Myelin Synthesis Research Articles

Central nervous system demyelination and remyelination in multiple sclerosis and viral models of disease

The mechanisms of myelin injury and repair were studied in acute multiple sclerosis lesions and in a murine model of demyelination induced by a virus. Injury to oligodendrocytes resulting in degeneration of inner glial loops and inner myelin lamellae (dying-back oligodendrogliopathy) was observed by electron microscopy in brain biopsies of acute demyelinating lesions. Attempts at central nervous system remyelination as manifested by thinly myelinated axons and proliferation of oligodendrocytes were observed at the edge of many acute plaques. To develop therapeutic strategies to inhibit demyelination or promote remyelination, mice infected intracranially with Theiler's virus (a picornavirus) were studied. Experimental manipulation of Theiler's virus-infected mice by treatment during chronic demyelinating disease with immunoglobulins directed at normal spinal cord antigens or with monoclonal antibodies which deplete CD4 or CD8-positive T cells reslted in augmentation of new myelin synthesis. These observations suggest that disturbances in the myelinating function of oligodendrocytes, events not accompanied by death of these cells, may be among the earliest pathological events in multiple sclerosis. Experiments using the Theiler's virus model of demyelination indicate that manipulation of the immune response has the potential to promote central nervous system remyelination and functional recovery in multiple sclerosis.

Multiple sclerosis and vitamin B12 metabolism

Multiple sclerosis (MS) is occasionally associated with vitamin B12 deficiency. Recent studies have shown and increase risk of macrocytosis, low serum and/or CSF vitamin B12 levels, raised plasma homocysteine and raised unsaturated R-binder capacity in MS. The aetiology of the vitamin B12 deficiency in MS is often uncertain and a disorder of vitamin B12 binding or transport is suspected. The nature of the association of vitamin B12 deficiency and MS is unclear but is likely to be more than coincidental. There is a remarkable similarity in the epidemiology of MS and pernicious anaemia. Vitamin B12 deficiency should always be looked for in MS. The deficiency may aggravate MS or impair recovery. There is evidence that vitamin B12 is important for myelin synthesis and integrity but further basic studies are required.

Primary demyelination induced by exposure to tellurium alters Schwann cell gene expression: a model for intracellular targeting of NGF receptor

Exposure of developing rats to tellurium results in a highly synchronous segmental demyelination of peripheral nerves with sparing of axons; this demyelination is followed closely by a period of rapid remyelination. Demyelination occurs subsequent to a tellurium-induced block in the synthesis of cholesterol, the major myelin lipid. We utilized the techniques of Northern blotting, in situ hybridization, and immunocytochemistry to examine temporal alterations in Schwann cell gene expression related to demyelination and remyelination. Tellurium-induced demyelination is associated with downregulation of myelin protein expression and a corresponding upregulation of NGF receptor (NGF-R) and glial fibrillary acidic protein (GFAP) expression. Steady-state mRNA levels (expressed on a "per nerve" basis) for P0, the major myelin protein, were decreased by about 50% after 5 d of tellurium exposure, while levels of mRNA for NGF-R and GFAP were markedly increased (about 15-fold). In situ hybridization of teased fibers suggested that the increase in steady-state mRNA levels for NGF-R was primarily associated with demyelinated internodes and not with adjacent unaffected internodes. Although P0 message was almost totally absent from demyelinating internodes, it was also reduced in normal-appearing internodes as well. This suggests that limiting the supply of a required membrane component (cholesterol) may lead to partial downregulation of myelin gene expression in all myelinating Schwann cells. In partially demyelinated internodes, NGF-R and GFAP immunofluorescence appeared largely confined to the demyelinated regions. This suggests specific targeting of these proteins to local areas of the Schwann cell where there is myelin loss. These results demonstrate that demyelination is associated with reversion of the affected Schwann cells to a precursor cell phenotype. Because axons remain intact, our results suggest that these changes in Schwann cell gene expression do not require input from a degenerating axon, but instead may depend on whether concerted synthesis of myelin is occurring.

