Long term culture of bovine oligodendroglia isolated with a percoll gradient

Oligodendroglia were isolated from calf central nervous system (CNS) white matter by trypsinization in phosphate buffered saline and separation by centrifugation through Percoll. Using antisera to phenotypic markers and double labelling experiments we were able to identify essentially all cells in the cultures. The cells obtained were: (1) viable; (2) had intact plasma membranes and well preserved organelles, ribosomes and mitochondria; and (3) were greater than or equal to 95% oligodendroglia 16-20 h after isolation as determined by ability to bind antigalactocerebroside antibodies (anti-GalC). Oligodendroglia could be cultured for several weeks to months. Oligodendroglia established and maintained processes which bound anti-GalC. Myelin basic protein could be demonstrated in the cytoplasm of 40-60% of oligodendroglia cell bodies but not in the processes.     

OLN-93: A new permanent oligodendroglia cell line derived from primary rat brain glial cultures

We have established a new permanent cell line (OLN-93), derived from spontaneously transformed cells in primary rat brain glial cultures. In growth medium supplemented with 10% fetal calf serum a doubling time of 16–18 hr was determined. OLN-93 cells in their antigenic properties resemble primary oligodendrocytes in culture. As analyzed by indirect immunofluorescence, the A2B5 surface marker is absent, they express galactocerebroside and myelin-specific proteins, such as myelin basic protein (MBP), myelin-associated glycoprotein (MAG), proteolipidprotein (PLP), and Wolfgram protein (WP), but do not exhibit astrocytic properties, such as the expression of vimentin or the glial fibrillary acidic protein (GFAP). In their morphological features they resemble bipolar O-2A-progenitor cells and, when grown at low density or on poly-L-lysine-coated culture dishes under low serum conditions, immature oligodendrocytes with a more arborized cell morphology. The cellular processes contain microfilaments, while N-CAM/D2 immunoreactivity is localized on the cell surface of the somata and processes. Immunoblot analysis further confirmed the presence of MAG, WP and MBP immunoreactivity, and the absence of vimentin and GFAP. Only a single MBP isoform (∼14 kDa) was detectable in the cellular extracts. PLP mRNA expression was studied by RT-PCR. The two proteolipid-specific mRNAs, DM20 and PLP, were present in OLN-93 cell extracts. Comparisons with embryonic rat cerebral cells in culture and primary oligodendrocytes suggest that OLN-93 cells in their morphological features and their antigenic properties resemble 5- to 10-day-old (postnatal time) cultured rat brain oligodendrocytes. Thus, the new cell line described in this study should provide a useful model system to investigate the specific mechanisms regulating the proliferation and differentiation of oligodendrocytes in vitro, and the molecular interactions with other cells of the nervous system. © 1996 Wiley-Liss, Inc.     

Purification and characterization of cultures of oligodendroglia from rat brain

It is possible to purify small dark cells from rat brain which by ultrastructural criteria are oligodendroglia. The modified technique involves the use of an isolation medium less damaging to the initial cell suspension and then differential plating of a stratified primary cell culture obtained from 1-3-day-old rat brain which has been described. Five percent serum encourages the growth of oligodendroglia. In this system, the oligodendroglia can be harvested several times from the primary cultures. To retain the homogeneous populations of oligodendroglia, it is necessary to constantly manipulate the cultures by hitting the flasks and replating the suspended cells. Electron microscopy reveals that the small dark cells are differentiated oligodendroglia with prominent microtubules. Immunofluorescence staining of the cells shows that the majority of the cells are negative for known surface markers which include cerebrosides and 04 antigen (markers for oligodendroglia), glial fibrillary acidic protein, and fibronectin. Interestingly, it is possible to increase the number of 04-positive cells by the addition of dibutyryl cyclic AMP to the medium. Rat oligodendroglia are able to incorporate about 20% of the radioactivity into cerebrosides, using the substrate, [3H]galactose; this is a biochemical feature unique to oligodendroglia. The expression of all the oligodendroglial antigens should be remarkably enhanced when the cells are induced to produce myelin.     

Expression of antigenic markers during the development of oligodendrocytes in mouse brain cell cultures

The expression of myelin basic protein (MBP) and galactocerebroside (GC), two antigenic markers for oligodendrocytes, was chekced on 7-, 14-, 21- and 28-day-old dissociated mouse brain cell cultures (BCC) by using the indirect immunofluorescence method with double staining.

