Dystrobrevin Localization in Photoreceptor Axon Terminals and at Blood–Ocular Barrier Sites

Abstract

purpose. Dystrobrevin is a newly discovered dystrophin-associated protein with multiple sites for phosphorylation on tyrosine residues. In the present study, the cellular distribution and subcellular localization of dystrobrevin were examined in the adult rat retina, cornea, lens, iris, ciliary body, and cultured Müller cells.

methods. Immunoblot analysis, confocal laser scanning microscopy, and immunoelectron microscopy were used to examine dystrobrevin expression.

results. Immunoblot analysis showed that an approximately 87-kDa band was expressed predominantly in the lens, retina, iris and ciliary body, whereas an approximately 60-kDa band was expressed in cultured Müller cells, cornea, retina, iris, and ciliary body. Confocal microscopy demonstrated dystrobrevin in the inner limiting membrane, outer plexiform layer, and retinal pigment epithelium and around blood vessels in the retina. At the ultrastructural level, dystrobrevin was localized under cell membranes of rod spherules and cone pedicles of photoreceptor cell terminals but often was found in the cytoplasm of endothelial cells and Müller cells. Furthermore, dystrobrevin was colocalized with β-dystroglycan in corneal endothelium; lens, iris, and ciliary epithelia; and cultured Müller cells.

conclusions. The present study demonstrates that dystrobrevin is expressed in neurons, glia, and endothelial cells in the rat retina. In addition, dystrobrevin is localized at the blood–ocular barrier sites in extraocular tissue. These data suggest that dystrobrevin plays an important role in visual function.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormality of the DMD gene product dystrophin. Dystrophin is a submembranous cytoskeletal protein with a 427-kDa molecular weight that is expressed not only in muscle tissues but also in nervous systems including the retina. Dystrophin has several truncated isoforms in addition to full-length dystrophin, including Dp260, Dp140, Dp116, and Dp71, which are expressed in a tissue-specific manner. Moreover, many dystrophin-associated proteins (DAPs), such as utrophin, dystroglycans, sarcoglycans, syntrophins, and dystrobrevins, have been identified so far. ^{1 2 3 4 5 6 7}  

The major clinical manifestations of DMD-BMD are progressive muscle weakness and nonprogressive cognitive impairment. Until several years ago there had been no reports of ocular manifestations, such as visual disturbance or morphologic abnormality of the retina, in patients with DMD-BMD. However, recent studies have shown that abnormal electroretinogram (ERG) patterns with a reduced b-wave amplitude under dark-adaptation conditions are seen in patients with DMD-BMD. ^{8 9 10 11 12} Moreover, dystrophin-mutant mice without Dp260 or Dp71 showed reduction of the b-wave amplitude ^{13 14} or prolonged implicit time ¹⁵ of the scotopic ERG. 

Subcellular localization of dystrophin and β-dystroglycan in the retina has been extensively examined. Dystrophin was immunohistochemically detected in the outer plexiform layer (OPL), ^{16 17 18} and immunoelectron microscopy showed that dystrophin was located in the presynaptic sites (i.e., rod spherules and cone pedicles of photoreceptor cells). ^{18 19 20} Recent studies have demonstrated that Dp71 is localized in the inner limiting membrane (ILM) and around blood vessels, ²¹ whereas Dp260 is expressed in the OPL, ^{15 21 22} indicating that dystrophin isoforms are localized in a cell-specific manner.β -Dystroglycan, a 43-kDa transmembrane protein, is also expressed in the ILM and OPL and around blood vessels. At the ultrastructural level,β -dystroglycan is localized in Müller cell processes and photoreceptor cell terminals in the retina. ^{23 24 25 26 27}  

It is generally assumed that dystrophin stabilizes muscle fibers with DAPs by linking the sarcolemma to the basement membrane, but the molecular mechanism associating dystrophin and DAPs with neurotransmission in the retina remains elusive. In addition, it remains unclear whether other DAPs such as sarcoglycans or syntrophins are expressed in the retina or in other ocular tissues. Recently, dystrobrevin, a newly discovered dystrophin-associated protein, was cloned and localized in skeletal muscle and brain. ^{28 29 30 31 32} Dystrobrevin contains multiple phosphorylation sites and is assumed to be associated with signal transduction. In the present study, we examined dystrobrevin expression in the rat retina, cornea, lens, iris, ciliary body, and cultured Müller cells by immunoblot analysis and immunohistochemistry. 

