We developed a method to extract differentially chondroitin sulfate proteoglycans (CSPGs) that are diffusely present in the central nervous system (CNS) matrix and CSPGs that are present in the condensed matrix of perineuronal nets (PNNs). Adult rat brain was sequentially extracted with Tris-buffered saline (TBS), TBS-containing detergent, 1 m NaCl, and 6 m urea. Extracting tissue sections with these buffers showed that the diffuse and membrane-bound CSPGs were extracted in the first three buffers, but PNN-associated CSPGs remained and were only removed by 6 m urea. Most of the CSPGs were extracted to some degree with all the buffers, with neurocan, brevican, aggrecan, and versican particularly associated with the stable urea-extractable PNNs. The CSPGs in stable complexes only extractable in urea buffer are found from postnatal day 7–14 coinciding with PNN formation. Disaccharide composition analysis indicated a different glycosaminoglycan (GAG) composition for PGs strongly associated with extracellular matrix (ECM). For CS/dermatan sulfate (DS)-GAG the content of nonsulfated, 6-O-sulfated, 2,6-O-disulfated, and 4,6-O-disulfated disaccharides were higher and for heparan sulfate (HS)-GAG, the content of 6-O-sulfated, 2-N-, 6-O-disulfated, 2-O-, 2-N-disulfated, and 2-O-, 2-N-, 6-O-trisulfated disaccharides were higher in urea extract compared with other buffer extracts. Digestions with chondroitinase ABC and hyaluronidase indicated that aggrecan, versican, neurocan, brevican, and phosphacan are retained in PNNs through binding to hyaluronan (HA). A comparison of the brain and spinal cord ECM with respect to CSPGs indicated that the PNNs in both parts of the CNS have the same composition. We developed a method to extract differentially chondroitin sulfate proteoglycans (CSPGs) that are diffusely present in the central nervous system (CNS) matrix and CSPGs that are present in the condensed matrix of perineuronal nets (PNNs). Adult rat brain was sequentially extracted with Tris-buffered saline (TBS), TBS-containing detergent, 1 m NaCl, and 6 m urea. Extracting tissue sections with these buffers showed that the diffuse and membrane-bound CSPGs were extracted in the first three buffers, but PNN-associated CSPGs remained and were only removed by 6 m urea. Most of the CSPGs were extracted to some degree with all the buffers, with neurocan, brevican, aggrecan, and versican particularly associated with the stable urea-extractable PNNs. The CSPGs in stable complexes only extractable in urea buffer are found from postnatal day 7–14 coinciding with PNN formation. Disaccharide composition analysis indicated a different glycosaminoglycan (GAG) composition for PGs strongly associated with extracellular matrix (ECM). For CS/dermatan sulfate (DS)-GAG the content of nonsulfated, 6-O-sulfated, 2,6-O-disulfated, and 4,6-O-disulfated disaccharides were higher and for heparan sulfate (HS)-GAG, the content of 6-O-sulfated, 2-N-, 6-O-disulfated, 2-O-, 2-N-disulfated, and 2-O-, 2-N-, 6-O-trisulfated disaccharides were higher in urea extract compared with other buffer extracts. Digestions with chondroitinase ABC and hyaluronidase indicated that aggrecan, versican, neurocan, brevican, and phosphacan are retained in PNNs through binding to hyaluronan (HA). A comparison of the brain and spinal cord ECM with respect to CSPGs indicated that the PNNs in both parts of the CNS have the same composition. In the adult brain, ECM 2The abbreviations used are: ECM, extracellular matrix; PG, proteoglycan; CS, chondroitin sulfate; HS, heparin sulfate; DS, dermatan sulfate; HA, hyaluronan; GAG, glycosaminoglycan; GlcUA, d-glucuronic acid; GalNAc, N-acetyl-d-galactosamine; TN-R, tenascin-R; CNS, central nervous system; PNN, perineuronal net; RPTPβ, receptor-type protein-tyrosine phosphatase β; PBS, phosphate-buffered saline; 2-AB, 2-aminobenzamide; WFA, Wisteria floribunda agglutinin; HABP, hyaluronan-binding protein; HPLC, high performance liquid chromatography; PFA, paraformaldehyde; NHS, normal horse serum; TRU, turbidityreducingunit; GPI, glycosylphosphatidylinositol; CSPG, chondroitin sulfate proteoglycans. 