The number of GC positive cells increased between the 7th and the 14th day culture before a steady state was reached. In contrast to this, the MBP-positive cells appeared only on the 14th day oulture, and their number increased with the age of the culture. In double staining, the serum produced against isolated oligodendrocytes shows the same picture as the anti-GC serum, while only a part of GC-positive cells showed also the presence of MBP. Our data suggest that the GC appears very early on the membrane of the oligodendrocytes during development while cells exhibiting both GC and MBP probably represent a more differentiated oligodendrocytes population.     

Biosynthesis and expression of the myelin-associated glycoprotein in cultured oligodendrocytes from adult bovine brain.

The biosynthesis and expression of myelin-associated glycoprotein (MAG) were investigated in cultured oligodendrocytes isolated from adult bovine brain. Western blotting revealed two prominent MAG bands that were present in comparable amounts; the larger component electrophoresed above the 97 kD standard but was slightly smaller than the MAG band in purified bovine myelin, and the smaller component electrophoresed below the 97 kD standard. In comparison to other precursors of oligosaccharides, inorganic [35S]sulfate was a relatively specific isotope for labeling MAG relative to other glycoproteins in the cells. Sulfate labeled only the larger of the two MAG components, which contains complex N-linked oligosaccharides, but which appears to be glycosylated to a lesser extent than MAG in vivo. The smaller MAG band in the cells is a form with high-mannose oligosaccharides and was not detected in purified bovine myelin. Both the large and small MAG components were expressed on the oligodendrocyte surface as indicated by their sensitivity to neuraminidase and/or trypsin treatment of live cells. MAG was also released by the oligodendrocytes into the culture medium. The MAG in the medium was slightly smaller than that in the cells, suggesting that it may be released from the cell surface by limited proteolysis. The release of MAG by myelin-forming cells could be relevant to physiological roles that have been postulated for soluble forms of MAG and other adhesion proteins.     

In vitro changes in the fine structure and protein composition of light myelin fractions isolated from guinea pig brain

To find out if in vitro maintenance produces changes in the electron microscopic appearance, protein composition and phosphorylation properties of guinea pig CNS myelin fractions, we incubated them for 10 min, 4 hr, 24 hr, and 48 hr in phosphate-buffered saline (pH 7.4) or in 20 mM Hepes, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM dithiothreitol, and 20 mM NaCl at 4 and 30 degree C. Aliquots were processed for electron microscopic study, were analyzed for protein content by gel electrophoresis, and were assayed for endogenous protein phosphorylation. Before incubation, electron micrographs of fractions contained two types of multilamellar whorls with the periodicity of CNS myelin sheaths. The first type of whorl was separated from nearby whorls; the other type had surface lamellae that were connected to other multilayered membrane fragments. After incubation at 4 degree C for 24 hr, the number of both types of multilamellar whorls in micrographs had increased approximately 3- to 4- fold. Counts per unit area showed that the observed increase was both time- and temperature-dependent. In aliquots studied by gel electrophoresis, only minor degradation of myelin proteins was observed. The endogenous protein phosphorylation properties of the myelin fragments also remained functional, suggesting that the activities of protein phosphotransferases were not altered. We conclude that the incubation conditions described here favor interactions of proteins and lipids that lead to the formation of multilayered aggregates of CNS myelin membranes.     

Isolation, culture, and characterization of adult rat oligodendrocytes.

Investigation into CNS demyelinating diseases, which usually occur in adults, can be facilitated by the use of a good in vitro model. We have established a methodology whereby oligodendrocytes from adult rat CNS can be cultured in vitro, and we have characterized these cultures morphologically and immunologically. Approximately 1 g of spinal cord and brainstem per adult rat was removed and dissociated mechanically and enzymatically. After filtration of the white matter homogenate, myelin was removed by 0.9 M sucrose density centrifugation. The cells were further purified by centrifugation through a 0.3%/4% discontinuous gradient of bovine serum albumin (BSA). The pellet was resuspended and placed in an untreated 6-well culture dish overnight to allow the astrocytes to attach. The non-adherent cells were replated on poly-l-lysine-treated coverslips. Approximately 8.25 x 10(5) cells were recovered per animal. The adult oligodendrocytes initially appeared as rounded cell bodies, but after 2-5 days in vitro (DIV), the oligodendrocytes extended 6-10 thick processes. A membrane sheath between these processes was immunostainable with either anti-galactocerebroside (GC), anti-O4, anti-myelin basic protein (MBP), or anti-2'3' cyclic nucleotide 3' phosphohydrolase (CNPase) and was also evident in scanning EM. Older cultures (up to 60 DIV) maintained whorls of myelin and transmission EM revealed a major dense line distance of approximately 103 A with up to 11 concentric layers of membrane. Immunologically, the adult oligodendrocytes are GC+, O4+, MBP+, CNPase+, and GFAP-. The method described will allow adult rat oligodendrocytes to be isolated and maintained in culture; these cultures retain the characteristics of differentiated adult oligodendrocytes.     