Materials and Methods

Animal and Tissue Preparation

All animals used in this study were cared for and handled in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Adult Sprague–Dawley rats were anesthetized with diethyl ether and sodium pentobarbital and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) through the heart. Eyeballs were removed and immersed in fixative overnight at 4°C. After they were rinsed in phosphate-buffered saline (PBS), the eyeballs were immersed in 30% sucrose for dehydration overnight at 4°C. Subsequently, they were embedded in optimal cutting temperature compound and sectioned at 10-μm thickness in a cryostat (Leica, Heidelberg, Germany) for immunohistochemistry. 

Müller Cell Culture

Eyes from five infant Sprague–Dawley rats 3 to 5 days of age were rapidly enucleated into Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mM glutamine and 0.1% penicillin-streptomycin and stored overnight at room temperature in the dark. Eyes were treated according to the method of Hicks and Courtois. ³³ Briefly, intact globes were incubated in DMEM containing 0.1% trypsin and 70 U/ml collagenase, 0.5 ml per eye at 37°C for 60 minutes. They were subsequently placed in a petri dish containing DMEM supplemented with 10% fetal calf serum (FCS). The retinas were removed and either mechanically dissociated into small aggregates with a sterile Pasteur pipette or chopped into 1-mm² fragments and seeded onto 10-cm Falcon culture dishes (Falcon Labware, Oxnard, CA) containing sterile glass coverslips, approximately six to eight retinas per dish. Medium was left unchanged for 5 to 6 days and then replenished every 3 to 4 days. All cultures were maintained at 37°C in a 5% CO₂-95% air atmosphere in a humidified incubator. 

Cell Passaging

Cell growth was first detectable 1 to 5 days after seeding (passage 1). When cell outgrowth had attained semiconfluence (5–7 days), retinal aggregates and debris were removed by forcibly pipetting medium onto the dish. Repeating this operation three to five times dislodged all aggregates and resulted in a purified, flat cell population. Cells proliferated rapidly, becoming fully confluent within 4 to 8 days after initial appearance. At this time cells could be easily passaged after they were rinsed twice with Ca²⁺-free PBS followed by a brief incubation in PBS containing 0.05% trypsin, 1 mM EDTA, and 1 mg/ml glucose (2–5 minutes at 37°C). The suspension was centrifuged at 800g for 5 minutes and cells resuspended and seeded at 1 to 2 × 10⁵ cells cm² in fresh DMEM with 10% FCS (passage 2). The same procedure was performed two more times to purify Müller cells (passage 4). Müller cells were easily distinguished from other cell types morphologically or immunocytochemically using anti-glutamine synthetase or anti-carbonic anhydrase II antibodies. 

Antibodies

Mouse monoclonal anti-dystrobrevin antibody (D62320) was purchased from Transduction Laboratories (Lexington, KY). The anti-dystrobrevin antibody was produced from an immunogen that corresponded with amino acids 249 to 403, a region that is highly homologous among all the dystrobrevin proteins. Thus, this antibody can detect both α- andβ -dystrobrevins. Mouse monoclonal anti-β-dystroglycan antibody (NCL-43DAG) was purchased from Novocastra (Newcastle-upon-Tyne, UK), and propidium iodide was purchased from Molecular Probes (Eugene, OR) for nuclear staining. Mouse monoclonal anti-glutamine synthetase antibody (MAB302) and rabbit polyclonal anti-carbonic anhydrase II antibody (AB1243; both from Chemicon, Temecula, CA) were used to identify Müller cells in culture. 

