ARTICLE |
Correspondence to: Tatsuo Suganuma, Dept. of Anatomy, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki 889-1692, Japan.
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Summary |
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We investigated the localization of polysialic acid (PSA), neural cell adhesion molecule (NCAM), and vesicular acetylcholine transporter (VAChT) in adult rat retina by using immunofluorescence with a confocal laser scanning microscope. Western blot analysis showed a typical broad smear of PSA and isoforms of NCAM (120, 140, and 180 kD). PSA immunofluorescence revealed multistratification in the inner plexiform layer (IPL). Dual immunostaining for PSA and NCAM exhibited the selective co-expression of PSA and NCAM on Müller cells. Moreover, dual immunolabeling for PSA and VAChT completely separated the five strata in the IPL. Strata 1, 3, and 5 were immunoreactive for PSA and Strata 2 and 4 for VAChT. These results suggest the possibility that PSA molecules on Müller cells are spatially related to ON and OFF retinal channels in the IPL. (J Histochem Cytochem 47:919927, 1999)
Key Words: polysialic acid, neural cell adhesion molecule, vesicular acetylcholine, transporter, rat, retina, Müller cell, cholinergic neuron, confocal laser scanning, microscopy
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Introduction |
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Polysialic acid (PSA) is a linear homopolymer of -2,8-linked N-acetylneuraminic acid (
All vertebrate retinas consist of a neuronal network organized according to the same basic plan: three nuclear layers and two intercalated plexiform (synaptic) layers. The structural and neurochemical diversity at the level of the inner plexiform layer (IPL) is particularly perplexing and as yet not well understood.
Müller cells are radial glial cells in the retina, which extend from the external to the internal limiting membrane. They are characterized by extensive cytoplasmic expansions, which fill all intercellular spaces and envelop the cell bodies of the neurons. As the principal glial cells, they conserve the structural alignment of its neuronal elements (
In this study we have investigated the expression of PSA in the adult rat retina using a monoclonal antibody against PSA. Immunofluorescence for PSA confirms the distinctive expression of PSA on Müller cells and demonstrates the multistratification in the IPL. To identify the PSA-immunoreactive strata, we have employed the dual immunostaining technique with a confocal laser scanning microscope. Dual staining for PSA and NCAM shows selective co-localization of PSA and NCAM on Müller cells. Interestingly, the five strata comprising the IPL are very clearly discernible by dual immunostaining for PSA and VAChT.
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Materials and Methods |
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Antibodies
The mouse IgG monoclonal antibody (designated MAb 735) was a kind gift from Dr. D. BitterSuermann (Institute of Medical Microbiology, School of Medicine; Hannover, Germany) (-2,8-linked polysialic acid at least more than eight residues in length (
Immunohistochemistry for PSA
Male Wistar rats, 810 weeks old, were deeply anesthetized with diethylether and then perfused with 3% paraformaldehyde0.1% glutaraldehyde in PBS. The eyes were removed and further fixed by immersion in the above fixative at 4C overnight. After washing in PBS for 2 hr, the eyes were cryoprotected in 15% sucrose in PBS for 2 hr and 30% sucrose in PBS at 4C overnight. They were then embedded in OCT compound (Miles; Elkhart, IN) and then cut into 12-µm sections with a cryostat. After several rinses in PBS, the sections were incubated with 50 mM NH4Cl in PBS for 30 min to block free aldehyde groups. After washing in PBS, the sections were treated with 5% normal goat serum (NGS) and 1% bovine serum albumin (BSA) in PBS for 10 min and then incubated with MAb 735 (diluted 1:2000 with 1% BSA in PBS) at 4C overnight. After washing in PBS, the sections were incubated with fluorescein isothiocyanate (FITC) conjugated to horse anti-mouse IgG (Vector Laboratories; Burlingame, CA; diluted 1:200 with 1% BSA in PBS) for 2 hr. After washing in PBS, the sections were briefly rinsed with water and mounted in Gel/mount (Biomeda; Foster City, CA). Immunofluorescence labelings were examined with a confocal laser scanning microscope (TCS4D; Leica, Welzlar, Germany). As controls, the primary antibody was omitted from the procedure or replaced by normal mouse IgG.
