Immunocytochemical localization of basic protein in major dense line regions of central and peripheral myelin.

To localize basic protein (BP) in the lamellar structure of central and peripheral myelin, we perfused newborn and 7-11-day rat pups with a phosphate-buffered fixative that contained 4% paraformaldehyde and 0.05 or 0.2% glutaraldehyde. Teased, longitudinally split or "brush" preparations of optic and trigeminal nerves were made by gently teasing apart groups of myelinated fibers with fine forceps or needles. Some of these preparations were immunostained without pretreatment in phosphate-buffered antiserum to BP according to the peroxidase-antiperoxidase method. Others were pretreated in ethanol before immunostaining. Then, all of them were dehydrated, embedded in Epon, and sectioned for electron microscopic study. In optic and trigeminal nerves that were not pretreated, myelin, glial cells, and their organelles were well preserved. BP immunostaining was present on cytoplasmic faces of oligodendroglial and Schwann cell membranes that formed mesaxons and loose myelin spirals. In compact central and peripheral myelin, reaction product was located in major dense line regions, and the myelin periodicity was the same as that observed in unstained control myelin that had been treated with preimmune serum. In ethanol-pretreated tissue, the myelin periodicity was reduced but dense line staining still was present. Our immunocytochemical demonstration of dense line localization of BP in both CNS and PNS myelin that was not disrupted or pretreated with solvents is important because of conflicting evidence in earlier immunostaining studies. Our results also support biochemical and histochemical evidence suggesting that BP exists in vivo as a membrane protein interacting with lipids on the cytoplasmic side of the bilayer in the spirally wrapped compact myelin membrane.

Myelin instability and oligodendrocyte metabolism in myelin-deficient mutant mice.

During the active phase of myelination in myelin-deficient mutant mice (mld), myelin basic protein (MBP) synthesis is defective and the myelin lamellae are uncompacted. In these mutants, we found a fast metabolism of the myelin-associated glycoprotein (MAG) and of sulfatides, and the presence of cholesterol esters and a degradation product of MAG, dMAG, indicating that mld myelin was unstable. The increased synthesis of MAG and Wolfgram protein, two proteins present in uncompacted myelin sheath and paranodal loops, was demonstrated by high levels of messengers. Simultaneously, we found an accumulation of inclusion bodies, vacuoles, and rough endoplasmic reticulum in mld oligodendrocytes. This material was heavily immunostained for MAG. Furthermore, the developmental change between the two molecular forms of MAG (p72MAG/p67MAG) was delayed in mld mice. In 85-d-old mld mice, the MBP content increased and myelin lamellae became better compacted. In these mutants, dMAG was absent and MAG mRNAs were found in normal amounts. Furthermore, the fine structure of mld oligodendrocytes was normal and the MAG immunostaining was similar to age-matched controls. These results support a functional role for MBP in maintaining the metabolic stability and the compact structure of myelin. Furthermore, in the absence of MBP and myelin compaction, the regulation of the synthesis of at least two membrane proteins related to myelin cannot proceed.

Expression of myelin-associated glycoprotein by small neurons of the dorsal root ganglion in chickens.

Biochemical and immunocytochemical investigations have shown that myelin-associated glycoprotein (MAG) is exclusively related to myelin and myelin-forming cells in mammals. In the present study it was found that dorsal root ganglia in young chickens display MAG-immunoreactive material in most small sensory neurons. The presence of MAG at the surface of small sensory neurons raises the question of whether this glycoprotein acts as a cell adhesion molecule in lower vertebrates.

Myelin and glial membrane structures in the optic nerve of normal and jimpy mouse. A freeze-etching study.

In order to investigate the myelin and the glial cell membranes in the optic nerve of the mutant mouse "Jimpy," the method of freeze-etching was applied. The compact myelin lamellae and the first two glial membranes of the mutant, as compared to the normal mouse, show several abnormalities: absence of intramembraneous particles on the P-face, myelin lamellae separated by cytoplasmic layers, vesicular protrusions forming irregular invaginations, and elevations and tight junctions with a discontinuous, zigzag course. Some of these characteristics were found in the membrane of the oligodendroglia cell of the pathological animal, as well. The astrocytic membranes of both normal and Jimpy mice contain two types of gap junctions. The occurrence of one type seems to be increased in the mutant. Freeze-fractured internodal regions of the axolemma are not significantly different from those of normal animals.

Myelin-associated glycoprotein immunoreactive material: an early neuronal marker of dorsal root ganglion cells during chick development.

Immunostaining of myelin-associated glycoprotein (MAG) was performed in chick dorsal root ganglia (DRG) during development. The MAG-immunoreactive material appeared first around 7 days of incubation in immature neurons of DRG. Immunoprecipitates first confined to one pole of nucleus were gradually redistributed in the perinuclear Golgi apparatus of small DRG cells. Thus MAG may be used in the chick embryo as an early marker of primary sensory neurons of class B.

Myelination in the CNS of mld mutant mice: comparison between composition and structure.