Western Blot Analysis

Immunoblot analysis was performed according to the protocol of the manufacturer’s anti-dystrobrevin antibody instruction sheet. Rat retina, iris, ciliary body, cornea, and lens were removed and stored at− 80°C until use. They were separately homogenized in lysis buffer containing 10 mM Tris, 1 mM sodium vanadate, and 1% sodium dodecyl sulfate (SDS), which was adjusted to pH 7.4 with HCl. They were centrifuged at 12,000g for 5 minutes at room temperature, and the supernatant was boiled for 3 minutes with sample buffer (1% SDS, 125 mM Tris, 30% glycerol, 5% 2-mercaptoethanol, and 0.02% bromophenol blue). Subsequently, the samples were loaded onto 10% SDS-polyacrylamide gels for electrophoresis and blotted on polyvinylidene fluoride membranes (Immobilon; Millipore, Bedford, MA). The blots were pretreated with 5% skim milk in PBS containing 0.1% Triton X-100 (PBST) for 60 minutes and incubated with anti-dystrobrevin antibody (diluted 1:1000) in PBST overnight at 4°C and in biotinylated rabbit anti-mouse immunoglobulin antibody (1:300; Amersham, Buckinghamshire, UK) and streptavidin conjugated to horseradish peroxidase (1:3000; Amersham) for 60 minutes at room temperature. Subsequently, the reaction products were visualized by enhanced chemiluminescence (ECL Plus; Amersham). Immunoblot analysis of cultured Müller cells was performed in the same manner. For the control, rat brain lysate supplied by the manufacturer (Transduction Laboratories) was used. 

Immunoperoxidase Staining

Cryosections were treated with PBS containing 1% Triton X-100 and 1% H₂O₂ for 30 minutes, 10% rabbit serum for 30 minutes, and primary antibodies (anti-dystrobrevin or anti-β-dystroglycan antibody) at 1:50 dilution for 2 hours each at room temperature. They were then incubated with an ABC kit (Histofine; Nichirei, Tokyo, Japan) according to the manufacturer’s protocol and treated with a metal-enhanced 3,3′-diaminobenzidine kit (DAB; Pierce, Rockford, IL) and 0.04% osmium tetroxide for 10 seconds. The sections were observed with a light microscope (BX50; Olympus, Tokyo, Japan). 

Confocal Laser Scanning Microscopy

Cryosections or coverslips on which Müller cells were cultured were incubated with primary antibodies overnight at 4°C, biotinylated anti-mouse IgG for 30 minutes at room temperature, and Alexa 488 coupled to streptavidin (Molecular Probes) together with propidium iodide at 1:1000 dilution for 60 minutes and mounted (FluoroGuard; Bio-Rad, Hercules, CA). A confocal laser scanning microscope (TCS4D; Leica) was used with a ×63 oil-immersion objective lens. For the control, the primary antibodies were omitted. 

Conventional Electron Microscopy

Conventional electron microscopy preparation has been described previously. ²⁷ Briefly, anesthetized rats were perfused with 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M PB through the heart, and eyeballs were immersed in the fixative overnight at 4°C. After they were rinses in PBS, the tissues were chopped into small pieces, treated with 1% osmium tetroxide for 60 minutes, dehydrated in a graded series of ethanol and acetone, and embedded in Epon (Nisshin EM, Tokyo, Japan). Ultrathin sections at 75-nm thickness were counterstained with uranyl acetate and lead citrate and observed in an electron microscope (H-7500; Hitachi, Tokyo, Japan). 

Immunoelectron Microscopy

Cryosections at 10-μm thickness, treated with 1% Triton X-100 and 1% H₂O₂ in PBS for 30 minutes and 10% rabbit serum for 30 minutes, were incubated with the primary antibody (dystrobrevin, 1:20) for 2 hours at room temperature or overnight at 4°C. Subsequently, sections were treated as described before ²⁷ : 1% osmium tetroxide for 60 minutes, dehydration in a graded series of ethanols, and embedding in Epon by the inverted gelatin capsule method. Ultrathin sections at 70 nm were stained with only uranyl acetate and observed in an electron microscope (Hitachi). 