For immunoelectron microscopy using the pre-embedding staining procedure, vibratome sections (90 µm thick) were incubated with 5% NGS and 1% BSA in PBS for 30 min to block nonspecific binding and were then incubated with MAb 735 (1:2000) for 2 days at 4C. After washing in PBS, the sections were incubated with anti-mouse IgGHRP (MBL, Nagoya, Japan; diluted 1:40) at 4C overnight. After washing in PBS, the sections were fixed with 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (CB, pH 7.4), containing 5% sucrose for 1 hr. After washing in 50 mM Tris-HCl buffer (TB, pH 7.4), the sections were incubated in 0.05% 3',3'-diaminobenzidine (DAB) in TB for 20 min, then in the complete DAB solution containing 0.005% H2O2 for 10 min. The sections were postfixed with 1% OsO4 in CB containing 1% potassium ferrocyanide for 1 hr, dehydrated with a graded series of ethanol, and then embedded in epoxy resin mixture. Ultrathin sections were contrasted with lead citrate for 20 sec and examined with a JEOL 1200 EX electron microscope (JEOL; Tokyo, Japan).
Enzyme Digestion with Neuraminidase
To confirm the specificity of MAb 735, the sections were treated with neuraminidase to remove the sialic acid residues. After briefly rinsing in PBS, the cryostat sections were incubated with 0.1 M acetate buffer, pH 5.3, for 5 min at room temperature (RT), and then were digested with highly purified neuraminidase from Arthrobacter ureafaciens (Nacalai Tesque, Kyoto, Japan; 0.5 U/ml in 0.1 M acetate buffer, pH 5.3) at 37C overnight. After washing in PBS, the sections were stained with MAb 735 as described above.
Western Blot Analysis
Immunoblotting was done essentially as described by
Dual Immunostaining for PSA and NCAM
The sections were incubated with MAb 735 (1:1000) mixed with antibody against NCAM (1:1000) at 4C overnight. After washing in PBS, the sections were incubated with a mixture of FITC-conjugated horse anti-mouse IgG (Vector; diluted 1:100 with 1% BSA in PBS) and Texas Red-conjugated goat anti-rabbit IgG (Southern Biotechnology Associates, Birmingham, AL; diluted 1:50 with 1% BSA in PBS) for 2 hr at RT. After washing in PBS, the sections were briefly rinsed with water and mounted in Gel/mount. Dual immunofluorescence labelings were simultaneously scanned by a confocal laser scanning microscope.
Dual Immunostaining for PSA and VAChT
The sections were preincubated with 5% normal horse serum in PBS and then incubated with the mixture of MAb 735 (1:1000) and antiserum to rat VAChT (1:1000) at 4C overnight. After washing in PBS, the sections were incubated with a mixture of FITC-conjugated horse anti-mouse IgG (Vector; diluted 1:100 with 1% BSA in PBS) and biotinylated horse anti-goat IgG (Vector; diluted 1:100 with 1% BSA in PBS) for 2 hr at RT. After washing in PBS, the sections were incubated with streptavidinTexas Red conjugate (Bethesda Research Laboratories, Gaithersburg, MD; diluted 1:200 with 1% BSA in PBS) for 30 min at RT. After washing in PBS, the sections were briefly rinsed with water and mounted in Gel/mount. Dual immunofluorescence labelings were simultaneously scanned by a confocal laser scanning microscope.
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Results |
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Light Microscopy for PSA Immunostaining
In the adult rat retina, immunoreactivity for MAb 735 was observed throughout the entire thickness of the neural retina, from the inner limiting membranes to the outer limiting membranes (Figure 1A and Figure 1B). In particular, dense labeling occurred in the outer plexiform layer (OPL) and the IPL. In the latter, MAb 735 clearly demonstrated multistratified structures (Figure 1B). The staining pattern was composed of the dense network of fine processes in the IPL and in the OPL, the decoration of the cell bodies in the INL and in the ONL, and the outer and inner limiting membranes, probably representing microvilli and end-feet of Müller cells, respectively. In addition to the neural retina, MAb 735 immunoreactivity was also detected in the choroid, particularly in the larger vessels and loose connective tissue (Figure 1A). Intense labeling was concentrated in the perivascular regions. It appears that MAb 735 labels the axons of the vasomotor nerves in the choroidal stroma. As controls, omission of MAb 735 or normal mouse IgG instead of the primary antibody revealed no immunofluorescence (not shown).