Myelination was studied between 15 and 135 days postnatally in the brain and optic nerves of myelin deficient (mld) mutant mice. Between 15 and 30 days almost no myelin basic protein (MBP) could be detected in mld myelin. The axons were loosely wrapped by membranes which only fused at the extracellular sites forming the intraperiod line. At this age the major dense line was absent. At 25-30 days, purified myelin contained extremely high 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) (EC 3.1.4.37) activities which could be related to the redundant paranodal-like structures observed at this age in mld CNS. Therefore, it can be suggested that CNP is probably localized in such paranodal loops. After the active phase of myelin deposition was completed in controls, mld mutants showed important increases of MBP concentration in myelin with the concomitant appearance of the major electron dense line and better compaction of the myelin lamellae. The yield of myelin increased from 5 to 14% of control values during the period of 30 to 135 days. Since the recovery phase occurred at the time when myelin lipid synthesizing enzymes are at low residual activities, the myelin deficit could only be partially corrected. This study indicates that there is a delay of MBP synthesis in mld mice and the decrease of other myelin proteins could be secondary to the assumed primary defect involving MBP.

Optic disc and optic nerve of the blind cape mole-rat (Georychus capensis): a proposed model for naturally occurring reactive gliosis.

Recent studies of the visual system of animal species that live in a subterranean environment show not only regressive but also progressive morphological features. In this regard the aim of the present investigation is to describe the structural organisation of the eye and optic nerve of the adult Cape mole-rat, with special emphasis on both glial cell population and myelination. The main results are: (a) astrocytes show identical features to those occurring in reactive gliosis; (b) optic fibers vary greatly in diameter; (c) very small axons are myelinated and are often surrounded by a thicker sheath than larger optic fibers; (d) a large onion bulb-like structure composed of optic fibers, glia, and ganglion cells is found within the choriocapillary layer. These results suggest that the Cape mole-rat and probably other subterranean rodents may serve as a model to study spontaneous gliosis as well as mechanisms involved in myelination and degenerative processes.

Cells with neuronal characteristics differentiate and persist for one year in rat optic nerve explants cultures.

Evidence concerning the presence or absence of common neuronglia lineages in the postnatal mammalian central nervous system is still a matter of speculation. We address this problem using optic nerve explants, which show an extremely long survival in culture. Morphological, immunocytochemical and immunochemical methods were applied. The results obtained from in vitro tissue were compared with optic nerves (ONs) and whole-brain samples from animals of different ages. Newborn rat ONs represented the starting material of our tissue culture; they are composed of unmyelinated axons, astrocytes and progenitor cells but devoid of neuronal cell bodies. At this age, Western blots of ONs were positively stained by neurofilament and synapsin I specific antibodies. These bands increased in intensity during postnatal in situ development. In explant cultures, the glia cells reach a stage of functional differentiation and they maintain, together with undifferentiated cells, a complex histotypic organization. After 6 days in vitro, neurofilaments and synapsin I could not be detected on immunoblots, indicating that 1) axonal degeneration was completed, and 2) neuronal somata were absent at the time. Surprisingly, after about 4-5 weeks in culture, a new cell type appeared, which showed characteristics typical of neurons. After 406 days in vitro, neurofilaments and synapsin I were unequivocally detectable on Western blots. Furthermore, both immunocytochemical staining and light and electron microscopic examinations corroborated the presence of this earlier-observed cell type. These in vitro results clearly show the high developmental plasticity of ON progenitor cells, even late in development. The existence of a common neuron-glia precursor, which never gives rise to neurons in situ, is suggested.

Minisegments of newborn rat optic nerves in vitro: gliogenesis and myelination.

The question of whether the development of CNS glial cells requires the presence of axons or not can be studied with in vitro systems. In order to compare the differentiation of glial cells during development in vitro with that in situ, we have selected the optic nerve, which is anatomically as well as histotypically a well defined structure. For the in vitro investigations, small explants, called minisegments, of newborn rat optic nerves were cultivated taking four major conditions into account: the regular size of the minisegments should guarantee a permanent exchange of the culture medium in order to avoid cell death, neither mechanical nor enzymatic dissociation of the tissue were applied, the minisegments were explanted into flasks without substrate for cell adhesion and the minisegments were under constant gyratory agitation. The following in situ results were obtained: optic nerves of newborn rats are morphologically characterized by the presence of naked axons, astrocytes, glial precursors, and the absence of both differentiated oligodendrocytes and myelin. At postnatal day 5 myelin sheaths are still absent. Two weeks after birth, differentiated oligodendrocytes and microglial cells are present and numerous axons are surrounded by compact myelin. The in vitro experiments show the following main results, which were obtained after 14 h, 2 d, 5 d and 14 d in culture: during time in culture, the shape of minisegment of newborn rat optic nerves undergoes drastic changes, which indicate high cellular dynamics. After 14 h in vitro, axonal profiles, cells with pyknotic nuclei as well as clusters of astrocytes and glial precursors are present. After 2 days in culture the axonal profiles disappeared and the number of degenerating cells decreased drastically. Many large cells, probably phagocytes containing inclusions and more cells are differentiated. At the stage of 5 d in vitro 4 major types of cells can be distinguished: differentiated oligodendrocytes, which form compact and loose myelin, astrocytes, large and small glioblasts and phagocytes. Immunoprecipitates for myelin basic protein and/or myelin associated glycoprotein were found in oligodendrocytes, in their processes and associated to the myelin. Processes of some astrocytes showed immunoreactive products of glial fibrillary acidic protein. After two weeks in culture, the minisegments were mostly composed of astrocytes, whereas oligodendrocytes became rare and phagocytes disappeared. It can be concluded that CNS glial cells can attain their structural and immunocytochemical characteristics in the total absence of neuronal cell bodies and axons.(ABSTRACT TRUNCATED AT 400 WORDS)