Results

Immunoblot Analysis of Dystrobrevin Expression In Vivo

Dystrobrevin is classified into two isoforms, α and β, which undergo extensive alternative splicing. ^{28 29 30 31 34} α -Dystrobrevin consists of α-dystrobrevin-1 of 78 to 84 kDa andα -dystrobrevin-2 of approximately 60 kDa, which are detected in muscle, ^{28 31 32} whereas β-dystrobrevin, of 61 kDa ²⁹ or 71 kDa, ³¹ is rich in brain, but not in muscle. ²⁹ Our immunoblot findings demonstrated that retinal and iris-ciliary body lanes showed quite similar patterns with bands of approximately 87, approximately 80, and 65 kDa (Figs. 1B 1C ). Cornea or lens lanes presented different banding patterns. Cornea contained much more 65-kDa protein than 87-kDa protein, whereas lens showed doublet bands with approximately 90-kDa proteins and much less 65-kDa protein (Figs. 1D 1E) . These differences of molecular size in various tissues were probably due to phosphorylation because dystrobrevin has multiple phosphorylation sites. Iris-ciliary body and lens lanes additionally showed approximately 30-kDa bands that may have been produced by posttranslational modifications or degradation during sample preparation. 

Dystrobrevin Expression in the Retinal Pigment Epithelium, Müller Cells, and Endothelial Cells of Blood Vessels in the Rat Retina

Our group ²⁷ and others ^{23 24 26} have demonstrated that β-dystroglycan is localized in the photoreceptor axon terminals and paravitreous or perivascular end feet of Müller cells. In this study, confocal microscopy showed that dystrobrevin was expressed in the ILM, OPL, and retinal pigment epithelium (RPE) and around blood vessels in the rat retina (Fig. 2) . The labeling pattern of dystrobrevin was consistent with that ofβ -dystroglycan, but, unlike β-dystroglycan, dystrobrevin was also detected in the ganglion cell layer (GCL) in a punctate pattern (Fig. 2B) . β-Dystroglycan was localized in perivascular Müller cell end feet but not in endothelial cells. ²⁷ To clarify whether dystrobrevin is expressed in endothelial cells, immunoelectron microscopy was used. Immunoelectron micrographs demonstrated that dystrobrevin was localized in the cytoplasm of both Müller cells and vascular endothelial cells (Fig. 3E ). Furthermore, dystrobrevin was detected in paravitreous Müller cell end feet, not only under the cell membrane but also in the cytoplasm (data not shown), which is consistent with the linear and punctate labeling pattern in Figure 2B . 

Dystrobrevin Localization in Rod Spherules and Cone Pedicles Similar to Dystrophin and β-Dystroglycan

Confocal images of dystrobrevin localization in the OPL (Fig. 2C) were similar to that of dystrophin and β-dystroglycan demonstrated previously, ^{18 19 20 27} suggesting that dystrobrevin is localized in the rod spherules and cone pedicles. To address the subcellular localization of dystrobrevin, immunoelectron microscopy was used. Similar to dystrophin and β-dystroglycan, dystrobrevin was localized in the electron-dense region apposing bipolar cell processes in the rod and cone photoreceptor terminals (Figs. 3B 3D) . No immunoreactive signals were detected in photoreceptor cell membranes facing horizontal cell processes. Therefore, these data are consistent with the hypothesis that dystrobrevin forms a complex with dystrophin and β-dystroglycan in the photoreceptor axon terminal. 