2The abbreviations used are: ECM, extracellular matrix; PG, proteoglycan; CS, chondroitin sulfate; HS, heparin sulfate; DS, dermatan sulfate; HA, hyaluronan; GAG, glycosaminoglycan; GlcUA, d-glucuronic acid; GalNAc, N-acetyl-d-galactosamine; TN-R, tenascin-R; CNS, central nervous system; PNN, perineuronal net; RPTPβ, receptor-type protein-tyrosine phosphatase β; PBS, phosphate-buffered saline; 2-AB, 2-aminobenzamide; WFA, Wisteria floribunda agglutinin; HABP, hyaluronan-binding protein; HPLC, high performance liquid chromatography; PFA, paraformaldehyde; NHS, normal horse serum; TRU, turbidityreducingunit; GPI, glycosylphosphatidylinositol; CSPG, chondroitin sulfate proteoglycans. is mainly present in the intercellular spaces between neurons and glial cells. Whereas most of this matrix is amorphous, there are specialized structures of dense organized matrix called PNNs around many neurons with holes at the sites of synaptic contacts (1Hockfield S. McKay R.D. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5758-5761Crossref PubMed Scopus (146) Google Scholar, 2Celio M.R. Blumcke I. Brain Res. Brain Res. Rev. 1994; 19: 128-145Crossref PubMed Scopus (336) Google Scholar, 3Celio M.R. Spreafico R. De Biasi S. Vitellaro-Zuccarello L. Trends Neurosci. 1998; 21: 510-515Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). PNNs are composed of CSPGs versican, brevican, neurocan, cat-301 antigen (aggrecan), phosphacan (DSD-1-PG), HA, tenascin-C, tenascin-R (TN-R), and link proteins (4Jaworski D.M. Kelly G.M. Hockfield S.J. Cell Biol. 1994; 125: 495-509Crossref PubMed Scopus (85) Google Scholar, 5Asher R.A. Scheibe R.J. Keiser H.D. Bignami A. Glia. 1995; 13: 294-308Crossref PubMed Scopus (92) Google Scholar, 6Yamaguchi Y. Cell Mol. 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The CSPGs in the brain can be grouped into secreted, membrane-bound, and GPI-anchored forms. Versican, aggrecan, neurocan, and brevican constitute a family of HA binding PGs called lecticans (17Ruoslahti E. Glycobiology. 1996; 6: 489-492Crossref PubMed Scopus (368) Google Scholar, 18Oohira A. Matsui F. Tokita Y. Yamauchi S. Aono S. Arch. Biochem. Biophys. 2000; 374: 24-34Crossref PubMed Scopus (151) Google Scholar, 19Rauch U. Cell Mol. Life Sci. 2004; 61: 2031-2045Crossref PubMed Scopus (166) Google Scholar). The lecticans share a common structure with an N-terminal hyaluronan binding domain, C-terminal lectin binding domain, and GAG-carrying middle portion of variable length (6Yamaguchi Y. Cell Mol. Life Sci. 2000; 57: 276-289Crossref PubMed Scopus (516) Google Scholar, 18Oohira A. Matsui F. Tokita Y. Yamauchi S. Aono S. Arch. Biochem. Biophys. 2000; 374: 24-34Crossref PubMed Scopus (151) Google Scholar, 20Bandtlow D.R. Zimmermann C.E. Physiol. Rev. 2000; 80: 1267-1290Crossref PubMed Scopus (538) Google Scholar). Except for the GPI-anchored splice variant of brevican, all lecticans are secreted. Phosphacan is a secreted CSPG, representing the entire extracellular domain of receptor-type protein-tyrosine phosphatase β (RPTPβ), which is a transmembrane PG in the brain (21Maurel P. Rauch U. Flad M. Margolis R.K. Margolis R.U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2512-2516Crossref PubMed Scopus (259) Google Scholar). NG2 and neuroglycan C are two other transmembrane CSPGs expressed in the brain (22Levine J.M. Nishiyama A. Perspect. Dev. Neurobiol. 