The use of networks of dissociated rat dorsal root ganglion neurons to induce myelination by oligodendrocytes in culture

A neuronal culture system has been developed that has demonstrated to induce myelin formation by added oligodendrocytes. Networks of dissociated dorsal root ganglion neurons were prepared by suppressing non-neuronal cells (i.e. fibroblasts and Schwann cells) with a continuous 2 week exposure to 10⁻⁵ M fluorodeoxyuridine in the culture medium. After drug withdrawal, neuroglial cells were introduced in optic nerve implants from 1–2 week-old rats. These added glial cells migrated extensively over the unensheathed neurities and central myelin was formed by 2 weeks after the implant addition.     

Synthesis of myelin glycosphingolipids by isolated oligodendrocytes in tissue culture

Isolated lamb oligodendrocytes in tissue culture were tested for their ability to express differentiated functions related to the synthesis of myelin. Galactocerebrosides and sulfatides are highly enriched in myelin and are recognized as markers for myelination. Hence, we followed the synthesis of these glycosphingolipids. At selected times, monolayer cultures of cells were double labeled for 60–72 h with [³H]galactose and H₂³⁵SO₄. Glycosphingolipids were separated and identified by thin layer chromatography. During the first week in tissue culture, there was gradual decline in incorporation of both isotopes relative to the values obtained for freshly isolated cells. However, by 3 weeks high levels of H₂³⁵SO₄ incorporation into sulfatide and [³H]galactose into cerebrosides were found. A complex pattern of incorporation ensued after this peak which varied for each particular glycosphingolipid. It is concluded that: (1) cultured oligodendrocytes retain the capacity to synthesize components of myelin; (2) since the isolated cells have already been involved in myelination, these cultures afford a unique model to study remyelination; and (3) these cells provide a good system to study the properties of oligodendrocytes.     

Survival,development, and electrical activity of central nervous system myelinated axons exposed to tumor necrosis factor in vitro

Spinal cord explants from CD-1 mouse embryos were cultured in Maximow slide assemblies to promote myelin development. At about 20 days in vitro, recombinant human or mouse tumor necrosis factor alpha (TNFa) was added. Observed 3–8 days later, myelin was largely intact. The myelin blistering and oligodendrocyte damage seen in other strains were generally absent. Axonal conduction was measured optically through the use of a voltage-sensitive dye. Action potential shape, conduction velocity, and refractory period were all unchanged by exposure to TNFa. Two series of explants were grown with TNFa present continuously throughout the culture period. Observed with light and electron microscopy, myelin developed in at least 50% of the explants treated with recombinant mouse TNFα and 80% of those exposed to recombinant human TNFα. Optically recorded action potentials were of normal shape and refractory period. Conduction velocities were slightly lower than controls. CD-1 mouse central nervous system contains TNFα receptors and yet was resistant to myelin damage. The apparent strain specificity of TNFα disruption of myelin may result from more indirect modes of action, including interaction with other cytokines produced by glial cells. Survival of axonal conduction suggest that Na⁺ channel function remains intact following TNFα exposure. © 1995 Wiley-Liss, Inc.     

Phagocytosis of myelin by microglia in vitro

Previous experiments from this laboratory have shown that peritoneal macrophages in culture will phagocytize myelin. Myelin preopsonized with myelin antibodies is phagocytized to a much greater extent than untreated myelin, indicating that macrophages ingest myelin by an Fc receptor. The present work was undertaken to determine the characteristics of myelin phagocytosis by microglia, the resident macrophages of the central nervous system. Microglia isolated from 4–5 day primary cultures of newborn rat brains were shown to bind and phagocytize myelin labeled in the lipids by ¹⁴C-acetate. Both binding and phagocytosis as shown by the appearance of ¹⁴C-cholesterol ester were greatly increased if labeled myelin was preopsonized with antiserum to myelin basic protein or galactocerebroside. Both preopsonized and untreated myelin were phagocytized more actively by microglia than by peritoneal macrophages under the same culture conditions. Microglia cultured in the presence of GM-CSF showed slightly increased cholesterol ester production from opsonized myelin, but the effect of GM-CSF was significantly greater than myelin pretreated with control serum (34% increase) or untreated myelin (154% increase). There was no significant effect of GM-CSF on myelin phagocytosis by peritoneal macrophages. Cerebrospinal fluid containing immunoglobulin drawn from rabbits with acute EAE also opsonized myelin to increase phago cytosis by microglia, as has been previously shown with peritoneal macrophages. These results indicate that microglia may actively participate in myelin destruction in demyelinating diseases where myelin antibodies or a source of GM-CSF may be present. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Differentiation of oligodendrocytes cultured from developing rat brain is enhanced by exogenous GM3 ganglioside

