellnuclei was determined in a 200 mm wide areaclose to the optic nerve head in control andpostischemic retinas. B) Number of cells immu-nopositive for calretinin and PKCa. C) Relativenumber (percentage) of surviving cells in retinas7 d after transient ischemia. Data are means 6SEM. The number of cells decreased significantlyafter ischemia in all cases (not indicated here, forclarity). Differences were also observed betweenWT and GFAP2/2Vim2/2 mice in the GCL ofcontrol retinas (A) and in the number of survivingcells in the INL and GCL after ischemia (B, C).Significant differences between the indicatedvalues: dP , 0.05; ddP , 0.01; dddP , 0.001.Significant differences to the respective value ofthe WT: *P , 0.05; ***P , 0.001. Control andischemic eyes from 8 WT and 5 GFAP2/2Vim2/2

mice and 4–6 central sections from each animalwere analyzed.

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produceGFAP andVim affected the in vivo function of theretina, changed the expression and properties of Kirchannels on Muller cells, and caused a slow loss of retinalganglion cells. Moreover, in a model of retinal ischemia–reperfusion, the lack of glial cytoplasmic IF proteinsaccelerated retinal cell death.

The ERG recordings showed that the amplitude of themesopic b-wave was increased in GFAP2/2Vim2/2 mice,whereas the a-wave was unchanged. There were no sim-ilar functional alterations of the cone system responsesunder photopic conditions, and therefore, we concludethat the likely causes of the phenotype observed in theGFAP2/2Vim2/2 mice were alterations in Muller cells,which subsequently affected the rod system. The ERGresponses to flicker stimulations were similarly altered inGFAP2/2Vim2/2 mice, where the top and the trailingedges of the positive-going response (b-wave analog) wereincreased and its return to the baseline slowed down.Withincreasing stimulus frequency, however, the responses ofGFAP2/2Vim2/2 mice became smaller than those of WTmice. The decline in amplitude occurred in the frequencyrange usually attributed to the cone system (35). However,since there was no notable alteration in cone system re-sponses in the LA single-flash ERG, there are at least2 possible interpretations of this functional phenotype inGFAP2/2Vim2/2 mice. First, the cone system functionalitywas not greatly affected; therefore, the alteration was

detected only during continuous repetitive stimuli but notafter a single stimulation. Second, neurons of the conesystem were normal, but the cone system signaling wasdisturbed, as the retinal network (including cone path-ways) became saturated because of the prolonged photo-responses of the rod system [for a similar situation, seeSeeliger et al. (40)]. In this study, we couldnot discriminatebetween these 2 possibilities because, to our knowledge, itis not possible to distinguish them by changing the ERGrecording parameters.

The main source of the b-wave is depolarizing bipolarcells (i.e., rod bipolar cells andON cone bipolar cells), andincreases in amplitude could occur if the output from rodsor cones is increased or the number or responsiveness ofthe bipolar cells is increased. We found that the distribu-tion of proteins involved in the rod and cone pathway sys-tems was comparable in WT and GFAP2/2Vim2/2 miceand that the photoreceptor responses (a-wave) were nor-mal in the latter. Further, the number of cells expressingPKCa, a rod bipolar cell–specific marker) and PKCa pro-tein levels were similar in both genotypes, suggesting thatmajor alterations in this bipolar cell population cannotexplain the increased b-wave amplitudes observed undermesopic conditions.

We found that thenumber of calretinin-labeled cells wasreduced in theGCL inGFAP2/2Vim2/2mice. Thisfindingsuggests that the expression of GFAP and Vim is essential

Figure 5. Immunohistochemical localization ofglial proteins in WT and GFAP2/2Vim2/2

(dKO) mice at PN60. A and B) Sox9 proteinstaining in the nuclei of Muller cells in the INL.C and D) CRALBP staining in Muller glial cellbodies and processes extending from the OLMto the ILM in both genotypes. Note thatstaining in the proximal processes (C, arrows)appears slightly reduced in the knockout mice(D). E and F) GLAST staining in Muller cellsand in their radial processes, in the OLM, as wellas in the OPL and IPL. G and H) AQP4 stainingin Muller cells and associated with vessels (arrow-heads). Note the disruption of the vitreoretinalsurface in some sections of GFAP2/2Vim2/2 mice(D, F, asterisks). Scale bar, 25 mm.