1996; 3: 245-259PubMed Google Scholar, 23Watanabe E. Maeda N. Matsui F. Kushima Y. Noda M. Oohira A. J. Biol. Chem. 1995; 270: 26876-26882Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). HA and the extracellular matrix glycoprotein TN-R are two ligands of lecticans (24Bignami A. Asher R. Perides G. Brain Res. 1992; 579: 173-177Crossref PubMed Scopus (53) Google Scholar, 25Aspberg A. Miura R. Bourdoulous S. Shimonaka M. Heinegard D. Schachner M. Ruoslahti E. Yamaguchi Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10116-10121Crossref PubMed Scopus (240) Google Scholar). Much of the binding and biological activity of the CSPGs depends on the CS-GAGs, composed of repeating disaccharide units of d-glucuronic acid (GlcUA) and N-acetyl-d-galactosamine (GalNAc). In mammals, the CS disaccharides can be disulfated in the positions 2 and 4 of GlcUA and GalNAc, respectively (CS-D) and in positions 4 and 6 of GalNAc (CS-E) or monosulfated in positions 4 or 6 of GalNAc (CS-A or CS-C, respectively) or nonsulfated (26Sugahara K. Yamada S. Trends Glycosci. Glycotechnol. 2000; 12: 321-349Crossref Scopus (95) Google Scholar). The differential arrangement of these units results in the structural diversity of CS chains and defines the charge patterns that give the GAGs their binding properties (27Deepa S.S. Umehara Y. Higashiyama S. Itoh N. Sugahara K. J. Biol. Chem. 2002; 277: 43707-43716Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 28Maeda N. He J. Yajima Y. Mikami T. Sugahara K. Yabe T. J. Biol. Chem. 2003; 278: 35805-35811Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). CSPGs in the CNS can interact with various growth factors and cell adhesion molecules, playing a significant role in development (18Oohira A. Matsui F. Tokita Y. Yamauchi S. Aono S. Arch. Biochem. Biophys. 2000; 374: 24-34Crossref PubMed Scopus (151) Google Scholar, 20Bandtlow D.R. Zimmermann C.E. Physiol. Rev. 2000; 80: 1267-1290Crossref PubMed Scopus (538) Google Scholar). They mostly have an inhibitory effect toward neurite outgrowth and regeneration, either via their CS chains or core proteins (29Rhodes K.E. Fawcett J.W. J. Anat. 2004; 204: 33-48Crossref PubMed Scopus (249) Google Scholar). They are up-regulated after CNS injury (30Asher R.A. Morgenstern D.A. Fidler P.S. Adcock K.H. Oohira A. Braistead J.E. Levine J.M. Margolis R.U. Rogers J.H. Fawcett J.W. J. Neurosci. 2000; 20: 2427-2438Crossref PubMed Google Scholar, 31Asher R.A. Morgenstern D.A. Shearer M.C. Adcock K.H. Pesheva P. Fawcett J.W. J. Neurosci. 2002; 22: 2225-2236Crossref PubMed Google Scholar, 32Thon N. Haas C.A. Rauch U. Merten T. Fassler R. Frotscher M. Deller T. Eur. J. Neurosci. 2000; 12: 2547-2558Crossref PubMed Scopus (100) Google Scholar, 33Jones L.L. Yamaguchi Y. Stallcup W.B. Tuszynski M.H. J. Neurosci. 2002; 22: 2792-2803Crossref PubMed Google Scholar), and enzymatic removal of GAG chains from CSPGs with chondroitinase ABC improves axon regeneration and functional recovery (34Moon L.D. Asher R.A. Rhodes K.E. Fawcett J.W. Nat. Neurosci. 2001; 4: 465-466Crossref PubMed Scopus (497) Google Scholar, 35Bradbury E.J. Moon L.D. Popat R.J. King V.R. Bennett G.S. Patel P.N. Fawcett J.W. McMahon S.B. Nature. 2002; 416: 636-640Crossref PubMed Scopus (1901) Google Scholar). Degradation of CSPGs induces sprouting of Purkinje axons in the cerebellum (36Corvetti L. Rossi F. J. Neurosci. 2005; 25: 7150-7158Crossref PubMed Scopus (119) Google Scholar) and promotes retinal fiber sprouting after denervation of the superior colliculus in adult rats (37Tropea D. Caleo M. Maffei L. J. Neurosci. 2003; 23: 7034-7744Crossref PubMed Google Scholar). PNNs form late in development, surrounding particular classes of neurons. Their time of appearance corresponds with the termination of plasticity at the end of critical periods in many parts of the CNS, and their appearance in the visual cortex can be delayed by dark rearing, which prolongs plasticity. Chondroitinase digestion of CSPGs in the PNNs in the adult visual cortex reactivates plasticity after the critical period (11Pizzorusso T. Medini P. Berardi N. Chierzi S. Fawcett J.W. Maffei L. Science. 