5'-nucleotidase localization in the brains and spinal cords of adult normal and dysmyelinating mutant (shiverer) mice

Immunocytochemical staining with the antibody against mouse liver 5'-nucleotidase revealed 5'-nucleotidase antigenicity in myelinated fibers in the brains and in myelinated fibers and some interfascicular oligodendroglia in the spinal cords of normal adult mice. Although the 5'-nucleotidase specific activity in adult shiverer mouse CNS tissue homogenates had been shown to be normal, immunocytochemical staining with anti-mouse-5'-nucleotidase could be demonstrated in CNS tissue sections from only 2 out of 10 of the mutant animals. In tissue from these animals the staining, which was relatively faint, was localized specifically to cell-bodies, usually arranged in rows, and to material oriented parallel to nerve fibers. This pattern of immunostaining with anti-5'-nucleotidase resembled the immunostaining with anti-carbonic anhydrase but not with anti-glial-fibrillary-acidic-protein. This suggested that the rows of cells were oligodendrocytes, not astrocytes, and that the material parallel to nerve fibers might consist of oligodendrocyte processes wrapped loosely around axons. The antibody against rat 5'-nucleotidase, as distinguished from mouse, immunostained only the blood vessels in the shiverer mouse CNS, a finding similar to a previous observation in the normal mouse CNS. From these findings it was inferred that the primary loci of 5'-nucleotidase in the shiverer mouse CNS were interfascicular oligodendrocytes, their processes, and blood vessels, and in the normal mouse CNS, the myelin in some tracts, the blood vessels, and some interfascicular oligodendrocytes.     

Immunocytochemical localization of 5'-nucleotidase in oligodendroglia and myelinated fibers in the central nervous system of adult and young rats

Rat central nervous system (CNS) tissue sections were immunostained by the peroxidase-anti-peroxidase (PAP) method using a rabbit serum directed against rat liver 5'-nucleotidase. In paraffin sections from the brains of 60-day-old rats 5'-nucleotidase immunoreactivity occurred in the same white-matter regions as myelin-basic protein immunoreactivity and histological staining of myelin. The immunostaining of cerebral white matter for 5'-nucleotidase was more intense and wide-spread at the age of 120 days than at 60 days, and the choroid plexus and blood vessels were stained consistently. In the paraffin sections from the brains of younger (20-day-old) rats the staining of 5'-nucleotidase in the white matter was faint and patchy. In paraffin sections from spinal cord, 5'-nucleotidase immunoreactivity was observed throughout the lateral white-matter columns and, frequently, in the cell bodies of interfascicular oligodendroglia. Interfascicular oligodendroglia also showed 5'-nucleotidase immunoreactivity in vibratome sections from the CNS tissue of young and adult rats. The findings were consistent with histochemical and biochemical evidence for 5'-nucleotidase in rat brain myelin and oligodendroglia, with substantial increases in activity in the myelin as rats develop from the ages of 20 to 120 days. 5'-Nucleotidase immunoreactivity was not observed in any astrocytes or in oligodendrocytes in the gray matter; however, the enzyme may occur in those glial cells at levels lower than were detectable using the present method.     

Identification of antibodies in anti-CNS and anti-PNS myelin sera by immunoblot, characterization by immunohistochemistry, and their effect in tissue culture

Immunoblot analysis of antiserum to rat central nervous system (CNS) myelin revealed antibodies to myelin basic protein (MBP), proteolipid protein (PLP), and numerous high molecular weight proteins. In addition, anti-CNS myelin serum exclusively immunostained 4 basic proteins of rat peripheral nervous system (PNS) myelin. Similarly, anti-PNS myelin sera immunostained many high molecular weight proteins in both CNS and PNS myelin in addition to P0 and 4 basic proteins. Purified MBP and PLP were immunostained by anti-CNS myelin sera and MBP and P0 by anti-PNS myelin sera, indicating that antigenic sites are preserved during protein purification. Immunohistochemical localization with antisera was confined to the myelin sheath except that antisera to CNS myelin also stained oligodendrocytes during the active period of myelination. While anti-CNS myelin sera specifically demyelinated centrally myelinated fibers in culture, none of the anti-PNS myelin sera used here demyelinated organotypic spinal cord-dorsal root ganglion cultures.     