IFs IN RETINAL CELL FUNCTION AND SURVIVAL 9

for the survival of ganglion cells. Unchallenged retinas ofGFAP2/2Vim2/2 mice exhibit an overall normal mor-phology, except for alterations of the vitreoretinal border,which,uponexertionofmoderate force, gets sheared fromthe rest of the retina (26, 27, 29). This phenomenon wasobserved in the present study in retinas stained with H&Eand with some of the Muller cell markers and is a likelyconsequence of the processing of the tissue (26, 27). Suchfragility is not surprising, as studies have shown that me-chanical properties of glial cells, such as stiffness and elas-ticity, aredependent on the amount of IFproteins (30, 41).

It is therefore possible that a lower number of calretinin-labeled cells was found in the GCL of theGFAP2/2Vim2/2

mice because of the tearing of this layer. However, it hasbeen shown that after retinal detachment in these animals,neurofilament proteins are expressed in ganglion cell so-mata only in regions where shearing of the vitreoretinalborder is observed, which implies that the shearing oc-curred in vivo (i.e.,before theprocessingof the tissue) (29).This possibility suggests that the innermost layers of theGFAP2/2Vim2/2 retina are morphologically, and possiblyalso functionally, less stable.

Figure 6. Immunohistochemical localizationof Kir channels in WT, GFAP2/2Vim2/2 (dKO)and single-knockout mice. A and B) Kir4.1 atPN60. C and D) Kir4.1 at PN270. E) Kir4.1 atPN60 in GFAP single-knockout (GFAP KO)mice. F) Kir4.1 at PN60 in Vim single-knockout(Vim KO) mice. In retinas of WT and GFAPKO mice, labeling is seen in the distal andproximal (arrows) radial processes of Mullercells, at the level of the OLM and ILM, and inassociation with vessels (arrowheads). In dKOmice and Vim KO mice, the overall labelingpattern is comparable (B, D, F), except thatstaining in the proximal processes and endfeet of Muller cells and in the ILM is reduced.G and H) Kir2.1 at PN60 is seen in Mullercell bodies and processes and at the level ofthe GCL. Note the disruption of the innerlimiting membrane in dKO (H, asterisks).Bottom: Representative examples of Westernblots showing the expression of Kir2.1 andKir4.1 in retinas of WT and GFAP2/2Vim2/2

(dKO) mice at PN7, -14, -21, and -60. Tenmicrograms of protein was loaded in eachlane, except for Kir4.1 at PN7 where 20 mg wasloaded. The housekeeping proteins GAPDHand tubulin served as loading controls. Scalebar, 25 mm (all micrographs).

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The expression of the Kir4.1 and Kir2.1 channels,expressed by Muller cells and necessary for spatial K+