2002; 298: 1248-1251Crossref PubMed Scopus (1252) Google Scholar). This and other evidence has led to the hypothesis that PNNs are involved in the control of plasticity in the CNS. The identity of the CSPGs in PNNs that are responsible for the control of plasticity has not been determined, but investigations of animals lacking brevican and neurocan have revealed abnormalities in one form of plasticity, long term potentiation (38Brakebusch C. Seidenbecher C.I. Asztely F. Rauch U. Matthies H. Meyer H. Krug M. Bockers T.M. Zhou X. Kreutz M.R. Montag D. Gundelfinger E.D. Fassler R. Mol. Cell. Biol. 2002; 21: 7417-7427Crossref Scopus (198) Google Scholar). Because enhancement of plasticity promotes recovery after damage to the CNS, development of methods to control neural plasticity is a priority. We have therefore investigated the composition and properties of PNNs. We have developed a method to differentially extract the CSPGs that are diffusely present in CNS parenchyma and those that are present in the condensed matrix of PNNs. This enabled us to examine the composition of PNNs and changes in development; to analyze the GAG chain composition of the different extracts in the adult brain; to study how the various components are retained within the net structure and to compare the properties of the rather different PNNs from brain and spinal cord. Materials—Chondroitinase ABC from Proteus vulgaris (EC 4.2.2.4) and CS disaccharides were purchased from Seikagaku Corp., Tokyo, Japan. Pronase was obtained from Kaken Pharmaceutical Co., Tokyo, Japan. Hyaluronidase (EC 4.2.2.1) from Streptomyces hyalurolyticus was purchased from Calbiochem, La Jolla, CA. Protease-free preparation of chondroitinase ABC from P. vulgaris (EC 4.2.2.4) and biotinylated HABP were obtained from Seikagaku, Falmouth, MA. Hiprep 16/10 DEAE-FF column, Cy3 streptavidin, and ECL™ Western blotting detection reagents were obtained from Amersham Biosciences UK Ltd., Chalfont St.Giles, UK. Heparinase I (EC 4.2.2.7) and III (EC 4.2.2.8) from Flavobacterium heparinum were obtained from IBEX Pharmaceuticals Inc., Montreal, Canada. BCA protein assay kit was from Pierce. Peroxidase-conjugated anti-mouse IgG, biotinylated horse anti-mouse IgG and Vectastain ABC elite kit were from Vector, Peterborough, UK. Biotinylated Wisteria floribunda agglutinin (WFA) and bisbenzimide fluorescent dye (Hoechst 33342) were purchased from Sigma. DPX-mounting medium was purchased from Lamb, Eastbourne, UK. Sodium pentobarbitone was from Rhone Merieux, Harlow, UK. 2-Aminobenzamide (2AB) was from Nacalai Tesque, Kyoto, Japan and amine-bound silica PA-03 column was from YMC Co., Kyoto, Japan. Complete protease inhibitor mixture tablets and pepstatin were from Roche Applied Science, Mannheim, Germany. Extraction of PGs from Rat Brain and Spinal Cord—Brains or spinal cords from 3-month-old Sprague-Dawley rats (Charles River, Margate, UK) were homogenized with a tight-fitting Potter glass homogenizer using 5 ml/brain or 3 ml/spinal cord extraction buffer containing 50 mm TBS, pH 7.0, 2 mm EDTA, 10 mm N-ethylmaleimide, and 2 mm phenylmethylsulfonyl fluoride as protease inhibitors (buffer 1). The homogenate was centrifuged at 15,000 rpm at 4 °C for 30 min. The supernatant was collected, and the pellet was re-extracted with buffer 1 twice, and the supernatants from the first and second extractions were pooled together (extract 1), whereas the supernatant from third extraction was discarded. The pellet obtained after centrifugation was further extracted with buffer 2 (buffer 1 containing 0.5% Triton X-100) three times to obtain extract 2, followed by extraction with buffer 3 (buffer 2 containing 1 m NaCl) and buffer 4 (buffer 2 containing 6 m urea) to obtain extracts 3 and 4, respectively. Extracts 3 and 4 were dialyzed against phosphate-buffered saline (PBS), and the protein content in all the four extracts was quantified by BCA protein assay kit. To investigate the release of CSPGs from PNNs, adult rat brain was sequentially extracted with buffers 1, 2, and 3. The precipitate obtained after three washes in buffer was resuspended in the digestion buffers for chondroitinase ABC (0.1 m Tris-HCl, pH 8.0 containing 0.03 m sodium acetate) and hyaluronidase (20 mm sodium acetate, pH 6.0 containing 0.15 m NaCl), separately and treated with 0.1 international unit (IU) of protease-free