Immunosuppression promotes CNS remyelination in chronic virus‐induced demyelinating disease

Immunosuppression using cyclophosphamide or anti-T cell monoclonal antibodies (mAbs) directed at CD4 or CD8 promoted remyelination of CNS axons in the spinal cords of mice infected chronically with Theiler's virus. Treatment with a mAb directed at class II major histocompatibility gene products did not increase the extent of CNS remyelination. Following immunosuppressive treatment, quantitative morphometry revealed a five- to sevenfold increase in new myelin synthesis. Proliferating nervous system cells were identified at the edges of remyelinated lesions by their incorporation of [3H]thymidine. CNS remyelination occurred in mice depleted of selected subsets of T lymphocytes despite the local persistence of viral antigen. These findings indicate that CNS remyelination occurs as a normal consequence of primary myelin injury, but factors associated with immune T cells somehow impair remyelination. Interference with the function of immune T cells enhances CNS remyelination by oligodendrocytes. Similar depletion of immune T cells may allow for enhanced remyelination in the CNS of patients with chronic multiple sclerosis.

Correlation of the ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine in the brain and cerebrospinal fluid of the pig: implications for the determination of this methylation ratio in human brain.

1. Pigs were maintained in air or in an atmosphere of nitrous oxide which dramatically changes the S-adenosyl-L-methionine to S-adenosyl-L-homocysteine ratio in neural tissues. Samples of cerebrospinal fluid, cortex, cerebellum and spinal cord were then extracted and analysed for S-adenosyl-L-methionine and S-adenosyl-L-homocysteine. Regression analyses were carried out on values obtained in cerebrospinal fluid and in neural tissues. 2. Highly significant correlations were obtained between levels of S-adenosyl-L-homocysteine (r2 = 0.42-0.69; P less than 0.001) and S-adenosyl-L-methionine/S-adenosyl-L-homocysteine ratios (r2 = 0.56-0.65; P less than 0.001) in cerebrospinal fluid and levels and ratios in cortex, cerebellum and spinal cord. The levels of S-adenosyl-L-methionine did not show a significant correlation. 3. We conclude that the ratio of these metabolites in the cerebrospinal fluid may reflect the ratio in the central nervous system and we suggest that this may also be true in human tissues. This finding will permit the determination of the probable methylation ratio in the central nervous system in human conditions, such as vitamin B12 deficiency and acquired immune deficiency syndrome, where a similar myelopathy occurs to that seen in the nitrous oxide-treated pig. All three myelopathies may arise from an inhibition of methyltransferases involved in the synthesis of myelin that would occur when the methylation ratio is reduced.

Insulin‐like growth factor I stimulates oligodendrocyte development and myelination in rat brain aggregate cultures

Insulin-like growth factor I (IGF-I) and high concentrations of insulin have been shown to stimulate an increase in the number of oligodendrocytes that appear in developing monolayer cultures of rat brain cells (McMorris et al., Proc Natl Acad Sci USA 83: 822-826, 1986; McMorris et al., Ann NY Acad Sci 605:101-109, 1990; McMorris and Dubois-Dalcq, J Neurosci Res 21:199-209, 1988). In the present study, we investigated whether IGF-I or insulin treatment induces a corresponding increase in the synthesis and accumulation of myelin. Aggregate cultures, established from 16-day-old fetal rat brains, were treated with either 100 ng/ml IGF-I or 5,000 ng/ml insulin and analyzed for the number of oligodendrocytes, activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), total amount of myelin, and synthesis rate of myelin proteins. Cultures treated with IGF-I beginning on day 2 after explantation contained 35-80% more oligodendrocytes and had 60-160% higher CNP activity than controls when tested on day 13, 20, or 27. By day 27, treated cultures had 35-90% more myelin than controls. Similar results were observed in response to 5,000 ng/ml insulin, a concentration at which insulin binds to IGF receptors and acts as an analogue of IGF-I. The synthesis rate of myelin proteins was measured in experiments using 5,000 ng/ml insulin. When treatment was begun at day 20 rather than day 2, cultures did not exhibit an increased number of oligodendrocytes over control during the following 4-6 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical staining of the RLM6 form of cytochrome P-450 in oligodendrocytes and myelin of rat brain.