Enrichment of Schwann cell cultures from neonatal rat sciatic nerve by differential adhesion

A novel method of Schwann cell purification from neonatal rat sciatic nerve has been developed using differential adhesion. After enzymatic and mechanical dissociation, the cell digest is allowed to settle on polylysine-coated glass coverslips for 30 min with intermittent shaking. After an 18-h incubation, bipolar cells comprise 95% of the non-adherent population. Indirect immunofluorescence with the cell-specific markers rabbit anti-galactocerebroside and rabbit anti-bovinr-P-2 basic protein antiserum confirmed light microscopic identification of these bipolar cells as Schwann cells. Rabbit anti-human fibronectin specifically labeled fibroblasts which comprised< 5% of the cell population, but did not bind to Schwann cells. Schwann cells isolated by differential adhesion were injected into a rabbit. When absorbed with cultured rat skin fibroblasts, serum from this rabbit specifically surface labeled 99% of the bipolar and round cells after 18 h and 5 days in vitro and also labeled Schwann cells in fetal rat dorsal root ganglia cultures, but not fibroblasts or neurons.     

Characterization of adult human astrocytes derived from explant culture

Four different human astrocytic cell lines established from either epilepsy surgical specimens or cerebral white matter obtained during thalamotomy for tremor in a patient with multiple sclerosis were characterized using morphologic analysis, ultrastructural attributes, growth characteristics, and immunocytochemical analysis. Immunocytochemical characterization of cultures indicated a mean of 84% of cells contained cytoplasmic glial fibrillary acidic protein (GFAP): to confirm that GFAP(+) cells also proliferated, bromo-deoxyuridine (BrdU) uptake was measured in cell line. Our method of simplified explant culture allows establishment of astrocytic cell lines from a variety of pathologic substrates using limited amounts of human material.     

Immunohistochemical localization of citrullinated proteins in adult rat brain

By using hybridoma technology, an IgM monoclonal antibody (F95) against multiple citrullinated synthetic and natural peptides was recently developed and used to stain immunohistochemically subsets of astrocytes and myelin basic protein (MBP) from selected regions of human brain (Nicholas and Whitaker [2002] Glia 37:328-336). With this antibody, the present study provides a more detailed localization of citrullinated epitopes in the central nervous system (CNS) by examining immunohistochemical staining patterns for F95 in the normal adult rat brain. Thus, immunohistochemical labeling for citrullinated epitopes was seen in white matter areas consistent with myelin staining; however, in general, it was more prominent and uniform in the caudal CNS (spinal cord, medulla oblongata, pons, and cerebellum) than in more rostral areas. F95 staining was also seen in cells and fibers often intimately associated with blood vessels and/or ventricular surfaces. By using dual-color immunofluorescence, the vast majority of this latter staining was colocalized within a subset of astrocytes also immunoreactive for glial fibrillary acidic protein (GFAP). By using Western blot analysis of rat brain proteins, multiple GFAP- and MBP-immunoreactive proteins and peptide fragments were seen, and many of them were also reactive with the F95 antibody. Thus, the present study not only demonstrates that citrullinated epitopes in normal rat brain are most concentrated in subsets of myelin and astrocytes but also provides evidence that GFAP, like MBP, may be present as multiple citrullinated isoforms.     