Cultures consisting primarily of O-2A progenitor cells and immature oligodendrocytes with a few microglia and astrocytes were obtained by shaking primary cultures from neonatal rat brain after 12--14 days in vitro. Addition of 50 μg/ml exogenous Neu-NAcα2-3Galβ1-1′ ceramide (GM3 ganglioside) to the cultures resulted in an increase in the number and thickness of cell processes that stained intensely for sulfatide and galactocerebroside (galC) in comparison to control cultures without added GM3. The treated cultures also contained fewer astrocytes than control cultures as revealed by immunostaining for glial fibrillary acidic protein (GFAP). Cells that immunostained for both GFAP and sulfatide/galC were very rare in control cultures but were frequently seen in the GM3-treated cultures, suggesting that these may represent cells changing their direction of differentiation away from type II astrocytes toward oligodendrocytes under the influence of GM3. These effects on the developing rat oligodendrocytes were specific for GM3 ganglioside and were not produced by adding GM1, GM2, GD3, or GD1a to the cultures. Lactosyl ceramide and neuraminyl lactose were also ineffective. When control cultures were initially plated on polylysine and incubated with [¹⁴C]galactose, GD3 was the principal labeled ganglioside. However, as the control cells differentiated over time in culture without the addition of exogenous GM3 and produced increasing amounts of myelin-related components, the incorporation of [¹⁴C]galactose into endogenous GM3 increased to become the predominant labeled ganglioside by 6 days after plating. Metabolic labeling of the GM3-treated oligodendrocytes with [¹⁴C]galactose revealed increased incorporation into galC and sulfatide in comparison to control cultures, but a decreased labeling of endogenous GM3. Similarly, incorporation of an amino acid precursor into the myelin-associated glycoprotein (MAG) was increased by GM3 treatment, but incorporation into myelin basic protein (MBP) was not affected. Although the overall effect of added GM3 was to decrease the phosphorylation of most proteins in the oligodendrocytes, including MBP, GM3 enhanced the phosphorylation of MAG. These findings indicate that GM3 ganglioside has an important role in the differentiation of cells of the O-2A lineage toward myelin production, since differentiation is associated with increased metabolic labeling of endogenous GM3 in control cultures and is enhanced by the addition of exogenous GM3. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Astrocytes and neurons regulate the expression of the neural recognition molecule janusin by cultured oligodendrocytes

Janusin (formerly designated J1–160/180) is an extracellular matrix glycoprotein highly homologous to tenascin, consisting of two major molecular forms of 160 and 180 kD expressed by oligodendrocytes and in myelin. Janusin expression is upregulated during myelination and in the adult it remains expressed at lower levels. It is also present at the node of Ranvier, where myelin, axon, and astrocytic process are in close contact. To gain an understanding of the regulatory mechanisms which may under-lie expression of janusin, the differentiation stage-dependent expression of janusin was studied in cultures enriched in mouse oligodendrocytes and their precursor cells. Expression of janusin by these cells was highest on both A2B5⁺ and O4⁺/O1^? oligodendroglial precursor cells and a subset of myelin associated glycoprotein-positive (MAG⁺) oligodendrocytes. Hardly any of the more differentiated O1⁺ or O10⁺ oligodendrocytes expressed janusin. Expression of janusin was influenced by co-culture with astrocytes or neurons. Astrocytes or astrocytic-conditioned culture supernatants elevated the expression of janusin by the more differentiated oligodendrocytes (O1⁺ or MAG⁺ cells), while its expression by oligodendroglial precursor cells was relatively unchanged. Platelet-derived growth factor, but not basic fibroblast growth factor, also elevated the expression of janusin by O1⁺ or O10⁺ oligodendrocytes. In contrast, co-culture with neurons originating from dorsal root ganglia or spinal cord decreased the expression of cell-bound janusin by oligodendrocytes and their precursor cells. These observations indicate that expression of janusin on these cells in culture is susceptible to opposing regulatory influences from astrocytes and neurons. Such influences may modulate the temporal and spatial distribution of janusin in the developing and adult central nervous system. © 1993 Wiley-Liss, Inc.     