buffering, was reduced in the GFAP2/2Vim2/2 mice, andthese cells displayed smaller Kir-mediated inward currentsthan were found in the WT mice. Some Kir channel sub-units are found in Muller cells and contribute to the reg-ulation of extracellular K+ concentrations, allowing entryor exit ofK+, dependingon their specificdistribution alongthe Muller cell membrane and on the K+ levels generatedby neuronal activation. Kir4.1 channels are abundant inthe end feet and the apical and perivascular processes ofthe Muller cells, redirecting K+ to the vitreous, subretinalspace, and circulation, in amechanism of spatial bufferingdenominated K+ siphoning (39, 42). In addition, Kir2.1channels, which are more homogeneously distributedalong theMuller cell and possibly on rod and cone bipolarcell terminals, and Kir4.1/Kir5.1 heterotetrameric chan-nels are believed to mediate K+ influx at the level of thesynaptic layers (39, 43, 44). Alterations in the levels or ac-tivity of these channels candirectly and indirectly affect thefunctional state of Muller cells. A recent study has showna correlation also between lower Kir4.1 levels and reducedcell survival in a mouse model of Huntington’s disease(45). We found that the expression of Kir4.1 channels inGFAP2/2Vim2/2 mice was markedly reduced in the innerprocesses and end feet of the Muller cells and that the Kircurrent amplitudes recorded in these cells were smallerthan in WT mice. Therefore, it is plausible that the lowernumberofganglioncells found inGFAP2/2Vim2/2wasnotan artifact, but rather was secondary to the lower expres-sion of Kir channels (or to disruption of other regulatoryfunctions of Muller cells). Further, the retina containsa relatively small number of astrocytes that are restricted tothe innermost part of the retina and are in close contactwith the axons of ganglion cells (5, 6). These cells are alsodevoid of GFAP and Vim, and although it was not possibletodiscriminate between themand theMuller cell end feet,they are also likely to express lower levels of Kir channels,

which could contribute to increase the vulnerability ofthe ganglion cells.

After ischemia–reperfusion, we found cell loss in allretinal layers in the WT mice, confirming the results ofprevious studies (14, 38, 46). ComparedwithWTmice, thenumber of surviving cells in the GFAP2/2Vim2/2mice waslower in the GCL and INL, indicating that the absence ofthe cytoplasmic IF system enhances the susceptibility ofat least some retinal cells to ischemia. This observationis in contrast to that in a previous study, which showed abetter survival of photoreceptor cells after detachmentin GFAP2/2Vim2/2 vs. WT mice (28). Reduced gliotic re-sponse caused by the lack of the IF proteins appearsthus to have different effects on retinal cell vulnerability,depending on the underlying pathologic condition. Thisfinding is in agreement with our previous results showingthat GFAP2/2 mice respond as do WT mice to scrapie-induced neurodegeneration (47), whereas in Alzheimer’s(48) and Batten diseases (49), the disease progressionis facilitated and the extent of neurodegeneration andneuroinflammatory response is increased on theGFAP2/2

Vim2/2 background. The finding is also supported by theobservation that GFAP2/2Vim2/2 mice exhibit increasedtissue loss after ischemic stroke (50), but not after perinatalhypoxia–ischemia (51) or photothrombotic lesion (52).In the present study, cell death was accelerated in theinner retina in GFAP2/2Vim2/2 mice after ischemia–reperfusion, and the degree of photoreceptor cell loss wascomparable to that in WT mice. Together, these findingssuggest that the impact of IF deficiency on retinal cellsurvival is notonlydisease specificbutalso cell type specific.

An important alteration of Muller cells in postischemicretinas is the decrease of inward current amplitudes (14,46). In the present study, we found that inward currentsafter ischemia decreased to a similar level in WT andGFAP2/2Vim2/2mice. In an earlier study on the ischemicretina of mice deficient in the nucleotide receptor P2Y1, itwas observed that lack of P2Y1 attenuated the ischemia-induced down-regulation of inward currents. This obser-vation was accompanied by a better survival of (amacrine)cells in the inner retina. It was hypothesized that thepreserved K+ conductance of Muller cells in P2Y1-deficient mice stabilizes the ion and volume homeo-stasis in the inner retina where cell types generatingaction potentials are located (38). In contrast to thesedata, the smaller K+ conductance we found in Mullercells of GFAP2/2Vim2/2 mice may cause an impairedhomeostatic function and thus explain the increasedvulnerability of neurons in the inner retina, particularlyafter an ischemic insult.

