Glial differentiation in dissociated cell cultures of neonatal rat brain: noncoordinate and density-dependent regulation of oligodendroglial enzymes

The mixed glial system of primary cultures of cells dissociated from neonatal rat brain was utilized to study glial differentiation. The investigation was addressed specifically to the possibility of noncoordinate regulation of two manifestations of oligodendroglial differentiation, i.e., activities of glycerol-3-phosphate dehydrogenase (GPDH) and of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), as well as the effects of initial cell density on the time of onset and the intensity of expression of these aspects of oligodendroglial differentiation. Simultaneously, glutamine synthetase activity was studied to determine effects on astrocytic differentiation. GPDH exhibited a major developmental increase in specific activity between 20 and 32 days in culture. However, CNP activity exhibited a major developmental increase that commenced approximately 2 weeks earlier. The onset of these expressions of oligodendroglial differentiation was not affected by such environmental factors as initial cell density. However, the intensity of expression of the temporally separate increases in GPDH and CNP activities was markedly density-dependent. The highest activities were attained in cultures plated at the lowest cell densities. The astrocytic enzyme, glutamine synthetase, also exhibited a striking developmental increase (approximately tenfold between 13 and 30 days in culture), but initial cell density affected neither the time of onset nor the intensity of expression of this aspect of astrocytic differentiation. The data demonstrate a striking developmental increase in GPDH activity that is not coordinate with that in CNP. The noncoordinate manifestations of oligodendroglial differentiation commence as a function of time in culture, whereas the intensity of expression of this differentiation can be influenced by such environmental factors as initial cell density.(ABSTRACT TRUNCATED AT 250 WORDS)     

The distribution of 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in the CNS of normal (+/+) and shiverer (Shi/Shi) mice

Immunohistochemical techniques were utilized to localize the putative myelin enzyme 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in the central nervous system (CNS) of normal (+/+) and Shiverer (Shi/Shi) mice (Mus musculus). CNP appeared to be only associated with myelinated nerve fibers in the CNS (corpus callosum, subcortical white matter, caudate nucleus, cerebellum and medulla oblongata) of +/+ mice. However, little or no immunostaining was observed in the same regions of the CNS of Shi/Shi mice, although these mice have normal levels of a CNS CNP activity. Unexpectedly, oligodendrocytes (cell periphery) were not stained for CNP in either +/+ or Shi/Shi mice, although erythrocytes were immunostained.     

Immunocytochemical localization of glycerol-3-phosphate dehydrogenase in rat oligodendrocytes

In this study, two indirect immunoperoxidase staining procedures were used to investigate the cellular localization of rat brain glycerol-3-phosphate dehydrogenase (EC 1.1.1.8; GPDH). At the light and electron microscopic level, we found that the use of monospecific rabbit antibodies to GPDH consistently resulted in the specific staining of only one glial cell population. GPDH-positive cells in perineuronal, interfascicular and perivascular positions were identified as oligodendrocytes by classical morphological criteria. The specificity of GPDH antigen-antibody reaction was determined by qualitative and quantitative immunochemical methods and by immunocytochemical controls for immunologic and methodologic sources of non-specific reaction product. The illustrative data from this study serve to qualitatively define GPDH as a biochemical marker for oligodendrocytes in rat central nervous tissue. In view of the fact that the synthesis of rat brain GPDH is specifically regulated by glucocorticoids, the positive results obtained in this study further warrant the interpretation that rat oligodendrocytes are target cells for glucocorticoids.     

Biochemical study on the brain of the macular mutant mouse as a model of Menkes' kinky hair disease

The wet weights, contents of DNA, RNA and protein and 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) activities were examined in the cerebrum, brain stem and cerebellum from macular mutant mouse, as a model animal for Menkes' kinky hair disease (MKHD). The DNA, RNA and protein contents in the cerebellum from macular hemizygous males (Ml/y) were affected to a much greater extent than those in the cerebrum and the brain stem. This suggests that the main affected part of the brain in Ml/y mouse is the cerebellum. CNP activities in the Ml/y mouse showed a significant decrease in every part of the brain after day 10. This finding may coincide with the changes in myelination in human MKHD.     