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Enzyme Digestion with Neuraminidase
To confirm the specificity of MAb 735, tissue sections were preincubated with neuraminidase. The extensive enzyme digestion completely abolished MAb 735 immunoreactivity (Figure 1C).
Western Blot Analysis
Western blots of the adult rat retina with MAb 735 revealed a broad smear from 150 to 260 kD (Figure 2, Lane 1). Such a typical broad smear and the molecular mass range corresponded to the profile of PSA reported previously. Immunoreactivity with anti-NCAM appeared as discrete bands at 120, 140, and 180 kD. (Figure 2, Lane 2).
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Immunoelectron Microscopy
At the external limiting membrane portion of the retina, immunoreactivity was found only on Müller cell processes and their microvilli, which projected into the space between photoreceptor cell processes (Figure 3A). At the internal limiting membrane portion, immunoreactivity was found along the cell membrane of Müller cell processes and their expanded end-feet (Figure 3B). No immunoreactivity was found in any other neuronal elements in the retina. On the basis of these findings, we confirmed that Müller cells exhibited PSA immunoreactivity in the adult rat retina. In the choroid, strong immunoreactivity was also observed on the surface of unmyelinated nerve fibers (not shown). As control, normal mouse IgG instead of MAb 735 did not reveal any specific DAB reaction products.
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Dual Immunostaining for PSA and NCAM
PSA immunoreactivity demonstrated the multistratified structures in the IPL (Figure 4A). In contrast, NCAM immunofluorescence showed diffuse labeling of the retina without stratification in the IPL (Figure 4B). To examine the co-localization of PSA and NCAM, we performed dual immunostaining for PSA and NCAM. Three yellow stratifications could be distinguished in the IPL (Figure 4C). These results indicated the selective expression of PSA in the adult rat retina, especially in the IPL.
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Dual Immunostaining for PSA and VAChT
Immunostaining for VAChT, which has been recently reported as a novel marker for cholinergic neurons, demonstrated two prominent strata corresponding to Strata 2 and 4 in the IPL (Figure 5A). To identify which strata were immunoreactive for PSA, we performed dual immunostaining for PSA and VAChT. Figure 5B clearly demonstrates that the immunoreactive strata for PSA are completely distinguished from those for VAChT. It indicates that the PSA-immunoreactive multistrata were Strata 1, 3, and 5.
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Discussion |
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We have demonstrated the expression of PSA in the adult rat retina and choroid by using MAb 735 immunostaining. The pattern of the immunostaining is quite compatible with that of the adult mouse retina as reported previously (
Western blots analysis of the adult rat retina exhibited three major isoforms of NCAM (120, 140, and 180 kD), which are in agreement with previous reports (
Dual immunostaining for PSA and NCAM revealed the selective co-localization of PSA and NCAM in the adult rat retina, especially in the IPL. Previously, it has been estimated that the degree of sialylation is less than 10% in the adult (
VAChT has been recently shown to be a novel and unique marker for cholinergic neurons in the central and peripheral nervous systems (
In conclusion, PSA molecules on Müller cells, which continue to be expressed in the adulthood, exhibit multistratification in the IPL. Dual immunostaining for PSA and VAChT clearly identify the five strata in the IPL. Strata 1, 3, and 5 are immunoreactive for PSA, and Strata 2 and 4 for VAChT. These results imply that Müller cell processes are spatially related to ON and OFF retinal channels in the IPL, and that PSA molecules on Müller cells and cholinergic dendritic development may play an important role in the establishment of retinal organization.
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Acknowledgments |
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Supported in part by a Grant-in-Aid for Scientific Research (C) (08670024 to TS) from the Ministry of Education, Science, Sports and Culture, Japan.
We thank Dr D. BitterSuermann for providing MAb 735, Dr Hiroaki Kataoka (Department of Pathology, Miyazaki Medical College) for excellent technical advice, Yoshiteru Goto (Division of Electron Microscopy, Central Research Laboratories, Miyazaki Medical College) and Eiko Matsuura (Department of Anatomy, Miyazaki Medical College) for expert assistance.
Received for publication September 4, 1998; accepted February 9, 1999.
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