Differential myelinogenic capacity of specific developmental stages of the oligodendrocyte lineage upon transplantation into hypomyelinating hosts

The capacity of oligodendrocytes (OLs) and their progenitors to migrate, proliferate, and differentiate in vivo was evaluated by transplanting highly enriched populations of sequential stage of the OL lineage (A2B5⁺ O4^?, O4⁺ GalC^?, and GalC⁺) into the telencephalon of the hypomyelinating mouse, shiverer. The shiverer mouse neither expresses the major myelin basic protein (MBP) nor makes normal myelin due to a large deletion in the gene for MBP. Thirty days after transplantation, serial 225 μm sections of the host brain were immunostained with antiserum to MBP and analyzed by confocal microscopy. The presence of MBP⁺ patches of myelin in the otherwise MBP^? host brain allowed a retrospective analysis of the myelinogenic activity of the transplanted progenitors cells. Both the extent of MBP⁺ myelin and the location of MBP⁺ structures relative to the initial site of cell deposition were highly dependent on the developmental stage of the transplanted cells. Specifically, A2B5⁺ O4^? OL progenitors migrated distances of ≥ 600 μm and produced MBP⁺ patches in nearly every slice of the host brain. An average of over 250 separate patches were found per host brain, some of which had cross-sectional areas of > 250,000 μm² containing as many as 60 MBP⁺ OL cell bodies, and with densities of myelination rivaling that of normal brain. In marked contrast, transplantation of O4⁺ GalC^? cells produced only small (1,000–25,000 μm²), scattered (25–40 per brain) patches of MBP⁺ myelin containing one to five cell bodies, all of which were within 50 μm of the needle track or the nearest ventricular surface. GalC⁺ cells produced MBP⁺ myelin at a level similar to that of O4⁺ CalC^? cells. These data suggest that the developmental transition of OL progenitors from the O4^? to the O4⁺ pheno-type is accompanied by a dramatic reduction in the innate capacity of the cells to migrate and survive in vivo. The use of developmentally identified, enriched populations of OL progenitor cells offers the opportunity for more precise analyses of transplantation and remyelination behavior, and relates to clinically relevant studies indicating that contaminant cell types can seriously interfere with the stable integration of donor tissue into the host. © 1993 Wiley-Liss, Inc.     

Juvenile and adult olfactory ensheathing cells bundle and myelinate dorsal root ganglion axons in culture

Olfactory ensheathing cells (OEC), which normally associate closely with but do not myelinate axons in situ, myelinate axons in the adult mammalian spinal cord. They are of clinical interest as candidate cells for autologous transplantation but the ability of OEC to myelinate axons in vitro has been controversial. To clarify this issue, we isolated OEC from olfactory bulbs (OB) of juvenile and adult rats expressing GFP and analyzed their ability to myelinate axons. Using a well-defined assay for myelination of dorsal root ganglia (DRG) axons in culture, we found that OEC from juvenile pups associated with and then myelinated DRG axons. OEC assembled into bundles with the axons by 1 week and required more than a week before myelination on axons was detected. In contrast, rat Schwann cells did not bundle axons and they formed P0⁺ and MBP⁺ myelin segments after as little as 1 week. Most of the OEC in culture exhibited staining for calponin, a marker that was not found on Schwann cells in culture, whereas in both OEC and Schwann cell populations nearly all cells were positive for p75NTR and GFAP. These results confirm previous reports showing only subtle immunological differences between Schwann cells and OEC. Besides differences in the rate of myelination, we detected two additional functional differences in the interactions of OEC and Schwann cells with DRG axons. First, the diameter of OEC generated myelin was greater than for Schwann cell myelin on DRG axons. Second, OEC but not Schwann cells myelinated DRG axons in the absence of vitamin C. OEC isolated from adult OB were also found to bundle and myelinate DRG axons but the latter occurred only after incubation times of at least 3 weeks. The results indicate that adult OEC require longer incubation times than juvenile OEC to myelinate axons and suggest that patterns of myelination by OEC and Schwann cells are distinguishable at least on axons in vitro. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.     