Immunohistochemical study of myelin sheaths formed by oligodendrocytes interacting with dissociated dorsal root ganglion neurons in culture

The addition of central nervous system (CNS) glial cells to dissociated networks of rat dorsal root ganglion neurons in tissue culture provided a useful system for the study of CNS myelin sheath formation. The CNS myelin basic proteins (BP) and proteolipid protein (PLP) were demonstrable in these cultures by immunoperoxidase techniques. Both BP and PLP were detectable in myelinating oligodendrocytes and CNS myelin sheaths. Anti-BP serum and anti-PLP serum were useful immunohistochemical staining reagents for the identification of myelinating oligodendrocytes and CNS myelin sheaths in tissue culture.     

The impact of erythropoietin and iron status on brain myelination in the newborn rat

Erythropoietin (Epo) drives iron (Fe) utilization for erythropoiesis, but the potentially resultant tissue iron deficiency (ID) can also impede brain development. Conversely, Epo binds to Epo receptors (EpoR) on immature brain oligodendrocytes and neurons, promoting growth and differentiation. The objective of the study was to examine the interaction between Epo and Fe on myelination in brain development during daily Epo treatment. Male and female Sprague‐Dawley rats from postnatal day (P) P4‐P12 modeled premature newborns. Dam‐fed Fe‐sufficient (IS) or postnatal ID groups were given daily subcutaneous sham or erythropoietic Epo injections (425 U^.kg^?1.d^?1), ± oral Fe (6 mg^.kg^?1.d^?1). Tissues and blood were collected and studied at P12. Epo in the ID groups, in the absence of oral Fe, stimulated microcytic ID anemia along with raising inflammatory markers. Both the microcytic anemia and inflammation improved in the ID + Epo + Fe group. Fe treatment positively impacted erythropoiesis and body Fe (µg/g) in all groups. Relative brain Fe (µg/g rat) was improved in the IS + Epo + Fe group. Brain Fe was not worsened in +Epo groups. Brain weight and brain Fe were related to plasma Epo levels. Amount of myelination was impacted by feeding type, but was not inhibited by Epo. Expression of a protein in myelin, mylein basic protein, was greater in all +Fe groups than –Fe groups. With therapeutic Epo, available body Fe was prioritized for erythropoiesis instead of brain, but Epo did not worsen brain Fe and potentially Epo improved myelination and maturation in the brain.     

Noncoordinate regulation of myelinogenic parameters in primary cultures of dissociated fetal rat brain

The developmental regulation of sulfogalactosylceramide (sulfatide) synthesis, 2′,3′-cyclic nucleotide-3′-phosphohydrolase activity (CNP), and myelin basic protein (MBP) accumulation, three markers characteristic of myelinogenesis, were observed in dispersed cultures of fetal rat brain in spite of the absence of the formation of compact, multilamellar myelin. Sulfatide synthetic rate and CNP activity began to increase by 8 days in vitro (DIV) (approximately comparable to days post-natal) and reached their maxima by 20 DIV. MBP began to accumulate after 14 DIV and reached a maximum by 38 DIV. Thus, the temporal regulation of the onset of expression of these parameters, which is coordinate in vivo, has been dissociated into two sequential periods in vitro. Similarly, the regulatory mechanisms controlling the subsequent decline of the net expression of these three parameters were dissociated. Whereas, the increase in the net CNP activity ceased on schedule, the decline of sulfatide synthetic rate was delayed 20 days, and the accumulation of MBP underwent a net loss. These data suggest that there are multiple parallel but separate mechanisms of temporal regulation controlling myelinogenic gene expression, and that, among them, those factors that control MBP accumulation are more sensitive to disruption by the rigors of dissociated culture.     

Establishment of human adult astrocyte cultures derived from postmortem multiple sclerosis and control brain and spinal cord regions: immunophenotypical and functional characterization

We have successfully established highly enriched astrocyte cultures upon passaging of primary cultures derived from various regions of postmortem human adult brain and spinal cord. Tissues were collected at autopsies with relatively short postmortem times (3–9 hr) from multiple sclerosis (MS) and (normal) control cases. Immunocytochemical analysis showed that primary cultures were composed of colonies of oligoclonal cells that expressed the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin, as well as glutamine synthetase (GS). Passaging the astrocytes did not affect their proliferating capacity as monitored by bromodeoxyuridine (BrdU) incorporation. Astrocyte-specific markers were stably expressed for at least 12 passages per individual tissue sample. Large numbers of GFAP-positive astrocytes were obtained from each sample and could be stored frozen and recultured. Very few macrophages/microglial cells (1–3%) were present in the human adult astrocyte cultures, using a panel of macrophage-specific markers. However, the monoclonal antibodies (mAbs KP1, EBM11, 25F9) and lysozyme antiserum directed against lysosomal antigens strongly immunostained cultured astrocytes derived from MS and control cases, implicating that expression of these lysosomal antigens is not restricted to macrophages/microglial cells in human glial cell cultures. Interestingly, astrocytes derived from active demyelinated MS lesions showed an increased proliferating capacity compared to astrocytes derived from non-lesioned and normal brain and spinal cord regions, as shown with a microculture tetrazolium assay (MTT assay). J. Neurosci. Res. 49:342–354, 1997. © 1997 Wiley-Liss, Inc.     