Poor myelination in the central nervous system of "dilute-lethal mutant mice" (d1/d1)

The activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), as a marker for myelinogenesis, was measured in different parts of the nervous system of dilute-lethal mutant mice (d1/d1). The activity in terms of micromoles of 2'-AMP formed per milligram of protein of homogenate per minute was found to be exceedingly reduced in the cerebrum, brain stem, and medulla oblongata, slightly reduced in the cerebellum and cervical spinal cord, but not reduced in the thoracic spinal cord and the optic and sciatic nerves. These results clearly indicate that dilute-lethal mutant mice show poor myelination in the cerebrum, brain stem, and medulla oblongata.     

Effects of dexamethasone on the expression of myelin basic protein, proteolipid protein, and glial fibrillary acidic protein genes in developing rat brain.

Effects of dexamethasone (DEX) on the relative abundance of myelin basic protein (MBP), proteolipid protein (PLP) and glial fibrillary acidic protein (GFAP) mRNAs in the developing rat brain were examined. After DEX (1.0 mg/kg body weight) or saline was administered intraperitoneally to 3-day-old rats for 7 consecutive days, wet weight, DNA content and the relative abundance of the glia-specific mRNAs in cerebrum and cerebellum were analyzed at postnatal days (P) 10, 20 and 30. DEX decreased both wet weight and DNA content in cerebellum more profoundly than in cerebrum. The appearance of MBP, PLP and GFAP mRNAs in cerebellum preceded that in cerebrum in the control group. In cerebrum, the relative abundance of MBP and PLP mRNAs was significantly less in the DEX group than that in the control group at P20 and P30. The relative abundance of the GFAP mRNA was significantly less in the DEX group than in the control group at P10 and P20, but there was no significant difference at P30. In cerebellum, a significant decrease in the abundance of MBP, PLP and GFAP mRNAs in the DEX group was observed only at P10 but not at P20 and P30. Our findings indicate that DEX suppresses expression of genes related to glial functions, especially myelination when administered in the early postnatal period.     

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.     

Immunofluorescence demonstration of 2′:3′-cyclic-nucleotide 3′-phosphodiesterase in cultured oligodendrocytes of mouse, rat, calf and human

Previous biochemical studies have shown that high enzyme activity of 2′:3′-cyclic-nucleotide 3′-phosphodiesterase (CNP) is found in isolated myelin and oligodendrocytes. We report here the specific and intense immunofluorescence staining of cultured oligodendrocytes obtained from the brains of mouse, rat, calf and human by rabbit antiserum specific for purified bovine CNP. Astrocytes and fibroblasts present in the cultures were negative for the immunostaining. The specificity of the CNP immunoreactivity was confirmed by blocking the reaction by prior absorption of the antiserum by purified CNP.     

Gene expression of the central and peripheral nervous system myelin membrane 2', 3'-cyclic nucleotide 3'-phosphodiesterase in development

Determination of gene expression for the 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) during development shows that the amount of translatable RNA encoding this enzyme increases in parallel with myelination in the mouse brain. The two isoforms of this enzyme are developmentally regulated in parallel, in contrast to several other myelin proteins. By comparison, the amount of translatable RNA encoding CNP in the peripheral nervous system (rat sciatic nerve) remains constant during development. The two isoforms that are expressed are identical in apparent molecular weight to rat brain CNP. Moreover, no apparent differences were detected by proteolytic digestion (Cleveland mapping) of CNP 1 from the central or peripheral nervous systems.     

Effects of thyroid deficiency at birth on deoxyribonucleic acid synthesis and deoxythymidine kinase activity in the developing rat brain

In thyroid deficiency at birth, the endogenous pool sizes of cellular dTTP did not change much in the cerebellum, cerebrum and brain stem at the whole ages studied, but the specific radioactivities of dTTP, at 2 hours after the subcutaneous injection of [3H]-thymidine, apparently increased only in the cerebellum on the 14th and 21st days as compared with the normal controls. The highest rate of DNA synthesis in vivo, expressed in terms of the specific radioactivity ratio of DNA to dTTP, was observed at the four-day-old normal rat cerebellum, and in the thyroid deficiency it appeared to shift between seven and 14 days of age. On the other hand, no apparent effects of thyroid deprivation on the rates of both the cerebrum and brain stem were found. The results suggest that a temporal alteration of DNA synthesis as well as thymidine metabolism occurred by thyroid deficiency was confined to the cerebellum in the early postnatal development of rats. Indeed, cerebellar thymidine kinase activity, related to DNA synthesis, also displayed a concomitant delay in thyroid deficiency to the characteristic age-dependence of DNA synthesis. No significant difference between normal and thyroidectomized rats was revealed in this respect on both the cerebrum and brain stem throughout the experimental periods.     

