Immunocytochemical localization of a chondroitin sulfate proteoglycan in nervous tissue. II. Studies in developing brain.

Abstract

In contrast to the intracellular (cytoplasmic) localization of chondroitin sulfate proteoglycans in adult brain (Aquino, D. A., R. U. Margolis, and R. K. Margolis, 1984, J. Cell Biol. 99:940-952), immunoelectron microscopic studies in immature (7 d postnatal) rat cerebellum demonstrated almost exclusively extracellular staining in the granule cell and molecular layers. Staining was also extracellular and/or associated with plasma membranes in the region of the presumptive white matter. Axons, which are unmyelinated at this age, generally did not stain, although faint intracellular staining was present in some astrocytes. At 10 and 14 d postnatal there was a significant decrease in extracellular space and staining, and by 21 d distinct cytoplasmic staining of neurons and astrocytes appeared. This intracellular staining further increased by 33 d so as to closely resemble the pattern seen in adult brain. Analyses of the proteoglycans isolated from 7-d-old and adult brain demonstrated that they have essentially identical biochemical compositions, immunochemical reactivity, size, charge, and density. These findings indicate that the antibodies used in this study recognize the same macromolecule in both early postnatal and adult brain, and that the localization of this proteoglycan changes progressively from an extracellular to an intracellular location during brain development.In the preceding report (2) we have demonstrated by immunoelectron microscopy the intracellular (cytoplasmic) localization of a chondroitin sulfate proteoglycan in neurons and astrocytes of adult rat central nervous tissue. Its functional role in this location is still unknown, and although our immunocytochemical findings were not unexpected on the basis of previous biochemical studies of brain glycosaminoglycans, they are nevertheless quite different from the extracellular matrix or cell surface location of proteoglycans in other tissues (8,16,18,19).Since the possibility remained that during brain development this proteoglycan might be involved in extracellular processes associated with neural histogenesis and various types of cell-cell interactions, we also examined its localization in rat cerebellum during the period extending from 1 wk to l mo of postnatal age. These studies demonstrated that in contrast to the cytoplasmic localization observed in adult brain, the chondroitin sulfate proteoglycan is almost exclusively extraceUular in 7-d-old cerebellum, and progressively assumes its final intracellular location in neurons and astrocytes during the succeeding 3 wk. MATERIALS AND METHODSThe immunochemical and immunocytochcmical procedures employed were essentially as described in the preceding paper (2). However, for studies of immature brain (7-33 d postnatal), a two-step fixation procedure was used (3). After perfusion with 0.1 M sodium phosphate buffer (pH 7.4) containing 0.9% NaCl, rats were perfused with 4% freshly prepared formaldehyde and 0.1% glutaraldehyde in sodium phosphate buffer (pH 6.5), followed by the same aldehydes...

Brain-specific hyaluronate-binding protein: an immunohistological study with monoclonal antibodies of human and bovine central nervous system.

“…In fact, the topic is not even mentioned in two recent reviews ofthe subject (1,2). On the basis of immunohistological studies, several extracellular matrix proteins have been proposed as the substrate allowing cell migration and axonal growth in immature brain: fibronectin (3), laminin (4), and chondroitin sulfate proteoglycan (5). However, the findings have not been independently confirmed to date and negative results have been published concerning the presence of laminin and fibronectin in brain development (6,7).…”

Strongly Anionic Sites in Peripheral Axons of the Rat Sciatic Nerve: Light and Electron Microscopic Detection Using Cationic Colloidal Iron.

“…The presence of glycosaminoglycan on microtubules and associated filaments in axons has been demonstrated using lanthanum hydroxide (LANE and TREHERNE, 1970), ruthenium red (TANI and AMETANI, 1970) or alcian blue (HINKLEY, 1973). Moreover, AQUINO et al (1984) used an immunocytochemical method to suggest that proteoglycan exists in the microtubules and may have be involved in axonal transportation.…”

“…Reports have shown the presence of hyaluronic acid in the epineurium (MUKADAI et al, 1994), hyaluronic acid and chondroitin 4-6 sulfate proteoglycan in the endoneurium (TONA et al, 1993;MUKADAI et al, 1994;UZUKI and SAWAI,1994), heparan sulfate proteoglycan in the basement membrane of capillary vessels among nerve fibers (LAURIE et al, 1983), hyaluronic acid and chondroitin 4-6 sulfate proteoglycan (AQUINO et al, 1984;CAREY et al, 1986;TONA et al, 1993;MUKADAI et al, 1994) and heparan sulfate proteoglycan (HINKLEY, 1973;LAURIE et al, 1983;BANNERMAN et al, 1986;OOHIRA, 1989;TONA et al, 1993) in the basement membrane of Schwann cells. The present enzyme digestion study has comfirmed the presence of hyaluronic acid in the epineurium, heparan sulfate proteoglycan in the basement membrane of capillary vessels among nerve fibers, chondroitin sulfate proteoglycan in the endoneurium, and heparan sulfate proteoglycan and chondroitin sulfate proteoglycan in the besement membrane of Schwann cells.…”

“…Using an immunocytochemical method, AQUINO (1984) suggested that a significant portion of the chondroitin sulfate proteoglycan is present as a axoplasmic component of neurons. The staining pattern in axons supports the idea that the proteoglycans may be closely associated with microtubules, which are thought to play a role in axonal transport, mainly the fast component of axonal flow.…”

Proteoglycans in Perineuronal Nets.

“…Most of the perineuronal net-constituting molecules have also been reported to exist in the extracellular space between the neuronal cell surfaces and the glial processes [10,13]. However, some CSPGs have been reported to exist intracellularly in adult rat brains although they were not reported to form perineuronal nets [1,2,40]. In addition, hyaluronectin, a hyaluronic acid-binding protein, has been reported to exist in the cytoplasm and at the plasma membrane of the glial cell processes [11].…”