Expression of Dystrobrevin and β-Dystroglycan in Cultured Müller Cells

Approximately 80% of the cells at passage 2 and almost all cells after passage 3 were Müller cells. We checked Müller cells at passage 2 to see whether dystrobrevin was expressed in vitro, by using immunoblot analysis and immunocytochemistry. Immunoblot analysis of cultured Müller cells showed that an approximately 65-kDa protein was expressed much more frequently than an approximately 87-kDa protein (Fig. 4A ). At passage 2, Müller cells often formed clusters, and dystrobrevin was strongly expressed in a rather diffuse manner (Fig. 4B) . β-Dystroglycan was also expressed in Müller cells in a similar pattern (Fig. 4C) , but a linear labeling pattern was often noted in some parts of clustered Müller cells (data not shown). At passage 4, most of the cultured cells were dissociated Müller cells, and dystrobrevin was expressed in the cytoplasm as seen at passage 2 (data not shown). These data demonstrate that Müller cells expressed dystrobrevin and β-dystroglycan in vitro. 

β-Dystroglycan and Dystrobrevin Coexpression in the Cornea, Lens, Iris, and Ciliary Body

The cornea is avascular, and its central region depends on diffusion from the aqueous humor for its nourishment. Corneal endothelium rests on the basement membrane and Descemet’s membrane, and both β-dystroglycan and dystrobrevin were expressed in the basal portion of the corneal endothelium (Figs. 5A 5B ). The lens is covered by a capsule that is homogeneous, rich in collagen and proteoglycans, and held in position by a system of fibers constituting the ciliary zonule. The zonule fibers arise from the epithelium of the ciliary portion of the retina and attach to the capsule in front of the equator of the lens. A lens capsule attached to the vitreous body forms the hyaloideocapsular ligaments, where lens epithelium becomes flattened. Of note, both β-dystroglycan and dystrobrevin were detected in the posterior parts of lens epithelium where the tension should be loaded through zonule fibers and hyaloideocapsular ligaments (Figs. 5C 5D) , but not in the anterior sites of the lens (data not shown). 

Rat iris and ciliary epithelia contain no osmiophilic pigment granules and are composed of two layers (Figs. 5I 5J) . In the iris, the inner layer is a continuation of the inner ciliary epithelium, and the outer layer contains contractile elements and is called the myoepithelium of the dilator pupil. Both layers rest on the basement membrane (Fig. 5I) .β -Dystroglycan and dystrobrevin were expressed at the basal sites of both layers attaching to the basement membrane (Figs. 5E 5F , inset in 5I). In the ciliary body, inner epithelial cells rested on the basement membrane contiguous with the ILM, and outer epithelial cells rested on the basal lamina contiguous with the RPE of the retina. Inner and outer epithelia were separated by discontinuous intercellular spaces called ciliary channels and contained many mitochondria in their cytoplasm and a labyrinth of interdigitating processes in the basal and lateral regions (Fig. 5J) . β-Dystroglycan and dystrobrevin were localized in the basal regions apposing the basement membrane, as well as in the iris (Figs. 5G 5H , inset in 5J ). 

Altogether, these data demonstrate that β-dystroglycan and dystrobrevin are closely associated with the basement membrane in various ocular tissues other than the retina. 

Discussion

In the present study, we demonstrated that dystrobrevin is expressed in various ocular cells by using immunoblot analysis and immunohistochemistry. Immunoblot data show that retina, iris and ciliary body, cornea, and lens have dystrobrevin of different molecular sizes, although we cannot exclude the possibility of phosphorylation, posttranslational modifications, or degradation during sample preparation. Confocal microscopy indicated that dystrobrevin was expressed in the ILM, OPL, and RPE and around blood vessels in the retina, and immunoelectron microscopy demonstrated that it was localized in the vascular endothelial cells, Müller cells, and rod spherules and cone pedicles of photoreceptor cells. In addition, dystrobrevin was coexpressed with β-dystroglycan in the corneal endothelium, lens epithelium, iris epithelium, and ciliary epithelium. Considering how widespread dystrobrevin localization is along the basement membrane or at synaptic sites in various ocular tissues, dystrobrevin may play an important role in ocular tissues. 