Characterization of antibodies against major fish CNS myelin proteins: immunoblot analysis and immunohistochemical localization of 36K and IP2 proteins in trout nerve tissue

Antisera against the trout CNS myelin proteins 36K and IP2 were prepared in rabbits and characterized by immunoblot analysis and immunohistochemistry. The anti-36K antiserum exclusively stained its corresponding antigen from trout CNS myelin but failed to recognize any myelin polypeptide from either trout PNS or mammalian CNS and PNS. Antibodies against the IP2 glycoprotein specifically cross-reacted with related intermediate proteins (IP) of both CNS and PNS myelin from trout but only faintly labeled the PO protein of mouse peripheral nerve. Immunohistochemical localization of both antigens in the CNS of young trout was confined to the myelin sheath, except that anti-36K antiserum also stained oligodendrocytes. Nodes of Ranvier, neuronal cell bodies, and dendrites, as well as other glial elements, were negative. Specificity of the immunofluorescent reaction was established by crossed immunoadsorption experiments. Whereas on adjacent sections through trout brain both antigens exhibited a nearly identical distribution pattern, immunostaining in peripheral nerves was seen only with anti-IP2 antibodies.     

Rat brain 5'-nucleotidase: developmental changes in myelin and activities in subcellular fractions and myelin subfractions

The activities of 5'-nucleotidase, measured in brain homogenates and myelin isolated from rats at 21, 60 and greater than 90 days of age, were compared to values for two other myelin-associated enzymes, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and carbonic anhydrase. Whereas the activities of all 3 enzymes were higher in brain homogenates from 60-day-old rats than in those from 21-day-old rats, only 5'-nucleotidase increased significantly in specific activity in both homogenates and myelin after the age of 60 days. The ratios of 5'-nucleotidase to the myelin basic and proteolipid proteins in subcellular fractions from adult rat brain suggested that the microsomal fraction was the only fraction containing 5'-nucleotidase levels not attributable to contamination by myelin membranes. Like carbonic anhydrase, 5'-nucleotidase had a greater distribution than CNP into microsomes of adult rats. When purified myelin was fractionated on a density gradient, the specific activity of 5'-nucleotidase was highest in the heaviest subfraction, with recovery of significant activity occurring, however, in all 3 subfractions. In rats over 60 days of age the recovery of 5'-nucleotidase in myelin was almost as high as that of the relatively myelin-specific enzyme CNP, suggesting that myelin may be the predominant, although not exclusive locus of 5'-nucleotidase in the adult rat brain.     

Myelination by transplanted human and mouse central nervous system tissue after long-term cryopreservation

Human and mouse oligodendrocytes were transplanted, after a long period of cryostorage, into newborn mouse brain. Tissue fragments were obtained from brain and spinal cord of 10-week-old human fetuses and from the periventricular zone of embryonic and newborn mouse brains. Samples were stored at –180°C for periods of 3 days to over 5 years. Frozen or fresh fragments were transplanted into the brains of newborn shiverer mutant mice, which are deficient in myelin basic protein (MBP). Normal myelin, produced by grafted oligodendrocytes, was detected by immunohistochemistry with an anti-MBP antiserum. The best results were obtained with isospecific grafts. The timing of myelin appearance did not depend significantly on the species or age of the donor. Myelination obtained with mouse grafts was more profuse when the donor was younger (embryonic versus newborn). Cryopreservation over 5 years did not impede the graft's ability to produce myelin and can be considered for long-term storage of oligodendrocytes in view of cell therapy. Received: 17 February 1995 / Revised: 26 July 1995 / Accepted: 2 August 1995     

A new oligodendrocyte specific plasma membrane surface protein identified by a monoclonal antibody produced in vitro