Vesicular demyelination induced by raised intracellular calcium

Incubation of nerve with high concentrations of the divalent cation ionophore A23187 produces myelin vesiculation (Schlaepfer 1977). This observation has now been extended using segments of rat ventral or dorsal root incubated with high (19 μM, 10 μg/ml) or low (1–1.5 μM) concentrations of A23187, or another divalent ionophore, ionomycin. Low concentrations of A23187 induced no vesiculation within a 2-h period. However, subsequent incubation of these roots in fresh, ionophore-free medium for 20 h, resulted in a prominent vesicular demyelination at the Schmidt-Lanterman incisures and paranodes of many fibres. At this time (22 h) the Schwann cells associated with some demyelinating internodes appeared vital upon ultrastructural examination: the cells also excluded the nuclear dye nigrosin. High concentrations of A23187 induced a similar vesicular demyelination in affected fibres within only 15–20 min. While the Schwann cells continued to exclude nigrosin for a further 4 h, their ultrastructural appearance indicated that they were probably in the early stages of necrosis. Incubation of moribund root with the ionophore produced no myelin vesiculation. At all ionophore concentrations, the myelin vesiculation was dependent upon the presence of extracellular Ca²⁺, and could be modulated in severity by varying this concentration. Other divalent cations (Ba²⁺, Co²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Sr²⁺) could not substitute for Ca²⁺. The vesiculation induced by A23187 could be entirely prevented by the addition of Zn²⁺ (? 1 μM), Ni²⁺ (? 1–10 μM), Co²⁺ (? 100 μM) or Mn²⁺ (? 100 μM) to the bathing medium. A23187 applied to only part of an isolated internode resulted in a localization of the myelin disruption to that region. Ionomycin (? 1 μM), an ionophore with a greater selectivity for Ca²⁺ than A23187, also induced a prompt Ca²⁺-dependent myelin vesiculation. We conclude that vesicular demyelination can be initiated in vital Schwann cells by a raised intracellular Ca²⁺ concentration. Such demyelination does not necessarily lead to Schwann cell death. The possible relevance of the findings to vesicular demyelinating neuropathies is discussed, and a hypothesis regarding the mechanism of demyelination is advanced.     

Early neuron differentiation in the mouse of olfactory bulb. I. Light microscopy

The relation between the growth of axons and the development of their myelin sheaths was determined in rats for normal, myelinating sciatic nerves, and for an experimental model allowing to retard or accelerate axon growth. Axon caliber was measured, and sheath development was determined from measurements of thickness (light microscopy), from counts of the number of turns of myelin lamellae (electron micrographs), and from the rate of the incorporation of acetate-H³ into ether-ethanol extractable lipids. An excellent correlation between changes in sheath thickness and acetate-H³ incorporation was obtained for all experiments. For the myelinating nerves the changes in the rate of acetate-H³ incorporation were related specifically to the rate with which new length of myelin leaflet was added to the existing turns of sheath. The rate of axonal growth was manipulated by applying a snug ligature around the nerve by the fourteenth day, allowing the nerve to compress itself by its own growth. Most axons passed the constriction without interruption, but they were markedly hypoplastic distal to the constriction. After removal of the ligature these axons regrew to their normal caliber ranges. Examination of myelin sheath development in this model showed that retardation of axon growth retarded sheath growth, while acceleration of axon growth accelerated sheath growth. Thus, the rate of axon growth appeared to be the factor controlling the rate of myelin formation by the sheath cells. An appendix describes a model consisting of two interrelated feedback mechanisms by which expansion of the axon may directly control the number of turns of myelin lamellae formed by the sheath cell. The model correspons on all points to established features in the fine structure of fibers.     