In previous studies, investigators have reported that α-dystrobrevin is detected in muscle, ^{28 31 32} but β-dystrobrevin is rich in brain but not in muscle. ²⁹ Recently, Blake et al. ³⁵ reported that β-dystrobrevin is a neuronal protein, whereas α-dystrobrevin-1 is found in glial cells in brain. Although it remains to be determined which dystrobrevin is expressed, our present study showed that dystrobrevin was expressed in retinal neurons, photoreceptor cells, and in the retinal glia, in Müller cells. In addition, we demonstrated dystrobrevin localization in the astrocytic end feet around blood vessels and the pia mater in the rat cerebellum. ³⁶ Therefore, these findings demonstrate that dystrobrevin is widespread in the central nervous system including the retina. 

Physiological studies in some patients with DMD or variable dystrophin-mutant mice have shown abnormal ERG patterns, such as reduced amplitude or prolonged implicit time of b-wave, under dark-adapted conditions. ^{8 9 10 11 12 13 14 15} These results lead us to think that abnormal neurotransmission develops between photoreceptor and bipolar cells. Dystrophin and β-dystroglycan are actually localized in presynaptic sites of photoreceptor cell terminals. In the present study, dystrobrevin was shown to be localized in photoreceptor axon terminals, supporting the hypothesis that dystrobrevin is associated with neurotransmission as a component of the dystrophin complex. 

Biochemically, it has been assumed that the dystrophin complex is associated with signaling as well as structural roles, because dystrophin and β-dystroglycan bind signaling molecules calmodulin and Grb2, respectively. ^{37 38} Another dystrophin-associated protein, α1-syntrophin, interacts with neuronal nitric oxide synthase (nNOS) ³⁹ and voltage-gated sodium channels ^{40 41} through their PDZ domains in the skeletal muscle. In addition, α- and β-dystrobrevin bind both dystrophin and syntrophins. ^{32 35 42} Recently, Grady et al. ⁴³ reported that α-dystrobrevin–deficient mice have no nNOS but retain the dystrophin complex and concluded that α-dystrobrevin is involved in signaling but not structural functions in the skeletal muscle. Accordingly, although it remains unclear whether dystrobrevin binds nNOS or other signaling proteins directly, it is plausible that dystrobrevin functions as a signaling molecule. However, in the retina, no studies on syntrophin localization have been reported so far, ⁴³ and the dystrobrevin expression pattern is different from nNOS (Ueda, unpublished data, 1999). Therefore, further studies should be focused on a dystrobrevin signaling role in the retina. 

Dystrobrevin distribution in the eye is consistent with that of blood–ocular barriers, including RPE, the blood–retina barrier at perivascular sites in the retina, and the blood–aqueous barrier in the ciliary body. The lateral membrane of polarized epithelial cells contains two specialized structures: tight junctions and adherens junctions. ⁴⁵ Recent biochemical studies have demonstrated that the tight and adherens junctions are key structures of epithelial cells where numerous PDZ-containing proteins have been shown to be important. ^{46 47 48 49 50 51} However, our immunohistochemical findings in the iris and ciliary body suggested that dystrobrevin is localized at the basal sites of both ciliary epithelial layers, rather than at tight or adherens junction sites. In addition, dystrobrevin was not colocalized with ZO-1, which is a specific marker for tight junctions (Ueda, unpublished data, 1999). Thus, it is unlikely that dystrobrevin is involved in structural barrier functions. So far, no studies on the expression of other DAPs such as syntrophin or sarcoglycan in ocular tissues have been reported, ⁴⁴ but our recent studies suggest that sarcoglycans are expressed in the iris and ciliary body in a pattern similar to that of dystrobrevin (Ueda, unpublished data, 1999). Our previous study also suggested that dystrobrevin is associated with the blood–brain barrier. ³⁶ Taken together, these data suggest that the dystrophin complex including dystrobrevin plays a role in physiological barrier functions in the blood–ocular barrier. 

In conclusion, dystrobrevin is localized in photoreceptor axon terminals and blood–ocular barrier sites, and it may play an important role morphologically or physiologically in visual function. 

 Submitted for publication February 4, 2000; revised May 30 and June 23, 2000; accepted July 7, 2000.