preparation of chondroitinase ABC at 37 °C for 3 h or 20 turbidity reducing unit (TRU)/ml of hyaluronidase containing protease inhibitors and 2 μg/ml pepstatin at 37 °C for 3 h, separately. The released CSPGs were analyzed by Western blotting as described later. Four separate extractions and analyses were performed on brain tissue, two on spinal cord. Partial Purification of PGs and Analysis of CS/DS and HS-GAG Disaccharide Composition—Brains from two 3-month-old rats were sequentially extracted with buffers 1, 2, 3, and 4 and after dialyzing the extracts against 50 mm Tris, pH 7.5, 2 m urea, 0.2 m NaCl (wash buffer), the samples were separately applied to a Hiprep 16/10 DEAE-FF column (1.6 × 10 cm) pre-equilibrated with wash buffer. The unbound fraction was eluted with the wash buffer whereas the bound fraction was eluted with the same buffer containing 1 m NaCl. The eluted fractions were dialyzed against PBS, and the PGs were precipitated with 95% ethanol containing 1.3% potassium acetate for 16 h at 4 °C, and the precipitate was dried. Each buffer extract (2–3 mg as ethanol precipitate) was treated with Pronase at 60 °C for 20 h to degrade proteins. After incubation and subsequent treatment with 5% trichloroacetic acid, the GAG-containing fractions were recovered by ethanol precipitation. An aliquot of the resultant GAG preparation derived from each buffer extract was digested exhaustively with chondroitinase ABC or a mixture of heparinase I and III. The reaction mixtures were lyophilized and derivatized with a fluorophore, 2AB. After removal of excess 2AB reagent by repeated extraction with a water-chloroform 1:1 mixture (v/v), the water phase was dried. An aliquot of each 2AB-derivative was analyzed by anion exchange HPLC on an amine-bound silica column (39Kinoshita A. Sugahara K. Anal. Biochem. 1999; 269: 367-378Crossref PubMed Scopus (190) Google Scholar) to identify and quantify the resultant 2AB-labeled unsaturated CS or HS disaccharides. Western Blotting—The extracted proteoglycan fractions corresponding to 200 μg of protein was precipitated with 95% ethanol containing 1.3% potassium acetate for 1 h in ice, and the precipitate was recovered by centrifugation at 15,000 rpm for 10 min and digested with 10 international milliunits of a protease-free preparation of chondroitinase ABC in 0.1 m Tris-HCl, pH 8.0 containing 0.03 m sodium acetate for 3 h at 37 °C. The digest containing the core proteins were subjected to SDS-PAGE in a 5% gel under reducing conditions (except for versican, for which non-reducing condition was applied). The separated proteins were electrotransferred onto a nitrocellulose membrane for 16 h at a constant current of 175 mA at 4 °C. All the following steps were performed at room temperature. After washing with TBS-T (TBS containing 0.05% Tween-20), the membrane was incubated with anti-CSPG antibodies in TBS-T for 1 h. The antibodies used are listed in Table 1. After washing with TBS-T for 5 min five times, the blot membrane was treated with a 1:10,000 dilution of peroxidase-conjugated anti-mouse IgG for 1 h. The blots were developed using chemiluminescent substrate.TABLE 1Antibodies used for Western blotting and immunohistochemistryAntibodyAntigenHost speciesSourceaDSHB-Developmental Studies Hybridoma Bank (University of Iowa); Chemicon (Temecula, CA); BD Biosciences (Oxford, UK).Working dilutionWBbWB, Western blotting.IHCcIHC, immunohistochemistry.1F6NeurocanMouseDSHB1:1001:5Neurocan-N3F8PhosphacanMouseDSHB1:50—RPTPβ2B49PhosphacanMouseDSHB—1:2RPTPβCat-301AggrecanMouseChemicon1:10001:50012C5VersicanMouseDr. R. A. Asher (31Asher R.A. Morgenstern D.A. Shearer M.C. Adcock K.H. Pesheva P. Fawcett J.W. J. Neurosci. 