Glycosaminoglycans in nerve injury: I. Low doses glycosaminoglycans promote neurite formation

This study has shown that glycosaminoglycans added to the culture medium may affect neurite formation in SH-SY5Y neuroblastoma cells. The most effective glycosaminoglycans are heparin and COS 8, a preparation with low anticoagulant activity. Promotion of neuritogenesis was remarkable at concentrations as low as 10^-8 and 10^-10. When added at 10^-4 M both agents are inhibitory. Chondroitin-4 sulfate, dermatan sulfate, and heparan sulfate were also effective, the doses required were, however, as high as 10^-4 M for promoting and 10^-4 M for inhibiting neuritogenesis. Thereby low doses of glycosaminoglycans promote, while higher doses inhibit neurite formation. The effects were observed when neuritogenesis was promoted in neuroblastoma cultures either by deprivation of serum or by addition of retinoic acid, in the former case neuritogenesis occurred within 48 hr; in the latter, in 14 days. PC12 pheochromocytoma cells neuritogenesis was triggered by adding NGF to the culture medium. We have also observed that glycosaminoglycan supplementation to the culture medium lowered the quantity of NGF required to form neurites by PC12 cells. Glycosaminoglycans at the dose of 10^-8 M allow the formation of PC12 neurites even in presence of 1 ng/ml NGF, a dose that normally is ineffective. © 1996 Wiley-Liss, Inc.     

A role of peripheral myelin protein 2 in lipid homeostasis of myelinating schwann cells

Peripheral myelin protein 2 (Pmp2, P2 or Fabp8), a member of the fatty acid binding protein family, was originally described together with myelin basic protein (Mbp or P1) and myelin protein zero (Mpz or P0) as one of the most abundant myelin proteins in the peripheral nervous system (PNS). Although Pmp2 is predominantly expressed in myelinated Schwann cells, its role in glia is currently unknown. To study its function in PNS biology, we have generated a complete Pmp2 knockout mouse (Pmp2^‐/‐). Comprehensive characterization of Pmp2^‐/‐ mice revealed a temporary reduction in their motor nerve conduction velocity (MNCV). While this change was not accompanied by any defects in general myelin structure, we detected transitory alterations in the myelin lipid profile of Pmp2^‐/‐ mice. It was previously proposed that Pmp2 and Mbp have comparable functions in the PNS suggesting that the presence of Mbp can partially mask the Pmp2^‐/‐ phenotype. Indeed, we found that Mbp lacking Shi^‐/‐ mice, similar to Pmp2^‐/‐ animals, have preserved myelin structure and reduced MNCV, but this phenotype was not aggravated in Pmp2^‐/‐/Shi^‐/‐ mutants indicating that Pmp2 and Mbp do not substitute each other's functions in the PNS. These data, together with our observation that Pmp2 binds and transports fatty acids to membranes, uncover a role for Pmp2 in lipid homeostasis of myelinating Schwann cells. GLIA 2014;62:1502–1512     

Cellular reaction to an acute demyelinating/remyelinating lesion of the rat brain stem: Localisation of GD3 ganglioside immunoreactivity

We describe a simple and reproducible acute demyelinating lesion of the rat brain stem induced by injection of ethidium bromide into the cisterna magna of young adult rats. Using immunofluorescence with a panel of antibodies to cell-specific antigens we have studied the changes in cell populations that occur at various stages during lesion progression and repair. In particular we localized the expression of ganglioside G_D3 immunoreactivity, a marker for oligodendroglial progenitors in developing brain. Both astroglia (GFAP⁺) and oligodendroglia (CNP⁺) were destroyed during the early response to the ethidium bromide although axons were spared. Splitting of myelin lamellae occurred as early as 4 days post-injection (DPI), with extensive demyelination of the inferior cerebellar peduncle following by 6 DPI. Large numbers of ED1⁺ and OX-42⁺ macrophages were present in the lesion site at this stage. Astrogliosis occurred around the perimeter of the lesions. Two populations of G_D3⁺ cells appeared within and around the lesion sites during the demyelination. One population was identified by the phenotype G_D3⁺ ED1⁺ and thus probably belonged to the macrophage/microglial lineage. In these cells both antigens appeared cytoplasmic. The second population of G_D3⁺ cells exhibited cell membrane G_D3 immunoreactivity but did not express the ED1 antigen. These cells are suggested to be oligodendroglial progenitors generated in response to the demyelination. No such cells were seen in control tissue. G_D3⁺ cells were present within the lesion sites from 6 DPI until 10–12. Following the clearance of myelin debris from the lesions, remyelination was a relatively rapid event with thin MBP⁺ myelin sheaths first seen at 11–12 DPI. Remyelination, which was extensive by 25 DPI, was predominantly oligodendroglial in origin (MBP⁺Po^? myelin) with only small pockets of peripheral myelin (MBP⁺Po⁺ myelin) observed. The present study, in addition to identifying putative glial progenitors within a demyelinated lesion, also demonstrates the difficulties in unambiguously identifying such cells in the normal and damaged adult CNS. © 1993 Wiley-Liss, Inc.     