DysfunctionofMuller cells is also likely tounderlie the invivo electrophysiological alterations we found in the un-challenged GFAP2/2Vim2/2 mice, because these cellscontribute to the ERG. In a normal retina, there is a light-evoked reduction in extracellular K+ concentration inthe subretinal space that triggers a positive-going response(c-wave) formed by the RPE response and by negative-going signals [slow peak (P)III] resulting from the Mullercell K+ currents, which are usually not visible due to anoverlapof the largepositive-goingb-wave (53).As observedin aprevious study, alterations inKir4.1 channels can affecttheERGresponses.After total inactivationofKir4.1channelsin an otherwise intact mouse retina, Muller cells displayed

Figure 7. Membrane currents in Muller cells from WT andGFAP2/2Vim2/2 mice. Typical original recordings from 4different cells are shown. Currents were evoked by 20 mVincremental steps from a holding potential of 280 mV.Outward currents (evoked by depolarizing steps) turn up-ward, inward currents (evoked by hyperpolarizing steps) turndownward. The inward current amplitudes were significantlyreduced in cells from GFAP2/2Vim2/2 mice and in cells fromboth genotypes after ischemia (Table 1).

IFs IN RETINAL CELL FUNCTION AND SURVIVAL 11

normal morphology, but their resting membrane potentialwas depolarized relative to the normal value, and thepotassium conductance wasmarkedly reduced (54). Thelight-induced slow PIII response of the ERG was com-pletely absent in these mice, and the intraretinal b-waveamplitude was increased, which was attributed to the lossof the slow PIII. The same effect was observed after ad-dition of Ba2+ to block the K+ channels on Muller cells inpreparations of normal mouse retinas, confirming thatthe PIII component is the result of currents produced inthese cells (54). Homozygous Kir4.1 knockout mice dieduring the first postnatal weeks before retinal develop-ment is complete, and the effects observed in our study atabout 8 wk of age cannot be studied in those animals.Nevertheless, similar results have been obtained in olderanimals, heterozygous for the Kir4.1 deletion (55). In thecurrent study, the level of expression and the distributionand the properties of Kir channels were all found tobe altered in Muller cells of GFAP2/2Vim2/2 mice. Theb-wave abnormalities observed in these animals are thuslikely to reflect compromised Kir channel activity, whichled to a reduction of the slow PIII component andthereby in increased amplitude and prolonged decay.

It is unclear what causes the Kir channel alterationsobserved inGFAP2/2Vim2/2mice.Onepossiblemechanisminvolves the role that glial cytoplasmic IFs may play in thecellular distribution of the channels. Whereas GFAPbuilds abnormal IF bundles in Vim2/2 astrocytes, Vimcannot form IFs on its own unless nestin is present (33),with consequences predicted to go beyond a mere al-teration of the mechanical properties of the cell. Vim isexpressed by many cell types during development andappears to be an organizer of many important proteinsassociated with cell–cell adhesion, migration, and cellsignaling (56), and in its absence, important structuraland signaling functions may be compromised, leadingto an abnormal expression and distribution of the K+

channels. Another possibility is that the inability to pro-duce the IF proteins affects the maturation of glial cells.The cell morphology and expression profile of severalcell-specific markers appeared normal in Vim2/2 andGFAP2/2Vim2/2mice, suggesting that lack of Vimhas nodetectable impact on the development of the retina.However, electronmicroscopy has revealed a thinning ofMuller cell end feet in retinas of GFAP2/2Vim2/2 mice(29). Further, the amplitude of inward K+ currents inMuller cells has been shown to be dependent on the

degree of differentiation of these cells (57, 58). Ampli-tudes thus increase in an age-dependent manner, sup-ported by an increasing number of K+ channels (39, 58).In the present study, the expression of Kir2.1 andKir4.1 increased, as expected with age, in both WT andGFAP2/2Vim2/2 mice. However, the increase rate waslower in the latter. The lack of IF proteins also correlatedwith smaller Muller cell current amplitudes, which is inagreement with the hypothesis of disturbed develop-ment.We therefore cannot exclude the possibility that thematuration of the Muller cells is somewhat delayed orimpaired in GFAP2/2Vim2/2 mice, contributing to thereduction in the levels of K+ channels.