We used immunocytochemical staining to localize the RLM6 form of cytochrome P-450 in rat brain. Immunofluorescence staining in vibratome sections was positive in cells that resembled oligodendrocytes, which are the cells that synthesize and maintain myelin. Double immunofluorescence staining with anti-RLM6, plus mouse monoclonal antibodies (MAb) against 2',3'-cyclic nucleotide-3'-phosphohydrolase or galactocerebrosides, showed localization of each of these oligodendrocyte "markers" in the same cells as RLM6. In vibratome sections from brains of adult rats there was faint RLM6 immunostaining in some of the myelinated fibers as well as in oligodendrocytes. In paraffin sections from adult rat brains, myelinated tracts were RLM6 positive, as were oligodendrocytes and myelinated fibers in the gray matter. Oligodendrocytes were also shown to contain glucose-6-phosphate dehydrogenase. We suggest that RLM6, which is constitutive to liver, is also constitutive to brain and, via the acetone monooxygenase reaction, which also utilizes NADPH, may contribute to the conversion of ketone bodies to substrates that could provide energy for the synthesis and maintenance of myelin.

The pathogenesis of demyelinating disease: insights from cell biology

Cellular and humoral immune mechanisms have been implicated in the pathogenesis of human and experimental demyelinating diseases of the CNS 1−5. How these interact in the complex sequence of events that culminates in phagocytosis of myelin by macrophages has yet to be resolved. The relationship between leakage of the blood-brain barrier and demyelination, the reason why recurrent inflammatory demyelination occurs - seemingly in the absence of an antigen-specific immune response - and the lack of effective remyelination all require explanation if a coherent account of immunologically mediated demyelination is to be achieved. One approach to these problems is to study in vitro the developmental and cellular biology of oligodendrocytes - the glial cells responsible for the synthesis and maintenance of CNS myelin. This provides experimental opportunities not offered by more direct investigation of the intact nervous system, but carries the clear disadvantage that observations made in vitro cannot necessarily be extrapolated to humans.

Tellurium‐induced neuropathy: A model for reversible reductions in myelin protein gene expression

Inclusion of 1.1% tellurium in the diet of developing rats causes a highly synchronous primary demyelination of peripheral nerves, which is followed closely by a period of rapid remyelination. The demyelination is related to the inhibition of squalene epoxidase activity, which results in a block in cholesterol synthesis and accumulation of squalene. We now report that the demyelination resulting from this limiting of the supply of an intrinsic component of myelin (cholesterol) leads to repression of the expression of mRNA for myelin-specific proteins. Tellurium exposure resulted in an increase in total RNA (largely rRNA) in sciatic nerve, which could not be accounted for by cellular proliferation; these increased levels of rRNA may be a reactive response of Schwann cells to toxic insult and may relate to the higher levels of protein synthesis required during remyelination. In contrast, steady-state levels of mRNA, determined by Northern blot analysis, for P0 and myelin basic protein were markedly decreased (levels after 5 days of tellurium exposure were only 10-15% of control levels as a fraction of total RNA and 25-35% of control levels when the increased levels of total RNA were taken into account). Message levels increased during the subsequent period of remyelination and reached near-normal levels 30 days after beginning tellurium exposure. Although message levels for the myelin-associated glycoprotein showed a similar temporal pattern, levels did not decrease as greatly and subsequently increased sooner than did levels for P0 and myelin basic protein. The coordinate alterations in message levels for myelin proteins indicate that Schwann cells can down-regulate and then up-regulate the synthesis of myelin in response to alterations in the supply of membrane components.

Effects of thiamine deficiency on thiamine-dependent enzymes in regions of the brain of pregnant rats and their offspring

Thiamine needs in pregnancy and lactation are known to be increased. Previous studies suggest that developing rat brain is more susceptible to a lack of thiamine intake than is adult brain. In order to study the basis of this susceptibility, activities of the three thiamine-dependent enzymes [pyruvate dehydrogenase complex (PDHC), alpha-ketoglutarate dehydrogenase (alpha KGDH), and transketolase (TK)] were measured in homogenates of brain tissue from thiamine-deficient female rats and their offspring. The study revealed a more rapid progression of thiamine deficiency in pregnant thiamine-deficient rats compared to nonpregnant rats as seen by significantly increased "TPP effect" values. No differences in activities of the three thiamine-dependent enzymes in brain were observed between pregnant and nonpregnant animals. However, activities of all three thiamine-dependent enzymes were significantly reduced in cerebral cortex of the offspring of thiamine-deficient mothers 13 days postnatally. TK activities were also reduced in cerebellum and brain stem of these animals. Since thiamine-dependent enzymes are important for the establishment of adult patterns of cerebral energy metabolism and also in myelin synthesis, maternal thiamine deficiency resulting in reductions of activities of these enzymes at a vulnerable period in brain development could have serious metabolic consequences leading to permanent neurological sequellae in the offspring.