Proteolipid protein modulates preservation of peripheral axons and premature death when myelin protein zero is lacking

Protein zero (P0) is the major structural component of peripheral myelin. Lack of this adhesion protein from Schwann cells causes a severe dysmyelinating neuropathy with secondary axonal degeneration in humans with the neuropathy Dejerine‐Sottas syndrome (DSS) and in the corresponding mouse model (P0^null‐mice). In the mammalian CNS, the tetraspan‐membrane protein PLP is the major structural myelin constituent and required for the long‐term preservation of myelinated axons, which fails in hereditary spastic paraplegia (SPG type‐2) and the relevant mouse model (Plp^null‐mice). The Plp‐gene is also expressed in Schwann cells but PLP is of very low abundance in normal peripheral myelin; its function has thus remained enigmatic. Here we show that the abundance of PLP but not of other tetraspan myelin proteins is strongly increased in compact peripheral myelin of P0^null‐mice. To determine the functional relevance of PLP expression in the absence of P0, we generated P0^null*Plp^null‐double‐mutant mice. Compared with either single‐mutant, P0^null*Plp^null‐mice display impaired nerve conduction, reduced motor functions, and premature death. At the morphological level, axonal segments were frequently non‐myelinated but in a one‐to‐one relationship with a hypertrophic Schwann cell. Importantly, axonal numbers were reduced in the vital phrenic nerve of P0^null*Plp^null‐mice. In the absence of P0, thus, PLP also contributes to myelination by Schwann cells and to the preservation of peripheral axons. These data provide a link between the Schwann cell‐dependent support of peripheral axons and the oligodendrocyte‐dependent support of central axons. GLIA 2016;64:155–174     

Myelination ofjp,jp andqk axons by normal glia in vitro: Ultrastructural and autoradiographic evidence

Normal optic nerve glia were ‘injected’ into hypomyelinated mutantjp,jp^msd, andqk cerebellum by co-culturing explants in direct physical contact. Quantitative light microscopic studies demonstrated that such glial injection significantly increased the number of myelin profiles counted in cultures, suggesting that axons in all 3 mutants can accept myelination from competent glia when they are made available. In each mutant, the observed increase in myelination was independent of the ages of donor optic nerves and recipient cultures, but absolutely required positioning of the optic nerve so that direct contact occurred with the mutant cerebellar explants. The additional myelin found near the zone of fusion with the optic nerve morphologically resembled normal, not mutant myelin. Autoradiographs made after [³H]thymidine-labeled normal optic nerve was injected intojp^msd cultures showed that labeled cells had colonized the nearby mutant tissue. Labeled cells identified as oligodendrocytes by ultrastructural criteria were found adjacent to myelin segments near the fusion zone, but direct continuity between processes of these oligodendrocytes and myelin sheaths was not demonstrated. The astrocytes and phagocytic cells which were also labeled had no obvious relationship to myelinated axons. These results provide experimental evidence that the primary abnormalities produced by the three mutationsjp,jp^msd, andqk are inherent in their glial cells, probably although not definitely in the oligodendrocytes.     

