ARTICLE |
Correspondence to: Tamao Endo, Dept. of Glycobiology, Tokyo Metropolitan Inst. of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan. E-mail: endo@tmig.or.jp
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Summary |
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The histochemical distribution of sialoglycoconjugates in the CA1 region in the hippocampus formation of 9-week-old rats and 30-month-old rats was examined using electron microscopy in combination with two lectins, Maackia amurensis lectin, specific for Sia2-3Gal, and Sambucus sieboldiana agglutinin, specific for Sia
2-6Gal. Each lectin stained the plasma membranes of pyramidal cells, indicating that the Sia
2-3Gal and Sia
2-6Gal groups were expressed on their plasma membranes. These lectins also bound to synapses in the stratum lacunosum moleculare. The staining intensity of the lectins in the synapses in these layers was downregulated in the 30-month-old rats. These results indicated that both the Sia
2-3Gal and Sia
2-6Gal groups are expressed on these synapses and that the expression of these sialyl linkages decreases in the aged brain.
(J Histochem Cytochem 49:13111319, 2001)
Key Words: sialoglycoconjugate, hippocampus, aging, lectin, neuron, synapse
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Introduction |
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GLYCOSYLATION is one of the common post-translational modifications of a protein. Interest in glycosylation has been growing rapidly in recent years due to the growing recognition of its importance in cellcell and cellextracellular matrix interactions, as well as in intracellular events (23,
26, and
28 ketosidic linkages. These sialic acid moieties function as receptors and cell-adhesion molecules in a linkage-specific manner (
28-linked sialic acid. PSA is specifically found on neural cell adhesion molecules (NCAMs) and is believed to modulate the adhesive properties of NCAM and to regulate neurite outgrowth and neuronal cell migration (
28 sialyl linkage, the location and function of the
23 and
26 sialyl linkages in the nervous system remain unclear.
Although many studies have demonstrated the importance of structural change of glycans during development, very little information is available on changes in glycans during aging. Because the biosynthesis of glycans is not controlled by the interaction of a template and depends on the concerted action of glycosyltransferases, the structures of glycans are much more variable than those of proteins and nucleic acids. Therefore, the structures of glycans can be easily altered by physiological conditions of the cells. Accordingly, age-related alterations of the glycans are relevant to the understanding of physiological changes found in aged individuals. It is important to determine the molecular events that occur in glycoconjugates during aging. As a step in this direction, we have recently found differences in glycoproteins between young adult and older rat brains by a combination of SDS-PAGE and lectin blotting analyses (23 and
26 sialoglycoproteins were downregulated in several regions of the aged rat hippocampus (
23 and
26 sialoglycoproteins occurs in the aged rat hippocampus. The experiments were based on a histochemical analysis of MAL and SSA using electron microscopy.
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Materials and Methods |
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Animals
Five each of 9-week-old and 30-month-old Fischer female rats were used in this study. All experimental procedures using laboratory animals were approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute of Gerontology. Because the same results were found in all five rats in each group, only the data obtained for one sample in each group is described in detail here.
Lectin Staining for Light Microscopy
Under pentobarbital anesthesia, the brains were dissected out and immediately frozen in an OCT compound (Sakura; Tokyo, Japan) with dry ice. Rat brain coronal sections, perpendicular to the septotemporal axis at the approximate mid-point, were cut into 6-µm-thick sections on a cryostat and mounted on gelatin-coated slides. The sections were fixed by incubation in 4% formaldehyde in 10 mM Tris-HCl (pH 7.4), 0.14 M NaCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM MnCl2 (TBS) for 10 min. They were then immersed in 0.3% H2O2 in methanol for 30 min at room temperature (RT). Then the sections were washed three times for 10 min at RT with TBS and incubated with the biotinylated lectins (SSA 33.3 µg/ml; MAL 13.3 µg/ml) (Seikagaku; Tokyo, Japan) diluted in 3% bovine serum albumin (BSA)TBS for 1 hr at RT. The sections were washed again with TBS three times for 10 min and incubated for 1 hr at RT with horseradish peroxidase (HRP)-conjugated streptavidin (20 µg/ml) (DAKO; Carpinteria, CA). After further washes three times with TBS for 10 min, the sections were incubated with 3,3'-diaminobenzidine solution (0.5 mg/ml in TBS) containing 0.3% H2O2 for 10 min at RT and then washed with distilled water.
Lectin Staining for Electron Microscopy
Under pentobarbital anesthesia, the rats were perfused transcardially with 10 mM PBS, pH 7.4, and then with 4% formaldehydePBS at RT 20 min. The brains were immediately dissected out and placed in 4% formaldehydePBS at 4C for 2 hr. Lectin staining for electron microscopy was carried out according to
Morphological Study with Electron Microscopy
After perfused fixation with 4% formaldehydePBS, the brains were dissected out and fixed in 2.5% glutaraldehydePBS for 1 hr at 4C. They were postfixed in 1% OsO40.1 M phosphate buffer for 1 hr at 4C and dehydrated through a series of graded ethanol. After passage through propylene oxide, the specimens were embedded in Epon 812. Ultrathin sections were cut and stained with uranyl acetate and lead citrate and then examined with a transmission electron microscope (JEM-1010).