Long‐term depression of synaptic transmission in the adult mouse insular cortex in vitro

The insular cortex (IC) is known to play important roles in higher brain functions such as memory and pain. Activity‐dependent long‐term depression (LTD) is a major form of synaptic plasticity related to memory and chronic pain. Previous studies of LTD have mainly focused on the hippocampus, and no study in the IC has been reported. In this study, using a 64‐channel recording system, we show for the first time that repetitive low‐frequency stimulation (LFS) can elicit frequency‐dependent LTD of glutamate receptor‐mediated excitatory synaptic transmission in both superficial and deep layers of the IC of adult mice. The induction of LTD in the IC required activation of the N‐methyl‐d ‐aspartate (NMDA) receptor, metabotropic glutamate receptor (mGluR)5, and L‐type voltage‐gated calcium channel. Protein phosphatase 1/2A and endocannabinoid signaling are also critical for the induction of LTD. In contrast, inhibiting protein kinase C, protein kinase A, protein kinase Mζ or calcium/calmodulin‐dependent protein kinase II did not affect LFS‐evoked LTD in the IC. Bath application of the group I mGluR agonist (RS)‐3,5‐dihydroxyphenylglycine produced another form of LTD in the IC, which was NMDA receptor‐independent and could not be occluded by LFS‐induced LTD. Our studies have characterised the basic mechanisms of LTD in the IC at the network level, and suggest that two different forms of LTD may co‐exist in the same population of IC synapses.     

Trembler mouse Schwann cells in culture: Anomalies in the synthesis of lipids and proteins

Nerve growth factor (NGF) mRNA is widely distributed throughout peripheral and central rat tissues and throughout the human central nervous system. In the rat, high levels were found in cerebral cortex, hippocampus and thalamus/hypothalamus, medium levels in striatum and brainstem and low levels in cerebellum and spinal cord. The hippocampal levels did not change following the surgical transection of the septohippocampal pathway; similarly, the ibotenic acid-induced lesion of the nucleus basalis magnocellularis did not affect the amounts of NGF mRNA in the cerebral cortex. NGF mRNA was also present in high amounts in human cortex and hippocampus, with only low levels in septum/nucleus basalis magnocellularis, suggesting that NGF may also function as a retrograde trophic messenger in the human central nervous system. No evidence was obtained for an insufficient production of NGF mRNA in senile dementia of the Alzheimer type. In peripheral rat tissues, the highest concentrations of NGF mRNA were found in vas deferens, heart, sciatic nerve, submandibular gland and skin, with low levels in tissues such as trigeminal ganglion and pituitary gland.     

In bovine brain extracts,RNAs are active factors that stimulate the proliferation of chick neuroblasts in culture

Extract was prepared from bovine brain by treatment with chloroform-methanol, drying of the precipitate, and successive extractions of the powder with Tris buffer. Influence of this extract on neuroblast proliferation in culture was tested. Neuroblast cultures were prepared from brain hemispheres of 6-day-old chick embryos. Cell proliferation was quantified by incorporation of ³-thymidine. The brain extract was able to stimulate the proliferation of the neuroblasts in culture. In order to purify the factor responsible of this effect, the brain extract was subjected to diethylaminoethyl (ENAE)-cellulose chromatography and gel filtrations. By UV spectrography and destruction of the activity with RNAase and KOH, it was shown that in the extract RNAs are responsible for at least a part of the effect on the proliferation of neuroblasts in culture. Brain RNA is not specific for this effect, since mouse ribosomal RNA was also active. The effect could be actually elicited by some nucleotides.     