Galactose oxidase labeling of membrane proteins from human brain white matter

The use of galactose oxidase (EC 1.1.3.9) and tritiated sodium borohydride for labeling of membrane glycoproteins, described by Gahmberg and Hakomori², has previously been applied to the study of myelin glycoproteins of experimental animals⁸. Rat brain myelin glycoproteins have been studied by sequential lectin affinity chromatography¹² and recently the lectin-binding capacity of rat central nervous system myelin glycoproteins has been characterized⁷. Complex heterogeneity of the glycoprotein pattern of rat central nervous system myelin has been reported^7,8,12, and so a variety of glycoproteins can be expected to exist in human white matter membranes. Application of the galactose oxidase procedure to the study of human brain membranes could be useful in research concerning certain neurological diseases if the properties of autopsy brain material are taken into account. In this study, membrane proteins of human autopsy brain white matter were subjected to the galactose oxidase/NaB³H₄ labeling procedure and the membranes labeled by this method or by the [³H]acetic anhydride techniques⁶ were studied by lectin affinity chromatography using Lens culinaris phytohemagglutinin (lentil lectin) attached to Sepharose 4B beads.     

Oligodendroglial progenitors labeled with the O4 antibody persist in the adult rat cerebral cortex in vivo

The monoclonal antibody O4 has been used to define a biologically distinct stage of the oligodendroglial lineage in vitro. Furthermore, O4⁺ oligodendroglial progenitors have been found in cell cultures derived from mature tissue, leading to speculation about the presence of oligodendroglial progenitors in the adult central nervous system (CNS). However, the existence of adult oligodendroglial progenitors has yet to be conclusively demonstrated in the intact animal. We have investigated the expression of O4 immunoreactivity in the developing and mature rat forebrain and the relationship of these cells to cells expressing the early oligodendroglial progenitor markers G_D3 ganglioside and NG₂ chondroitin sulfate proteoglycan, and to differentiated galactocerebroside expressing oligodendroglia. By the day of birth O4⁺ cells were already widely distributed throughout the formative corpus callosum and increased in number in the white matter and cortical gray matter over the first 2 postnatal weeks. In contrast to cell culture observations, most O4⁺ cells seen over this period failed to express G_D3, although the majority did express NG₂. Beginning at postnatal day 4, NG₂⁺/O4⁻ progenitors in the corpus callosum and cerebral cortical gray matter underwent a wave of differentiation into NG₂⁺/O4⁺ cells and then into galactocerebroside-positive oligodendroglia. Interestingly, not all cells underwent this progression: a population remained as O4⁺/NG₂⁺ progenitors. Furthermore, this O4⁺/NG₂⁺ population persisted into adulthood and failed to express either G_D3, galactocerebroside, RIP, or myelin basic protein (MBP). They were also distinguishable from glial fibrillary acidic protein⁺ and glutamine synthetase⁺ astrocytes and OX-42⁺ microglia. We therefore propose that these O4⁺/NG₂⁺ cells represent adult oligodendroglial progenitors hitherto only described in cell culture. © 1997 Wiley-Liss Inc.     

Neuronal stimulation of [3H]thymidine incorporation by primary cultures of highly purified non-neuronal cells

A specific intercellular interaction has been demonstrated between neuronal and non-neuronal cells that appears to increase the rate of non-neuronal cell proliferation. Isolated and recombined primary cultures of both cell types were prepared from 11-day embryonic chick sympathetic ganglia by a method recently developed in this laboratory. When non-dividing neurons were added to an equal number of proliferating non-neuronal cells, the amount of [methyl-³H]thymidine incorporated by these mixed cultures was 230% greater than that incorporated by 99% pure non-neuronal cultures. Removal of all neurons from such non-neuronal cultures by a 48-h preincubation without nerve growth factor resulted in an even greater increase in [³H]thymidine incorporation upon addition of neurons (370%). When increasing numbers of isolated neurons were added to non-neuronal cell cultures, the amount of [³H]thymidine incorporation initially increased in a dose-dependent fashion until it reached a plateau. In contrast, the addition of increasing numbers of non-neuronal cells to a constant number of neurons resulted in a linear increase in [³H]thymidine incorporation. In some cases neurons and non-neuronal cells were not grown in direct physical contact but were only allowed to communicate with one another through the culture medium. Such indirect communication never resulted in a stimulation of [³H]thymidine incorporation. When neurons were added to cultures of embryonic chick fibroblasts, the neurons grew well but did not stimulate [³H]thymidine incorporation by the fibroblasts. These results suggest that embryonic sympathetic neurons selectively stimulate the proliferation of non-neuronal cells derived from the same source.