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Figure 1.

 

Immunoblot analysis of rat cerebellar lysate (lane A) for positive control, retina (lane B), iris and ciliary body (lane C), cornea (lane D), and lens (lane E). Retinal and iris and ciliary body lanes show similar banding patterns consisting of approximately 87-, 80-, and 65-kDa bands. Corneal lane prominently shows an approximately 65-kDa protein, but the lens lane strongly presents doublet bands with an approximately 90-kDa and a faint approximately 65-kDa band. In addition, the iris-ciliary body and lens show approximately 30-kDa bands.

Figure 2.

 

Dystrobrevin localization in the rat retina. Dystrobrevin revealed by DAB deposits is recognized in the GCL, IPL, and OPL and around the vessel (A). More highly magnified confocal images show dystrobrevin localization in more detail (B, C, and D). Dystrobrevin is detected in the ILM, GCL, and perivascular regions (B). Patch-like labeling (approximately 1 μm in diameter) with dystrobrevin is found in the OPL (C), and a granular labeling pattern is seen in the (RPE; D). V, blood vessel. Bar, (A) 10 μm; (B, C, and D) 5 μm.

Figure 3.

 

Electron micrographs of the rat retina. Conventional electron micrographs show electron-dense regions under the cell membrane of rod spherules around and between bipolar cell processes (A). (A, B; ∗) Rod spherule protrusions into the interspace between bipolar cell processes. Dystrobrevin immunoreactive deposits (arrowheads) were found in the rod spherule along with bipolar cell processes, but not around horizontal cell processes (B). Cone pedicles consist of flat and invaginated synapses with electron-dense regions apposing bipolar processes (C). Dystrobrevin was detected in cone pedicles in the same manner as in rod spherules (D). Arrowheads: Dystrobrevin immunoreactive products; arrows: synaptic ribbons (A through D). Immunoelectron micrographs of perivascular regions demonstrate that dystrobrevin is detected in the cytoplasm of Müller cells (E, arrowheads) and around the tight junctions of endothelial cells (E, arrows in inset); (∗) basement membranes. B, bipolar cell processes; EC, endothelial cell; H, horizontal cell processes; L, lumen of blood vessel; Mü, Müller cell. Bar, 200 nm.

Figure 4.

 

Immunoblot and confocal images of cultured Müller cells at passage 2. An approximately 65-kDa band was strongly expressed, and an approximately 87-kDa band was faintly detected (A). Double labeling with propidium iodide (red) and dystrobrevin (B, green) or β-dystroglycan (C, green) showed that both seemed to locate in the cytoplasm of Müller cells (arrows). Probable neuronal cells (arrowheads) strongly expressed dystrobrevin (B). Immunocontrol sections show no green signal (D). Bar, 20μ m.

Figure 5.

 

Light micrographs of rat cornea (A, B), lens (C, D), iris (E, F), and ciliary body (G, H) labeled with β-dystroglycan (A, C, E, and G) and dystrobrevin (B, D, F, and H) and electron micrographs of iris (I) and ciliary body (J). Corneal endothelium and lens, iris, and ciliary body epithelia were labeled with both β-dystroglycan and dystrobrevin (arrows; A through H). Arrows: Immunoreactive deposits. Electron micrographs show that iris (I) and ciliary body (J) epithelium consist of two layers. (I, J; insets) Confocal images labeled with dystrobrevin. Dystrobrevin expression areas in the inner epithelium of iris or ciliary body (white arrowheads) seem to be consistent with their cytoplasm close to the basement membranes (black arrowheads). (I, J; insets, white arrows) Outer positive signals demonstrate dystrobrevin localization in the outer epithelium. (I, J; black arrows) Myoepithelium; (∗) outer basement membranes. N, nucleus; V, blood vessel. Bar: (A through D) 50 μm; (E through H, insets I, J) 20 μm; (I, J), 500 nm.

The authors thank Yoko Iizuka for her excellent technical assistance in culture experiments.