Immunoglobulins promote remyelination in the central nervous system

Remyelination of central nervous system axons was promoted by systemic injection of serum immunoglobulin G (IgG) from donor mice hyperimmunized with homogenized spinal cord. In SJL/J mice infected with Theiler's virus, primary demyelination and inflammation in the spinal cord were extensive. Following treatment with IgG to spinal cord homogenate, new myelin synthesis as measured by quantitative morphometry increased sixfold. Cells in areas of remyelination that incorporated [3H]thymidine did not express differentiation markers of macrophages, oligodendrocytes, or astrocytes but were identified by electron microscopic autoradiography as progenitor glial cells. These findings raise the possibility that IgG secreted in demyelinating lesions might have the potential to promote myelin repair.

Conservative amino acid substitution in the myelin proteolipid protein of jimpymsd mice

The capacity for synthesizing and maintaining a compact myelin sheath is destroyed in a number of inborn errors of myelin metabolism. One class of hypomyelinating mutations, which displays an X-linked pattern of inheritance, is distinguished by marked disturbances in oligodendrocyte differentiation. We have defined the molecular defect in one such mutant that lacks mature oligodendrocytes, the X-linked jimpy myelin synthesis deficient (jpmsd) trait in mice. The structure of the gene encoding the most abundant myelin protein, proteolipid protein (PLP), was determined by mapping and partially sequencing genomic clones from jpmsd and wild-type mice. Jpmsd mice have a single base change in PLP, a C----T transition in exon 6 that would substitute a valine for alanine in both PLP and its alternatively spliced isoform, DM20. The mutation was confirmed by polymerase chain reaction-amplifying exon 6 from genomic DNA and then either sequencing the amplified DNA or directly probing exon 6 with oligonucleotides designed to detect a single base mismatch. The conservative amino acid replacement in PLP/DM20 of jpmsd mice results in a pleiotropic phenotype similar to that observed for the allelic mutation jimpy, in which a splicing defect has radically altered the PLP/DM20 protein. The accelerated turnover of oligodendrocytes in both mouse mutants suggests a function for PLP/DM20 in oligodendrocyte differentiation distinct from the role of these proteolipid proteins as structural components of the myelin sheath.

The initial events in myelin synthesis: orientation of proteolipid protein in the plasma membrane of cultured oligodendrocytes.

Proteolipid protein (PLP) is the most abundant transmembrane protein in myelin of the central nervous system. Conflicting models of PLP topology have been generated by computer predictions based on its primary sequence and experiments with purified myelin. We have examined the initial events in myelin synthesis, including the insertion and orientation of PLP in the plasma membrane, in rat oligodendrocytes which express PLP and the other myelin-specific proteins when cultured without neurons (Dubois-Dalcq, M., T. Behar, L. Hudson, and R. A. Lazzarini. 1986. J. Cell Biol. 102:384-392). These cells, identified by the presence of surface galactocerebroside, the major myelin glycolipid, were stained with six anti-peptide antibodies directed against hydrophilic or short hydrophobic sequences of PLP. Five of these anti-peptide antibodies specifically stained living oligodendrocytes. Staining was only seen approximately 10 d after PLP was first detected in the cytoplasm of fixed and permeabilized cells, suggesting that PLP is slowly transported from the RER to the cell surface. The presence of PLP domains on the extracellular surface was also confirmed by cleavage of such domains with proteases and by antibody-dependent complement-mediated lysis of living oligodendrocytes. Our results indicate that PLP has only two transmembrane domains and that the great majority of the protein, including its amino and carboxy termini, is located on the extracellular face of the oligodendrocyte plasma membrane. This disposition of the PLP molecule suggests that homophilic interactions between PLP molecules of apposed extracellular faces may mediate compaction of adjacent bilayers in the myelin sheath.