2002; 22: 2225-2236Crossref PubMed Google Scholar)1:201:2Anti-BrevicanBrevicanMouseBD-Biosciences1:1000—D31.10NG2MouseDr. J. Levine (85Stallcup W.B. Beasley L. Levine J. Cold Spring Harb. Symp. Quant. Biol. 1983; 48: 761-774Crossref PubMed Google Scholar)1:20—Anti-Tenascin-RTenascin-RMouseDr. P. Pesheva (40Probstmeier R. Stichel C.C. Muller H.W. Asou H. Pesheva P. J. Neurosci. Res. 2000; 60: 21-36Crossref PubMed Scopus (31) Google Scholar)—1:500a DSHB-Developmental Studies Hybridoma Bank (University of Iowa); Chemicon (Temecula, CA); BD Biosciences (Oxford, UK).b WB, Western blotting.c IHC, immunohistochemistry. Open table in a new tab Immunohistochemistry—Postnatal (P) 7, 14, 21, and adult female Sprague-Dawley rats, three at each time point, were terminally anesthetized with an intraperiteonal overdose of sodium pentobarbitone and perfused through the heart with 200 ml of PBS prewash (pH 7.4) followed by 200 ml of 4% paraformaldehyde (PFA). The brains were post-fixed overnight at 4 °C, transferred to 30% sucrose and then sectioned into 40-μm sagittal sections. Sections were quenched (10% methanol and 3% H2O2) for 5 min and blocked in 3% normal horse serum (NHS) in PBS with 0.02% Triton X-100 (TXPBS) for 1 h at room temperature. Sections were incubated overnight in anti-neurocan, anti-phosphacan, or anti-versican antibodies (see Table 1 for details) with 1% NHS in TXPBS at 4 °C, and then in biotinylated horse anti-mouse IgG (1:200) for 1 h at room temperature. Then they were incubated in ABC solution (Vectastain ABC elite kit) for 1 h at room temperature, and the staining was revealed using diaminobenzamide as a chromogen. For immunofluorescence on fresh frozen sections of adult cerebellum, adult rats were sacrificed by decapitation, and the cerebellum was immediately frozen on dry ice. Sagittal sections (16-μm thick) were cut on a cryostat, collected on SuperFrost® Plus slides, and air-dried at room temperature for 20–30 min. The slides were sequentially washed with buffers 1, 2, 3, and 4, each wash lasting 1 h, and then were fixed in 4% PFA for 10 min. WFA and HABP histochemistry were performed by incubating sections in biotinylated WFA (20 μg/ml) or biotinylated HABP (10 μg/ml) in TXPBS overnight, then in Cy3 streptavidin (1:500) for 1 h at room temperature. For CSPG immunostaining, sections were incubated overnight in primary antibodies (see Table 1) with 1.5% NHS in TXPBS at 4 °C, then in biotinylated horse anti-mouse IgG (1:200) in TXPBS containing 1.5% NHS for 1 h at room temperature and then in a solution made of Cy3 streptavidin and 10 μg/ml bisbenzimide fluorescent dye for 1 h at room temperature. In control experiments primary antibodies were omitted, yielding unstained sections. The slides were examined on a Leitz DMRD microscope. Digital images were produced using a Lucia imaging program (Nikon, Kingston upon Thames, UK) with a Nikon DXM 1200 digital camera. The images were imported into Adobe Photoshop (Adobe Systems Inc., San Jose, CA) in which size, contrast, and brightness were adjusted when necessary. For enzymatic digestions sagittal sections of adult rat cerebellum (16-μm thickness) was equilibrated either with 0.1 m Tris, pH 8.0 containing 0.03 m sodium acetate for chondroitinase or 20 mm sodium acetate, pH 6.0 containing 0.15 m NaCl for hyaluronidase digestions. A protease-free preparation of chondroitinase ABC (0.1 international units/ml) and hyaluronidase (10 TRU/ml) containing protease inhibitors and 2 μg/ml pepstatin were added to the slides separately and incubated at 37 °C for 60 min and 37 °C for 120 min, respectively. After digestion, the slides were washed with PBS and fixed in 4% PFA in PBS and the PNN components were visualized by immunofluorescence as described earlier. To extract and analyze CSPGs loosely associated in the ECM, those associated with membranes and those bound in stable ternary complexes in PNNs, adult rat brain was sequentially extracted with four different buffers as described under “Experimental Procedures.” The CSPGs were then characterized in each fraction. Concurrently we extracted CSPGs from tissue sections using the same sequential buffers so that we could visualize directly the locations of the CSPGs extracted by each step. In the present study we use the term PNN based on the evidence from immunohistochemical staining. However there is also condensed matrix containing CSPGs around the nodes of Ranvier in a perineuronal net-like structure (7Oohashi T. Hirakawa S. Bekku Y. Rauch U. Zimmermann D.R. Su W.D. Ohtsuka A. Murakami T. Ninomiya Y. Mol. Cell. Neurosci. 