This paper describes a novel monoclonal antibody (C1G5F2) derived from mice splenocytes immunized in vitro with a wheat germ agglutinin glycoprotein fraction isolated from bovine central nervous system (CNS) myelin. Immunohistochemical reactions with C1G5F2 were investigated on rat brain sections during the active period of myelination. From day 10 to 13 postnatally, no stained structures were observed throughout the whole brain. The first immunolabeled myelin fibers were detected within the pons at day 14, and the white matter areas in the cerebrum started to be stained some days later. White matter areas of the cerebellum were clearly immunopositive after the third week. There was a strong positive signal on myelin fibers in the cerebrum at day 30. By contrast, no immunolabeled cell bodies of oligodendrocytes were observed throughout the brain. The other neural cell types were also not labeled. This C1G5F2 monoclonal antibody bound mainly to the extracytosolic membrane surface of the processes of live cultured oligodendrocytes derived from newborn rat brain but was unreactive with live or fixed astrocytes and neurons maintained in culture. No immunostaining was detected in the peripheral nervous system or in the spleen, liver, or pancreas. The C1G5F2 epitope containing antigen may therefore be considered as a CNS myelin/oligodendrocyte specific molecule. Sodium deoxycholate-Tween 20 extracts of secondary oligodendrocyte cultures, biotinylated with biotin hydrazide, were used to attempt the purification of the antigen with C1G5F2 IgMs linked to antimouse IgM agarose. A main broad biotinylated protein band of 54–58 kDa molecular mass was noted. In a second approach, the antigen was immunopurified from cultured oligodendrocytes as an immune complex using biotinylated C1G5F2 IgMs. A distinct protein doublet of 53–56 kDa was also observed. It is postulated that this antigen may play an essential role in myelin formation and could be a possible target in diseases restricted to CNS myelin. © 1994 Wiley-Liss, Inc.     

Characterization of a novel monoclonal anti-myelin basic protein antibody: use in immunoblotting and immunohistochemical studies

Monoclonal antibodies (MAbs) to the myelin basic protein (MBP) were produced in CAF1 (BALB/c x A/J) mice immunized with intact bovine MBP. A number of MAbs were obtained, one of which was characterized in detail with respect to its isotype, antigenic determinant on the MBP, the spectrum of antigens with which it reacted in mouse brain, and its immunohistochemical staining characteristics. This monoclonal, GB-1 (an IgG1), recognized an epitope within residues 30-51 of bovine MBP. It also reacted with a family of MBP-related proteins present in brain homogenates of mice from 7-35 days. Immunohistochemically, GB-1 stained myelinated fibers and oligodendrocytes in the rodent CNS. A second monoclonal (GB-2, and IgM) was partially characterized. It reacted with intact MBP when it was immobilized to plastic or nitrocellulose, but it was not found to be useful for immunoblots or immunohistochemistry.     

Immunohistochemical study with an anti-myelin serum a marker for all glial cells except ‘dark’ oligodendrocytes

The usefulness of an anti-myelin antiserum as a possible marker for glial cells and related structures was investigated using rat brain. As expected, the myelin fibers were heavily stained but the neuronal cells and their processes were unreactive. The oligodendrocytes, identified on electron microscopy, revealed labelling of only the light and medium types, but not the dark cells. These results indicate that the suggested morphological classification of oligodendrocytes may be based on varying amounts of myelin antigen synthesis. Astrocytes from all areas, Golgi epithelial cells, Bergmann fibers and some subependymal cells also reacted with this anti-myelin antiserum but the staining was abolished completely by preabsorption with kidney powder. In contrast, the myelin fibers and the light and medium oligodendrocytes could still be labelled. We conclude that this anti-myelin antiserum should prove useful in studies of oligodendrocytes in the central nervous system.     

Transplantation of bulk-separated oligodendrocytes into the brains of shiverer mutant mice: Immunohistochemical and electron microscopic studies on the myelination

Normal oligodendrocytes were separated from 7-day-old mouse (BALB/c) brains by the Percoll gradient method. Immunohistochemical staining with an anti-galactocerebroside serum revealed that about 85% of the separated cells were oligodendrocytes. The oligodendrocytes were transplanted into the corpus striatum of 4-week-old shiverer mutant mice which are characterized by the lack of myelin basic protein (MBP). Myelination by the implanted oligodendrocytes was investigated immunohistochemically and electron microscopically 6 weeks after operation. Certain areas in the corpus striatum were intensely stained with antiserum to MBP. Electron microscopic examination showed that some axons were surrounded by normal type myelin sheaths with major dense lines. These results clearly indicate that matured oligodendrocytes are able to survive and myelinate the host axons even in the adult brain.     