Effects of maternal thiamine deficiency on the development of thiamine-dependent enzymes in regions of the rat brain

Previous studies suggest that developing rat brain is susceptible to reduced thiamine intake. In order to assess the metabolic basis for this susceptibility, activities of three thiamine-dependent enzymes (pyruvate dehydrogenase complex, ?-ketoglutarate dehydrogenase and transketolase) were measured in homogenates of brain tissue from the offspring of thiamine-deficient mothers. Control groups of animals were pair-fed to equal food consumption with the thiamine-deficient animals. The study revealed region-selective delays in the establishment of adult activities of thiamine-dependent enzymes as a result of maternal thiamine deficiency. Pyruvate dehydrogenase complex activities in cerebral cortex were significantly reduced (by 20% P < 0.05); ?-ketoglutarate dehydrogenase activities were also reduced in cerebral cortex (by 30% P < 0.05). In the case of transketolase, enzyme activities were significantly reduced in cerebral cortex, cerebellum and brainstem. Following thiamine replenishment, defective enzyme activities were restored to normal in all cases. However, since thiamine-dependent enzymes are important for the establishment of adult patterns of cerebral energy metabolism and also in myelin synthesis, maternal thiamine deficiency resulting in reductions of thiamine-dependent enzymes at a vulnerable period in brain development could have serious metabolic consequences leading to permanent neurological sequellae in the offspring.

RGD-containing peptides inhibit the synthesis of myelin-like membrane by cultured oligodendrocytes.

A synthetic peptide derived from the fibronectin cell-binding domain, GRGDSP, inhibits the adhesion of rat oligodendrocytes to a number of substrates. However, while GRGDSP inhibited the adhesion of cells in a short term adhesion assay, the presence of the peptide did not prevent cells from adhering and thriving in longer term culture. The morphological characteristics of individual cells cultured with 0.1 mg/ml GRGDSP were similar to untreated cultures; small rounded cell bodies radiating numerous fine processes. Peptide-treated cultures were inhibited in their ability to produce myelin specific components. The characteristic developmental peak in sulfolipid synthesis which occurs both in vivo and in vitro was completely inhibited when cells were cultured with GRGDSP. In addition, the synthesis of myelin basic protein was inhibited. Ultrastructurally, cells treated with GRGDSP showed a greatly reduced number of multilamellar myelin-like membrane figures than cells grown without peptide or those grown with GRADSP. Cultured oligodendrocytes did not become sensitive to inhibition of sulfolipid synthesis by GRGDSP until a period immediately preceding the peak in sulfolipid biosynthesis. The effects of pretreatment with peptide for 5 d before this time were completely reversible. Pretreatment which extended into the time of peak myelin synthesis resulted in permanent impairment in the cell's ability to synthesize sulfolipid. The oligodendrocyte's ability to synthesize a myelin-like membrane in culture is, in part, inherent since it occurs in the absence of neurons. The present results indicate that myelin membrane production is also subject to external control since it appears that occupancy of an RGD-dependent cell surface receptor during a critical period of in vitro development is required for the oligodendrocyte to produce myelin-like membrane.

Myelin Proteolipid Protein Gene Expression in Jimpy and Jimpymsd Mice

Proteolipid protein (PLP) gene expression was studied in the dysmyelinating mouse mutant jimpy(msd) (jpmsd; myelin synthesis deficient) and compared with that in wild-type mice and the allelic mutant, jimpy (jp). Southern analyses of genomic DNA from jpmsd mice revealed no major rearrangements of the PLP gene relative to the wild-type mouse PLP gene. PLP-specific mRNA levels were significantly reduced in these mutant mice, although both the 3.2- and 2.4-kilobase PLP-specific mRNAs were seen. Also, no size differences in either PLP or DM20 mRNAs were found by S1 nuclease assays of brain RNA from either jpmsd or wild-type mice. Both PLP and DM20 protein were detectable at low levels in jpmsd brain homogenates, and these proteins comigrated with PLP and DM20 protein from normal mice. Western analyses showed an altered PLP:DM20 ratio in jpmsd mice relative to wild-type mice; DM20 levels exceeded PLP levels. It is surprising that a similar pattern of expression was seen in normal mice at less than 10 days of age: DM20 protein expression preceding PLP expression. Thus, jpmsd mice are capable of synthesizing normal PLP and DM20 protein; however, the PLP gene defect has affected the normal developmental pattern of expression for these two proteins.