大鼠胎脑皮质神经干细胞体外培养及诱导分化的实验研究

目的 从孕龄15d SD胚胎鼠脑皮质中分离并培养神经干细胞（neural stem cells。NSCs），观察其生长、增殖及分化。方法 采用包含碱性成纤维细胞生长因子（bFGF）和表皮细胞生长因子（EGF）的无血清培养及单细胞克隆技术，对胚胎鼠脑皮质神经干细胞进行原代、传代培养及诱导其分化。用Nestin染色鉴定神经干细胞特性，用免疫组化方法（β-Ⅲ-tubulin、GFAP染色）检测神经干细胞分化为神经元及神经胶质细胞状况。结果 从孕龄15dSD胚胎鼠脑皮质中分离的组织，经原代及传代培养均可形成细胞克隆．切具有增殖能力。原代及传代培养细胞呈Nestin（神经上皮干细胞蛋白）表达阳性．诱导分化后的细胞表达神经元细胞、星形胶质细胞的特异性抗原。结论 本实验分离、培养的孕龄15dSD胚胎鼠脑皮质细胞Nestin表达阳性．分化后表达神经元和星形胶质细胞的标记物，是大鼠的神经干细胞，并具有多向分化潜能。     

Neurogenesis in explants from the walls of the lateral ventricle of adult bovine brain: role of endogenous IGF-1 as a survival factor

Previous studies have shown the existence of proliferating cells in explants from bovine (Bos Taurus) lateral ventricle walls that were maintained for several days in vitro in the absence of serum and growth factors. In this study we have characterized the nature of new cells and have assessed whether the insulin-like growth factor-1 (IGF-1) receptor regulates their survival and/or proliferation. The explants were composed of the ependymal layer and attached subependymal cells. Ependymal cells in culture were labelled with glial markers (S-100, vimentin, GFAP, BLBP, 3A7 and 3CB2) and did not incorporate bromodeoxiuridine when this molecule was added to the culture media. Most subependymal cells were immunoreactive for beta III-tubulin, a neuronal marker, and did incorporate bromodeoxiuridine. Subependymal neurons displayed immunoreactivity for IGF-1 and its receptor and expressed IGF-1 mRNA, indicating that IGF-1 is produced in the explants and may act on new neurons. Addition to the culture media of an IGF-1 receptor antagonist, the peptide JB1, did not affect the incorporation of bromodeoxiuridine to proliferating subependymal cells. However, JB1 significantly increased the number of TUNEL positive cells in the subependymal zone, suggesting that IGF-1 receptor is involved in the survival of subependymal neurons. In conclusion, these findings indicate that neurogenesis is maintained in explants from the lateral cerebral ventricle of adult bovine brains and that IGF-1 is locally produced in the explants and may regulate the survival of the proliferating neurons.     

Distribution of myelin-associated enzymes and myelin proteins into membrane fractions from the brains of adult shiverer and control (+/+) mice

The Shiverer, an autosomal recessive mutation in the mouse, is characterized by a severe deficiency in CNS myelin. The concentrations of the myelin basic and proteolipid proteins in the brain of two-month-old shiverer mice, although high enough to be measured, were much lower than in the control (+/+) brains. In contrast, the specific acitivies of teh myelin-associated enzymes, 2′, 3′-cyclic nucleotide-3′-phosphohydrolase (CNP), 5′-nucleotidase, and carbonic anhydrase, were close to normal in the brains of the mutants. The activities of these enzymes and the concentrations of the myelin large basic and proteolipid proteins were compared in membrane fractions prepared, by differential and density gradient centrifugation, from the brains of shiverer and +/+ control mice. In myelin purified from the brains of shiverer mice the specific activities of 5′-nucleotidase and CNP were close to normal, and the specific activities of all three enzymes were normal in a crude myelin fractions from brains of the mutants. However, in the shi/shi brains abnormally high proportions of the three myelin-associated enzymes were distributed into the P₃ (microsomal) fraction and into membrane fractions denser than myelin. The major myelin proteins, although low in total amounts in the mutants' brains, were distributed into the membrane fractions from control and shiverer brains in relative proportions similar to the relative proportions observed for the three enzymes. Thus, carbonic anhydrase, 5′-nucleotidase and CNP in the brains of shiverer mice are not truly dissociated from the major myelin proteins but are, rather, distributed for the most part into the same populations of membranes as are the residual, small amounts of the myelin basic and proteolipid proteins.