Demyelination in the central nervous system mediated by an anti-oligodendrocyte antibody

The factors responsible for the major demyelinating disease of the central nervous system (CNS), multiple sclerosis, are poorly defined. Although T-cell-mediated immune responses play a pivotal role in establishing the inflammatory response, humoral factors also may be critical in disease progress. We have isolated a mouse monoclonal antibody (mAb 2B10) that recognizes a cell-surface molecule expressed exclusively by rat oligodendrocytes, the cells responsible for the formation and maintenance of CNS myelin. In cultures of neonatal rat spinal cord, mAb 2B10 specifically mediated oligodendrocyte cell death in the absence of complement. In the current study, mAb 2B10–producing hybridoma cells were implanted into adult rat brain ventricles, and the effect of mAb 2B10 on CNS cytoarchitecture was examined. In the optic nerves of mAb 2B10–treated animals, there was significant focal myelin degeneration near the optic chiasm. Axons in the myelin degenerate regions were largely healthy. There was no significant infiltration of hematopoietic-derived cells into the affected regions, but microglia were activated focally and phagocytosed the collapsed myelin. This study demonstrates that an antibody directed against myelin-forming cells induces CNS demyelination and supports the hypothesis that autoantibodies may play a role in CNS demyelinating diseases. J. Neurosci. Res. 54:158–168, 1998. © 1998 Wiley-Liss, Inc.     

Connexin29 and connexin32 at oligodendrocyte and astrocyte gap junctions and in myelin of the mouse central nervous system

The cellular localization, relation to other glial connexins (Cx30, Cx32, and Cx43), and developmental expression of Cx29 were investigated in the mouse central nervous system (CNS) with an anti-Cx29 antibody. Cx29 was enriched in subcellular fractions of myelin, and immunofluorescence for Cx29 was localized to oligodendrocytes and myelinated fibers throughout the brain and spinal cord. Oligodendrocyte somata displayed minute Cx29-immunopositive puncta around their periphery and intracellularly. In developing brain, Cx29 levels increased during the first few postnatal weeks and were highest in the adult brain. Immunofluorescence labeling for Cx29 in oligodendrocyte somata was intense at young ages and was dramatically shifted in localization primarily to myelinated fibers in mature CNS. Labeling for Cx32 also was localized to oligodendrocyte somata and myelin and absent in Cx32 knockout mice. Cx29 and Cx32 were minimally colocalized on oligodendrocytes somata and partly colocalized along myelinated fibers. At gap junctions on oligodendrocyte somata, Cx43/Cx32 and Cx30/Cx32 were strongly associated, but there was minimal association of Cx29 and Cx43. Cx32 was very sparsely associated with astrocytic connexins along myelinated fibers. With Cx26, Cx30, and Cx43 expressed in astrocytes and Cx29, Cx32, and Cx47 expressed in oligodendrocytes, the number of connexins localized to gap junctions of glial cells is increased to six. The results suggested that Cx29 in mature CNS contributes minimally to gap junctional intercellular communication in oligodendrocyte cell bodies but rather is targeted to myelin, where it, with Cx32, may contribute to connexin-mediated communication between adjacent layers of uncompacted myelin.     

Localization of organ-specific antigens in the nervous system of the rat

Summary Localization of organ-specific brain antigens in the central nervous system of the rat has been studied by means of indirect immunofluorescence. Rabbit antiserum against homogenate of rat brain, previously absorbed with normal serum and homogenates of rat organs (kidney, liver, spleen), reacted with the water-soluble antigens of rat brain prepared by extraction with phosphate buffer (pH 7.3) and ultracentrifugation at 50000×g to give one band in the immunodiffusion test and 2–3 precipitation arcs in immunoelectrophoresis. There was also a positive reaction with peripheral nerve. The antigen was detectable in all regions of the CNS. Cells with distinct cytoplasmic immunofluorescence were most frequently observed in cerebellar white matter, pons, cerebellar pedunculi, longitudinal tracts of the brain stem. Positive immunofluorescence reaction has appeared in the outer plexiform layer and granular layer of the retina, satellite cells of the spinal root ganglia and Schwann cells. A similar reaction was observed in human, mouse and guinea pig brain slices. Both the morphological and immunochemical reactions are indicative of glial localization of this antigen.     

Antiserum induced myelination inhibition in vitro without complement

Exposure of neonatal rat cerebellum cultures to antiserum to whole spinal cord or galactocerebroside inhibited myelin formation regardless of whether guinea pig serum was added fresh or after heating to 56 degrees C for 1 h in order to achieve complete removal of hemolytic complement activity. Myelination followed removal of antisera from the culture media. This suggests that the inhibition of primary myelination by anti-CNS tissue antiserum occurs through some mechanism other than as the result of a cytotoxic reaction against oligodendrocytes mediated via the complement system.     