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Results |
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Alteration of Granule Cells in Dentate Gyrus in the Aged Brain
Two populations of granule cells were observed in the granule cell layer of the hippocampal formation of the 9-week-old rats (Fig 1A). Cells with high electron density were observed in the innermost portion of the granule cell layer (Fig 1A, indicated with H) and cells with low electron density were in the remaining portion (Fig 1A, indicated with L). On the other hand, the cells with low electron density were mainly observed in the granule cell layer of the 30-month-old rats (Fig 1B, indicated with L), suggesting that the cells with high electron density almost disappeared during aging. It is interesting that the cells with high electron density were observed not only in the granule cell layer but also in the stratum pyramidale of the CA1 region of the 9-week-old rats, and the numbers of these cells were downregulated in the 30-month-old rats (data not shown). The same results were found in all five rats in each group, indicating that the difference was not due to differences among individuals.
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Lectin Histochemistry
Cell Bodies of the Neurons in the Pyramidal Cell Layer and Their Dendrites in the Stratum Radiatum.
Because a histochemical study of the CA1 region at the light microscopic level revealed reactivity to the stratum radiatum (sr in Fig 2F2I) and low reactivity to the pyramidal cells (sp in Fig 2B2E), we analyzed both areas in detail by electron microscopy. The plasma membranes of neuronal cell bodies in the stratum pyramidale from 9-week-old (Fig 3A) and 30-month-old rats (Fig 3B) were stained by MAL (indicated by arrowheads). The stratum radiatum is next to the stratum pyramidale, and many dendrites that originated from the pyramidal cells in the stratum pyramidale were observed there. The plasma membranes of the dendrites in stratum radiatum were also reactive with MAL in the 9-week- and 30-month-old rats (arrowheads in Fig 4A and Fig 4B, respectively). Although all plasma membranes of cell bodies and dendrites were stained by MAL, no difference in staining between the 9-week-old and 30-month-old rats was observed. On the other hand, SSA showed similar reactivities to these plasma membranes (Fig 3C, Fig 3D, Fig 4C, and Fig 4D). The lectin binding was inhibited by the addition of a hapten sugar, 0.2 M N-acetylneuraminic acid. Nonspecific binding of HRPstreptavidin conjugates and endogenous peroxidase activity were scarcely detected (data not shown). These results indicated that the Sia23Gal and Sia
26Gal groups were expressed on the plasma membranes of the neuronal cell bodies and dendrites of pyramidal cells and suggested that the expression of these sialyl linkages did not change significantly during aging.
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It should be noted that conspicuous lipofuscin accumulation was observed only in the cytoplasm of the 30-month-old rats (arrows in Fig 3B). Lipofuscin is known to accumulate in the cytoplasm of aged cells only (
Synapses in the Stratum Lacunosum Moleculare of the CA1 Region.
The stratum lacunosum moleculare of the CA1 region is composed of stratum lacunosum and stratum moleculare, and is known to contain many synapses (
At first, the presynaptic membranes could be easily identified because of the presence of synaptic vesicles (arrowheads in Fig 5, Fig 6, and Fig 7). The reactivity of MAL was observed at the presynaptic membranes (arrowheads in Fig 5A and Fig 6A) and postsynaptic membranes (arrows) in the stratum lacunosum moleculare of the 9-week-old rats. On the other hand, when the synaptic membranes of the 30-month-old rats were stained by MAL, its reactivity was less intense than that in the synaptic membranes of the 9-week-old rats (Fig 5B and Fig 6B, indicated with arrowheads for the presynaptic membranes and arrows for the postsynaptic membranes). It is noteworthy that, in the 30-month-old rat samples, they showed similar intensity when they were stained with or without MAL (Fig 7). These results indicated that the reactivity of MAL decreased in the aged synapses. SSA also showed the same reactivity with these pre- and postsynaptic membranes (Fig 5C, Fig 5D, Fig 6C, and Fig 6D, indicated with arrowheads for the presynaptic membranes and arrows for the postsynaptic membranes). Binding of these lectins was also inhibited by the addition of 0.2 M N-acetylneuraminic acid (data not shown). On the basis of these results, we conclude that, in these regions, the Sia23Gal and Sia
26Gal groups were expressed on the pre- and postsynaptic membranes and that aging affected their expression.
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Discussion |
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Alteration of Granule Cells in the Dentate Gyrus During Aging
In this study we observed two kinds of cells (low and high electron density) in the granule cell layer of the 9-week-old rats, while in the 30-month-old rats we observed mainly low electron density cells (Fig 1). These results suggest that the high electron density cells almost disappeared during aging. These high electron density cells were localized at the innermost portion in the granule cell layer. The granule cells in the innermost region are known to be newly generated (
Lectin Histochemistry
The stratum lacunosum moleculare of the CA1 region is known to contain many synapses (23 and Sia
26 groups at synapses in the stratum lacunosum moleculare, and suggested that the expression of these sialyl groups decreased during aging. On the other hand, the expression of these sialyl groups did not change on the plasma membranes of pyramidal cells (Fig 3 and Fig 4). Therefore, the decrease in expression of sialyl linkages may be specific for synaptic membranes. The decrease of sialyl groups in the synapses in the aged brain could be due to a depression of the activity of sialyltransferases, such as
23 sialyltransferases which are involved in the formation of the Sia
23 group and/or in
26 sialyltransferases which are involved in the formation of the Sia
26 group (
In the cerebral cortex, the amounts of gangliosides (sialic acid-containing glycolipids) in the synaptosomes decrease during aging (23 and 26Gal groups in the synapses may be regulated in a complex manner, although the details remain unclear.
It was reported that cell adhesion molecules are crucially involved in the assembly and restructuring of synapses during development and that they are involved in synaptic plasticity (
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Acknowledgments |
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Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology and by the Mizutani Foundation.
We thank Mr M. Fukuda and Ms T. Shibata (Laboratory for Electron Microscopy and Department of Anatomy, Kyorin University School of Medicine) for technical assistance.
Received for publication December 5, 2000; accepted May 2, 2001.
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