Histopathologic and biochemical analysis of classic Pelizaeus-Merzbacher disease

An autopsy study of an 11-year-old boy with the classic type of Pelizaeus-Merzbacher disease is presented. He developed normally until 5 years of age when he began to deteriorate with scanning speech and gait abnormality. Auditory brainstem responses were normally preserved. At the age of 11 years, 8 months, he died of pneumonia while in a vegetative state. The neuropathologic findings suggested a typical classic type of Pelizaeus-Merzbacher disease and biochemical analysis of cerebral white matter demonstrated a decreased ratio of long-chain fatty acids (≥ C19) to short- and mediumchain fatty acids (< C19). These findings suggested defective myelin synthesis as the etiology of Pelizaeus-Merzbacher disease; comprehensive classifications of the disease are expected to include both pathologic and biochemical parameters.

Heterosis for myelin content is limited to the central nervous system.

We have recently described a murine model for studying aspects of myelination (Seyfried and Yu, 1980; Ebato et al., 1983; Miskimins et al., 1986). This mouse shows hypermyelination during the period of most active synthesis of myelin, 9 to 21 days post-natal. The myelin parameters showing an increase were all measured in the central nervous system. We investigated here whether this effect extends into the peripheral nervous system. Our results indicate that the hypermyelination is limited to the central nervous system.

Reduction by lead of hydrocortisone-induced glycerol phosphate dehydrogenase activity in cultured rat oligodendroglia.

Time- and dose-dependent toxic effects of lead (Pb) acetate on astroglia, oligodendroglia, and meningeal fibroblasts cultured from immature rat brain were measured. Cultures were exposed for 3 d to Pb (1, 10, and 100 microM) and then examined immediately (Day 0) or 3 or 10 d after Pb treatment was discontinued. The percentages of astroglia and fibroblasts excluding dye were unaffected by Pb, whereas the percentage of oligodendroglia excluding dye decreased significantly (P less than 0.01) at all time points after exposure to 100 microM Pb. Lead (100 microM) also reduced the total cell numbers of astroglia, oligodendroglia, and meningeal fibroblasts. Amino acid incorporation into protein by oligodendroglia was stimulated after exposure to 100 microM Pb at all time points and also by 1 and 10 microM on Day 3. Incorporation was stimulated in astroglia only on Day 0 by 10 and 100 microM. Hydrocortisone-stimulated glycerolphosphate dehydrogenase (GPDH) activity was assayed in oligodendroglia cultures. A significant decrease in specific activity was seen after a 4-d exposure to lead. Because oligodendroglia are responsible for myelin synthesis in the central nervous system, and GPDH may synthesize a precursor for myelin lipid synthesis, it was proposed that the hypomyelination observed in lead-intoxicated neonatal rats may result partially from a primary toxic effect on oligodendroglia. GPDH activity was not inhibited by Pb in mixed glial cultures containing both astroglia and oligodendroglia. This result suggests that astroglia in culture have the ability to delay the lead-induced inhibition of oligodendroglial GPDH activity and supports the hypothesis that astroglia in culture serve a protective function.

Myelinated, but not unmyelinated axons, reversibly down-regulate N-CAM in Schwann cells.

There is evidence from chicks and mice that N-CAM expression in Schwann cells is subject to significant regulation during development and following injury. In the present work, rat sciatic nerve and immunohistochemical methods have been used to study developmental and injury-related modulation of N-CAM in Schwann cells, using cell type specific markers to identify different Schwann cell populations, and cell counting to quantify their size. The study has sought to determine unambiguously whether immature Schwann cells in developing nerves and denervated Schwann cells in injured adult nerves express surface N-CAM, and has investigated the temporal relationship between the gradual loss of surface N-CAM and the differentiation of myelin-forming Schwann cells, monitored by the sequential appearance of the glycolipid galactocerebroside and the myelin-specific protein P0. Further points examined are whether this down-regulation of N-CAM is rapidly reversible following loss of axonal contact, and whether N-CAM reappearance in Schwann cells depends on protein synthesis. In nerves from 17- to 18-day embryos, 90% of the Schwann cells, identified with Ran-1 antibodies, expressed surface N-CAM. In nerves from newborn rats many cells are in the early stage of myelin synthesis and therefore express galactocerebroside, although they have not yet acquired P0. Suspension staining of dissociated cells from this nerve showed that 92% of the galactocerebroside-positive cells were also N-CAM positive. In suspension staining of nerves from 5-day, 10-day and adult rats, P0-positive cells were essentially N-CAM negative.(ABSTRACT TRUNCATED AT 250 WORDS)