Distribution of freed-serine in vertebrate brains

Freed-serine distribution in vertebrate brains was investigated. In various brain regions of the lower vertebrate species, carp, frog and chick, freed-serine levels were low. On the contrary, in the mammals, mouse, rat and bull, the contents of freed-serine were high in the forebrain (around 400 nmol/g wet weight, and the ratio ofd-serine tol-serine, was d/l = 0.4), and low in the hindbrain. In developing mice,d-serine levels in the cerebrum increased with age and attained the adult level (d/l = 0.40) 8 weeks after birth. In the cerebellum and brain stem, the freed-serine levels increased with age until 2 weeks, followed by a decrease to the adult levels: thed/l ratios remained constant until 2 weeks of age, then decreased to 0.03 in the cerebellum and 0.12 in the brain stem. Freed-serine was shown not to be of microbial origin using germ-free mice. In the rat forebrain,d-serine was evenly distributed in two cerebral regions, namely frontal and occipital lobes. Thed/l ratios in other regions of forebrain, hippocampus and hypothalamus, were comparable to the cerebrum (d/l = 4), while that in the olfactory bulb was lower (d/l = 0.12). In the rat cerebrum, thed-serine content in the grey matter was significantly higher than that in the white matter. The contents of freed-serine in bovine cerebrum and cerebellum were similar to those in other mammalian brains, but thed/l ratio for bovine cerebral grey matter was lower than that for the cerebral white matter. Thed-serine level was discussed in terms ofd-amino-acid oxidase activity.     

Effects of Thyroid Deficiency at Birth on Deoxyribonucleic Acid Synthesis and Deoxythymidine Kinase Activity in the Developing Rat Brain

In thyroid deficiency at birth, the endogenous pool sizes of cellular dTTP did not change much in the cerebellum, cerebrum and brain stem at the whole ages studied, but the specific radioactivities of dTTP, at 2 hours after the subcutaneous injection of [³H]-thymidine, apparently increased only in the cerebellum on the 14th and 21st days as compared with the normal controls. The highest rate of DNA synthesis in vivo, expressed in terms of the specific radioactivity ratio of DNA to dTTP, was observed at the four-day-old normal rat cerebellum, and in the thyroid deficiency it appeared to shift between seven and 14 days of age. On the other hand, no apparent effects of thyroid deprivation on the rates of both the cerebrum and brain stem were found. The results suggest that a temporal alteration of DNA synthesis as well as thymidine metabolism occurred by thyroid deficiency was confined to the cerebellum in the early postnatal development of rats. Indeed, cerebellar thymidine kinase activity, related to DNA synthesis, also displayed a concomitant delay in thyroid deficiency to the characteristic age-dependence of DNA synthesis. No significant difference between normal and thyroidectomized rats was revealed in this respect on both the cerebrum and brain stem throughout the experimental periods.     

Neuropathology of twitcher mice: examination by histochemistry,immunohistochemistry, lectin histochemistry and fourier transform infrared microspectroscopy

The twitcher mouse is an authentic animal model of globoid cell leukodystrophy, which is a genetic disease that affects the lysosomal enzyme galactocerebroside β-galactosidase. This enzyme deficiency causes one of its substrates, galactosylsphingosine (psychosine), to accumulate in myelin-forming cells, which eventually results in their death. In the central nervous system, the death of oligodendrocytes is thought to cause a series of secondary pathological changes. In this study, several techniques were utilized to examine the neuropathology of two different brain regions in the twitcher mouse—the hindbrain and the cerebrum. Neuropathological changes were as follows: (1) demyelination was detected in the hindbrain but not in the cerebrum, (2) a high density of periodic acid-Schiff-positive cells were detected in the hindbrain and to a lesser extent in the cerebrum, (3) astrocyte gliosis was pronounced in both the hindbrain and cerebrum, and (4) macrophages were abundant in both the hindbrain and the cerebrum. We found that Periodic acid-Schiff-positive cells, astrocyte gliosis and macrophage infiltration were present in white and gray matter regions of the cerebrum, while they were generally absent from the granule and molecular layers of the cerebellum. In addition to these studies, we utilized the technique of Fourier transform infrared (FT-IR) microspectroscopy to identify the in situ distribution of psychosine in the brains of twitcher mice. Evidence was obtained that indicates a large accumulation of psychosine in the hindbrain, and to a lesser extent in the white matter of the cerebrum in the twitcher mouse, but not the normal mouse. There was no evidence for the accumulation of psychosine in the molecular layer of the cerebellum from the twitcher or normal mouse. Our conclusions are as follows: (1) pathology is more advanced in the hindbrain compared to the cerebrum, which is likely due to the hindbrain becoming myelinated prior to the cerebrum, (2) demyelination is not necessary for the development of secondary pathological changes, (3) pathology is not limited to white matter in the cerebrum, (4) pathology is not present in all brain regions, i.e. the granule and molecular layers of the cerebellum are devoid of pathological changes, and (5) psychosine accumulates in both the cerebrum and hindbrain, but not in the molecular layer of the cerebellum in the twitcher mouse. This study demonstrates that FT-IR microspectroscopy can be used to correlate chemical changes to histopathological changes in brains from twitcher mice, which suggests that FT-IR microspectroscopy may be a useful tool for studies examining other brain diseases.     