ATPase activities in myelin and oligodendrocytes isolated from the brains of developing rats and from bovine brain white matter

Of the Na, K-ATPase activity in brain homogenates from 20-, 60-, and 120-day-old rats, 1.4 to 2.6% was recovered in myelin. The relative specific activities, at 0.2 to 0.3 times the specific activities in the rat brain homogenates, did not decrease during development, and myelin from bovine brain white matter had a similar relative specific activity. Oligodendrocytes from rat brains and bovine white matter had approximately one third the Na, K-ATPase specific activities found in myelin from the respective sources. The Mg-ATPase activity in rat brain myelin decreased during development and, in myelin from adult rats, was much lower than the Na, K-ATPase activity. Notably, oligodendrocytes from both the forebrains of 10- to 120-day-old rats and from bovine white matter had high Mg-ATPase activities. Whereas Na, K-ATPase may be intrinsic to certain regions of the myelin sheath, the Mg-ATPase in isolated myelin probably arises from fragments of oligodendrocyte membranes.     

Four-kilobase sequence of the mouse CNP gene directs spatial and temporal expression of lacZ in transgenic mice

The gene encoding 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) is one of the earliest myelin genes to be expressed in the brain. It is expressed at basal levels in some non-neural tissues but at much higher levels in the nervous system, and its relevance and mechanism are unknown. Using transgenic mice, we examined the expression pattern conferred by a 4-kilobase (-kb) 5′-flanking sequence of the mouse CNP gene coupled to the bacterial lacZ reporter gene. Here we report that this 4-kb fragment contains sufficient information to direct expression of the transgene to the tissue and/or cell type, in which CNP is normally expressed. In the central nervous system (CNS), CNP-lacZ expression was regulated in a temporal manner, consistent with endogenous CNP expression. Transgene expression was detected in embryonic brain and spinal cord in immature oligodendrocytes, and it significantly increased with age. In adult mice, β-galactosidase activity (which appeared to be oligodendrocyte specific) was found essentially in white matter areas of the CNS. Moreover, the transgene was expressed in peripheral nervous system, testis, and thymus—tissues that normally express CNP. Taken together, our results provide strong evidence that cis-acting regulatory elements, necessary to direct spatial and temporal expression of the transgene in oligodendrocytes, are located within the 4-kb 5′-flanking sequence of the mouse CNP gene. This promoter could be a valuable tool to target specific expression of other transgenes to oligodendrocytes, and may provide important new insights into myelination or dysmyelination. J. Neurosci. Res. 53:393–404, 1998. © 1998 Wiley-Liss, Inc.     

Oligodendrocytes and myelin sheaths in normal, quaking and shiverer brains are enriched in iron

Early studies employing iron histochemical techniques found a spatially restricted distribution of oligodendrocytes and myelin enriched in iron. In particular, oligodendrocytes and myelin positively stained for iron were found in sparsely myelinated brain regions but not in densely myelinated tracts. Subsequent studies modified the iron histochemical technique and demonstrated that oligodendrocytes and myelin were stained in densely myelinated brain regions but the staining occurred in patches rather than uniformly throughout densely myelinated tracts. This study further modified the iron histochemistry technique to establish that oligodendrocytes and myelin are enriched in iron throughout densely myelinated tracts. This finding supports biochemical studies that detected high levels of iron in white matter (Hallgren and Sourander, J Neurochem 3:41-51, 1958) and myelin fractions of brain homogenates (Rajan et al., Life Sci 18:423-432, 1976). Thus, this histochemical study and earlier biochemical studies indicate that white matter is a major site of iron concentration within the brain. The present study also examined the distribution of iron in oligodendrocytes and myelin from the dysmyelinating mutant mice quaking and shiverer. Results from these studies demonstrate that oligodendrocytes and myelin are enriched in iron in both quaking and shiverer brains. An unexpected finding was an intense staining of oval structures within the oligodendrocyte cytoplasm. This result indicates a concentration of iron in these structures and may be important for understanding how high concentrations of iron are processed by oligodendrocytes.     