In summary, in this study, genetic attenuation of glialactivation accelerated retinal cell death after an ischemicinsult, providing further support to the notion that theimpact of IF deficiency on neuronal cell vulnerabilitydepends on the type of insult. In addition, GFAP and Vimwere necessary to maintain basic glial homeostatic prop-erties and the inability to produce these proteins affectedretinal physiology and cell survival, even in the absenceof injury. These novel findings show that the IFs are im-portant, not only in the process of reactive gliosis, butalso under physiologic conditions. Further, given thatneuron–glia interactions are common outside the retina,other areas of theCNSmay be similarly affected by the lackof cytoplasmic IFs of astrocytes.

The authors thank B. Sandstrom for maintaining the animalcolonies; H. Abdshill for tissue sectioning; Drs. U. Wilhelmssonand Y. de Pablo for crossing, breeding and genotyping of someof the mice; and Dr. R. S. Molday (University of BritishColumbia, Vancouver, BC, Canada) for the generous gift ofantibodies against rhodopsin. This work was supported bygrants from the VELUX Stiftung (to M.T.P.); Crown PrincessMargareta’s Committee for the Blind (to M.T.P.), Stiftelse forSynskadade i fd Malmohus Lan (to M.T.P.), Thorsten och ElsaSegerfalks Stiftelse (to M.T.P.); the European Union projects020235-NEUROTRAIN (to M.T.P), 512036-GENORET (to M.T.P.),237956-EduGlia (to M.P.), and 279017-TargetBraIn (to M.P.);Deutsche Forschungsgemeinschaft Grants RE 849/16-1 (to A.R.and T.P.), SPP 1757 (to A.R.), KFO134-Se837/5-2 (to M.W.S. andN.T.), and Se837/6-2 (to M.W.S.); the PRO RETINA-Stiftung (to A.G.); the Center of Integrative Neuroscience (EXC 307), a Center ofExcellence of the German Research Council and the BernsteinCenter for Computational Neuroscience, Tubingen (to E.Z.);and Swedish Medical Research Council Grant 11548, Avtal omLakarutbildning och Forskning (ALF) Gothenburg (11392),the Arbetsmarknadens Forsakringsaktiebolag (AFA) Research

TABLE 1. Whole-cell patch-clamp data from WT and GFAP2/2Vim2/2 mice

Parameter Wild-type controlWild-type

postischemiaGFAP2/2 Vim2/2

controlGFAP2/2 Vim2/2

postischemia

Inward current (pA) 2644 6 691 967 6 771*** 1678 6 612††† 839 6 766***Relative inward current (%) 100 6 23 37 6 30*** 100 6 34 46 6 41***Membrane capacitance (pF) 41 6 10 48 6 15* 31 6 8††† 39 6 15*Current density (pA/pF) 70 6 25 21 6 15*** 58 6 22 21 6 17***Membrane potential (mV) 285 6 4 275 6 16*** 282 6 4† 270 6 19**Cells (n) 46 from 9 mice 49 from 9 mice 33 from 7 mice 31 from 6 mice

Data are means 6 SD. *P , 0.05; **P , 0.01; ***P , 0.001 (significant differences between values recorded in cells from control andpostischemic eyes of the same strain). †P , 0.05; †††P , 0.001 (significant differences between values recorded in cells from control eyes of bothstrains).

12 Vol. 29 December 2015 WUNDERLICH ET AL.The FASEB Journal x www.fasebj.org

Foundation, Soderbergs Foundations, Sten A. Olsson Foun-dation for Research and Culture, Hjarnfonden, Hagstromer’sFoundation Millennium, and NanoNet COST Action GrantBM1002 (to M.P.).

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Received for publication May 20, 2015.Accepted for publication July 27, 2015.

14 Vol. 29 December 2015 WUNDERLICH ET AL.The FASEB Journal x www.fasebj.org