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.     

(+)‐Methamphetamine‐induced monoamine reductions and impaired egocentric learning in adrenalectomized rats is independent of hyperthermia

Methamphetamine (MA) is widely abused and implicated in residual cognitive deficits. In rats, increases in plasma corticosterone and egocentric learning deficits are observed after a 1‐day binge regimen of MA (10 mg/kg × 4 at 2‐h intervals). The purpose of this experiment was to determine if adrenal inactivation during and following MA exposure would attenuate the egocentric learning deficits in the Cincinnati water maze (CWM). In the first experiment, the effects of adrenalectomy (ADX) or sham surgery (SHAM) on MA‐induced neurotoxicity at 72 h were determined. SHAM‐MA animals showed typical patterns of hyperthermia, whereas ADX‐MA animals were normothermic. Both SHAM‐MA‐ and ADX‐MA‐treated animals showed increased neostriatal glial fibrillary acidic protein and decreased monoamines in the neostriatum, hippocampus, and entorhinal cortex. In the second experiment, SHAM‐MA‐ and ADX‐MA‐treated groups showed equivalently impaired CWM performance 2 weeks post‐treatment (increased latencies, errors, and start returns) compared to SHAM‐saline (SAL) and ADX‐SAL groups with no effects on novel object recognition, elevated zero maze, or acoustic startle/prepulse inhibition. Post‐testing, monoamine levels remained decreased in both MA‐treated groups in all three brain regions, but were not as large as those observed at 72‐h post‐treatment. The data demonstrate that MA‐induced learning deficits can be dissociated from drug‐induced increases in plasma corticosterone or hyperthermia, but co‐occur with dopamine and serotonin reductions. Synapse 64:773–785, 2010. © 2010 Wiley‐Liss, Inc.     

Proteins of rat brain myelin in neonatal hypothyroidism.

The amount of myelin and its protein composition was studied in hypothyroid rats during the first 30 days after birth. Although the brain weight was 92% of that in the controls, the yield of myelin in hypothyroid animals was only 60% of that in controls. The protein and glycoprotein ocmposition of the isolated myelin was similar in hypothyroid and control rats. The 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity in whole brain from hypothyroid animals was only 60% of the controls and reflected the decrease in myelin formation. In isolated myelin the specific activity of CNP was not different in hypothyroid and control animals with the exception of very immature animals (10 days). The major fucose-labeled glycoprotein in myelin of the hypothyroid rats had a slightly higher apparent molecular weight than that in myelin from age matched controls, probably reflecting a retardation of brain maturation and myelin formation.     

Monoclonal antibody production to human and bovine 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNPase): high-specificity recognition in whole brain acetone powders and conservation of sequence between CNP1 and CNP2

Monoclonal antibodies against human and bovine 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) were generated by fusing FOX-NY myeloma cells with spleen cells from RBF/Dn mice previously immunized with the purified brain antigens. The enzyme isolated from bovine brain was quite basic, with an isoelectric point of 9.71 and both the bovine and human enzymes consisted of a closely spaced doublet at approximately 44 and 46 kDa on SDS-PAGE. Six monoclonals were identified as strongly recognizing the enzyme on both ELISA plates and on immunoblots of whole brain protein. Four monoclonals very weakly cross-reacted with guinea pig myelin basic protein. In contrast with two previous reports, some of our monoclonal antibodies did immunostain 2 or 3 protein bands in peripheral nerve, two bands closely corresponding to those immunostained in central nervous system (CNS) myelin, the Wolfgram protein fraction and in acetone powders of whole brain. Each of the 6 monoclonals reacting strongly on immunoblots recognized the enzyme in from 2 to 5 of the species examined (human, bovine, rat, mouse and rabbit). In addition, all 6 monoclonals that immunostained the enzyme in whole brain, myelin and Wolfgram protein immunoblots recognized both CNP1 (44 kDa) and CNP2 (46 kDa). The two closely spaced protein bands observed on SDS-PAGE and previously stained on immunoblots of CNS CNPase using polyvalent rabbit anti-bovine CNPase antisera, and now different monoclonal antibodies, appear to be immunologically related and to contain highly conserved sequences.     