The Critical Period for Repair of CNS of Neonatal Opossum (Monodelphis domestica) in Culture: Correlation with Development of Glial Cells, Myelin and Growth-inhibitory Molecules

A comparison was made of neurite growth across spinal cord lesions in the isolated central nervous system (CMS) of newborn opossums (Monodelphis domestica) at various stages of development. The aim was to define the critical period at which growth after injury ceases to occur, with emphasis on growth-inhibitory proteins, myelin and glial cells. In postnatal opossums 3–6 days old (P3–6), repair was observed 5 days after lesions were made in culture at the cervical level (C7) by crushing with forceps. Through-conduction of action potentials was re-established and axons stained by Oil grew into and beyond the crush. In a series of 66 animals 29 showed repair. In 28 animals at P11–12 with comparable lesions repair was observed in five preparations. At P13–14, the CMS was still viable in culture, but none of the 25 preparations examined showed any axonal growth into the crush or conduction through it. The rostra-caudal gradient of development permitted lesions to be made in mature cervical and immature lumbar regions of P11–12 spinal cord. Growth across crushes occurred in lumbar but not in cervical segments of the same preparation. The development of glial cells and myelin was assessed by electron microscopy and by staining with specific antibodies (Rip-1 and myelin-associated glycoprotein) in cervical segments of neonatal P6–14 opossums. At P8, oligodendrocytes and thin myelin sheaths started to appear followed at P9 by astrocytes stained with antibody against glial fibrillary acidic protein. By P14, astrocytes, oligodendrocytes and well-developed myelin sheaths were abundant. The cervical crush sites of P12 cords contained occasional astrocytes but no oligodendrocytes. Specific antibodies (IN-1) to neurite growth-inhibiting proteins (NI-35/250) associated with oligodendrocytes and myelin in the rat CNS cross-reacted with opossum proteins. Assays using the spreading of 3T3 fibroblasts and IN-1 showed that by P7 inhibitory proteins became apparent, particularly in the hindbrain and cervical spinal cord. The concentrations of NI-35/250 thereafter increased and became abundant in the adult opossum. Our finding of a well-defined critical period, encompassing only 5 days, in CNS preparations that can be maintained in culture offers advantages for analysing mechanisms that promote or prevent CNS repair.     

Growth of regenerating goldfish axons is inhibited by rat oligodendrocytes and CNS myelin but not but not by goldfish optic nerve tract oligodendrocytelike cells and fish CNS myelin

Encounters of regenerating goldfish retinal axons with oligodendrocytes and CNS myelin of mammals and fish were monitored in in vitro assays. Upon contact with highly branched rat oligodendrocytes, goldfish axons collapsed or grew around but never crossed these cells. However, in the presence of the antibody IN-1 against the oligodendrocyte-associated growth-inhibitory proteins, axons did grow over highly branched oligodencrocytes. In contrast to the mammalian oligodendrocytes, goldfish optic nerve/tract-derived oligodendrocytelike cells allowed the growth of axons across their surface and even along their processes. The fish growth cones avoided entering the region of rat CNS myelin applied to polylysine/laminin-coated coverslips or failed to elongate on this substrate. They were, however, able to pass over CNS myelin of fish. When exposed to rat CNS myelin as the sole substrate, axonal outgrowth from fish retinal explants was inhibited almost entirely. However, outgrowth on fish CNS myelin was substantial, but many more axons extended on fish or rat brain membranes that were depleted of myelin. Thus, goldfish retinal axons are sensitive to the axon-growth-inhibiting cell-surface molecules of mammalian oligodendrocytes as well as CNS myelin. Fish optic nerve oligodendrocytelike cells and fish CNS myelin lack these inhibitory properties and are growth permissive. These in vitro experiments suggest that the success of axonal regeneration in the fish optic nerve is causally related to the presence of growth-permissive properties and to the absence of growth inhibitors on fish optic nerve/tract oligodendrocytelike cells.     

Immunohistochemical localization of myelin-associated glycoprotein isoforms during the development in the mouse brain.

The developmental changes in localization of myelin-associated glycoprotein (MAG) isoforms in the mouse brain were demonstrated by an immunohistochemical method using antisera specific to two MAG isoforms. The antiserum to the large isoform of MAG (L-MAG) stained the myelin sheaths and the cytoplasm of oligodendroglia in the active myelinating stage in the mouse central nervous system. However, the antiserum to the small isoform of MAG (S-MAG) stained only myelin sheaths in the adult stage. These findings suggest that L-MAG plays an important role in active myelination.