2002; 19: 43-57Crossref PubMed Scopus (108) Google Scholar). The components of these structures are probably extracted in the same conditions as PNNs. Types and Location of CSPGs Extracted by the Sequential Buffers—Extraction of adult rat brain with buffer 1, which is normal saline, released most of the neurocan, brevican, phosphacan, and part of the aggrecan, but only a small proportion of versican V2 and NG2 (Fig. 1, lane 1). The monoclonal antibody 1F6 detects two forms of neurocan: the full-length neurocan (240-kDa core protein) and the proteolytically cleaved N-terminal neurocan, neurocan-N (130-kDa core protein), and both these forms were present in the saline extract. All the full-length neurocan was extracted in saline, because none appeared in the other buffers, but the proteolytically cleaved neurocan-N was bound to other structures, and appeared in the later buffers. Phosphacan was detected as a 400-kDa core protein, and there was a single isoform of brevican corresponding to 145-kDa core protein. Two strong bands were detected for the soluble form of aggrecan (>500-kDa core protein) together with three or more faintly stained bands (between 450 and 250 kDa). Among the three spliced variants of versican, only versican V2 (400 kDa protein) was detected in the soluble extract. For NG2, a 290-kDa core protein was detected. To monitor anatomically the location of the matrix molecules extracted by saline we performed immunohistochemistry on the deep cerebellar nuclei, the PNNs of which have been investigated in detail (9Carulli D. Rhodes K. E. Brown D.J. Bonnert T. P. Pollack S.J. Oliver K. Strata P. Fawcett J.W. J. Comp. Neurol. 2006; 494: 559-577Crossref PubMed Scopus (243) Google Scholar). The sections were fixed after buffer extraction to prevent further removal of matrix during staining. Before extraction there was staining for neurocan, aggrecan, versican, and phosphacan in the PNNs and also diffusely in the cerebellar ECM (Fig. 2, A–D). Brevican was not studied since the anti-brevican antibody gave high background on fresh frozen sections. Washing with normal saline resulted in a decrease in neurocan, aggrecan, and phosphacan staining in the diffuse ECM, whereas the staining in PNNs remained unchanged. WFA, which is a marker of PNNs did not show any decrease in the ECM staining after saline wash (Fig. 2E). The staining intensity of HABP and TN-R (Fig. 2, F and G) in the general ECM was decreased by saline wash, whereas their staining in PNNs remained unaltered. Extraction with buffer 2 (detergent buffer), released neurocan-N, phosphacan/RPTPβ, brevican, and aggrecan in smaller amounts than was released by saline buffer (Fig. 1, lane 2). Large amounts of the membrane-attached CSPG NG2 (300-kDa core protein) was released along with a small proportion of 260-kDa core protein. Versican V2 was released in very small amounts by detergent buffer. The results from immunostaining of cerebellar sections indicated that the PNN staining for neurocan, aggrecan, versican, HABP, WFA, and TN-R (Fig. 2, A–C, E, F, and G), remained unchanged whereas phosphacan staining (Fig. 2D) was completely removed by detergent wash. Extraction with buffer 3 (saline-containing detergent and 1 m NaCl), released small amounts of versican and aggrecan as shown in Fig. 1 (lane 3). Large amounts of neurocan comparable to detergent wash were released by high salt buffer, but barely detectable amounts of NG2, phosphacan, and brevican were released. Immunohistochemical staining of sections showed that the ECM staining for neurocan, aggrecan, versican, and HABP was further reduced (Fig. 2, A–C and F), but their staining in PNNs remained more or less the same. PNN staining for TN-R was slightly decreased by