Cell specific enzyme markers as indicators of neurotoxicity: effects of acute exposure to methylmercury

In order to assess the sensitivity of several cell specific enzyme markers (tyrosine hydroxylase (TH), glutamic acid decarboxylase, choline acetyltransferase, glutamine synthetase (GS), neuron specific and non-neuronal enolase and 2',3'-cyclic nucleotide phosphohydrolase (CNP] as indices of neurotoxicity, changes in their activities were monitored after rats were treated with two doses of the neurotoxic agent, methylmercury chloride (MMC). Comparisons were also made of any histopathological changes occurring in the tissues examined. At the low dose rate (3.36 mg Hg/kg, po, for 14 days), the rats exhibited less body weight gain compared to untreated animals. No change in either the neuronal or noneuronal enzyme markers was observed in brain but a significant increase in the myelin marker, CNP, and total enolase activity was seen in the optic nerve. Morphological evaluation by light microscopy indicated no discernible neuronal lesions in MMC-exposed animals. At the higher MMC dose (7.05 mg Hg/kg, po, for 7 days), there was about a 20% loss in the body weight of treated animals and partial hind limb paralysis was observed. Of all the neuronal marker enzymes examined, only TH was found to be decreased in the striatum. The proliferating astroglial marker, GS, was elevated only in the cerebellum. CNP was found to be decreased in both the optic and sciatic nerve. As in the lower dose group no pathological changes were observed at the light microscopic level in the brain of MMC-treated rats. These data suggest that of the cell specific marker enzymes studied, GS in the cerebellum and TH in the striatum may be useful biochemical markers for the neurotoxic action of MMC.     

Depletion of glutathione interferes with induction of glycerolphosphate dehydrogenase in the brains of young rats.

In the rat brain hydrocortisone induces the enzyme, glycerolphosphate dehydrogenase (GPDH), during the first postnatal week. The present studies focused on a hypothetical role for glutathione-S-transferase (GST) in that phenomenon. Two forms of GST, Yb and Yp, had been detected in glial cells in mature rat brains, and it was suggested that they might function in hormone transport. Now GSTs have also been observed in the brains of 1-day-old rats. Two glutathione-depleting agents, buthionine sulfoximine and cyclohexene-1-one, were administered to rats, along with hydrocortisone, during the first postnatal week. Hydrocortisone or a depleting agent alone was administered to control animals. During the early days of the experiment there were lower GPDH specific activities in brains from the animals given hydrocortisone plus a depleting agent than in those from animals given hydrocortisone alone. Depleting agents alone did not affect the specific activities of GPDH. It is suggested that one function of the GST in rat brain is transport of hydrocortisone between or within glial cells.     

2′, 3′-cyclic nucleotide 3′-phosphodiesterase in the nervous system. Electrophoretic properties and developmental studies

The activity of 2′, 3′-cyclic nucleotide 3′-phosphodiesterase in the sciatic nerve of rats was determined during the myelination phase of postnatal development. In contrast to the large increase in activity of this enzyme in myelinating cerebrum and cerebellum, the specific activity of the diesterase in sciatic nerve remains constant. It seems unlikely, therefore, that this diesterase is a component of the myelin sheath in sciatic nerve.

Determinations of cyclic nucleotide diesterase activity in demyelinated tissue (plaques) of multiple sclerosis patients showed that the enzyme is nearly completely absent from this tissue. These data confirm and support the view that the diesterase is a component of myelin in the CNS.

The diesterase was demonstrated in polyacrylamide gels containing the electrophoretically separated rat CNS myelin proteins by staining for enzymatic activity. The enzyme is tentatively identified as a high molecular weight minor protein component of myelin; it does not correspond in its electrophoretic behavior to either the basic (encephalitogenic) protein or the proteolipid protein of myelin.