Murine leukodystrophies as tools to study myelinogenesis in normal and pathological conditions.

Myelinogenesis is controlled by several genes. Therefore, the study of mutations affecting myelination should provide better understanding of the assembly and maintenance of myelin, and in the case of similitude with human diseases, a direct insight into the pathogenesis of these diseases. Murine mutants can be bred readily and sequential analyses allow an examination of the dynamic processes of myelination. In this study, we have selected certain aspects of four leukodystrophies in mice. The precise mechanisms leading to the important myelin deficit observed in the jimpy mutation, a sex-linked recessive trait, are still not completely elucidated. Our results show in jimpy mice severe astrocytic alterations prior to myelin formation. Therefore, abnormalities of oligodendrocytes and possibly axons could be a secondary phenomenon. Nevertheless, a defect involving neuroglia precursor cells cannot be ruled out. At the present time, Pelizaeus-Merzbacher is a disease in which mainly oligodendrocytes appear to be altered. In consequence, our findings in jimpy mice do not support the present contention that this mutation represents a model for Pelizaeus-Merzbacher disease. We propose that the jimpy mutation could be invaluable to study cell-cell interactions at an early stage of myelinogenesis. Interest in the myelin deficient (mld) mutant derives from the specific lack of one of the major myelin constituents, myelin basic protein, and the concomitant absence of the major electron dense line during the active phase of myelin deposition in the CNS. Our present knowledge points out a defect of gene regulation involving myelin basic protein synthesis. The absence of this extrinsic membrane component made it possible to catch a glimpse of its role in the formation and maintenance of the complex structure of the myelin sheath in the CNS. Quaking mice have been considered to represent an arrest of myelinogenesis. This commonly accepted view has to be reconsidered at the light of recent morphological and biochemical findings. An abnormal processing of the myelin-associated glycoprotein could prevent the normal compaction of myelin until the animals reach adulthood. Twitcher is characterized by extensive central and peripheral demyelination and the presence of intracellular inclusions in macrophages similar to human globoid cell leukodystrophy. In both human and murine diseases, a deficiency of galactosylceramidase activity was demonstrated.(ABSTRACT TRUNCATED AT 400 WORDS)

Abnormal cell relationships in Jimpy mice: electron microscopic and immunocytochemical findings.

The mutant mouse strain Jimpy is characterized by a deficiency of myelin formation throughout the C.N.S. The cause of this hypomyelination is unknown. Based on previous reports, astrocytes, axons and oligodendrocytes are all altered, but no single cell type can be unequivocally defined as the primary target. Jimpy and age-matched normal mice were investigated using thin sectioning, freeze-fracturing and immunocytochemistry. We examined optic nerves and cervical spinal cords of Jimpy to determine which cells were morphologically altered during the period which precedes the onset of myelination and which cellular alterations persisted during myelinogenesis. Abnormalities of astrocytes and axons were frequently observed in Jimpy not only during myelination but also in early postnatal development before mature oligodendrocytes were present. The early astrocytic changes included hyperplasia and alterations of both cytoplasm and plasma membrane. An unusually complex network of astrocytic processes divided the axons into very small groups. During myelination, astrocytic processes were found insinuated between the axons and myelin sheath and/or within the myelin lamellae. Immunocytochemical investigations also revealed a complex network of glial fibrillary acidic protein-positive processes in contact with the majority of the axons. At stages prior to myelination axonal alterations were detected. Most of the axons were not in close contact with one another and individual axons had an undulating and irregular course. In areas where axon separation by astrocytic processes occurred, axonal diameters were more variable than the homogeneously sized axons of the normal mice. Our immunocytochemical results at stages during myelination showed not only many myelin basic protein-positive processes around axons in Jimpy but also clearly immunostained myelin sheaths. This indicates that the myelinating glia present not only produce myelin basic protein but can also incorporate it into the myelin spiral. The presented results suggest that the mouse mutant Jimpy could be a model for disturbed cell interactions in the C.N.S. Therefore, the hypomyelination may not be attributed to a defect of a single cell but rather to a deficiency in both macroglial types and, perhaps, the axon as well.

Differentiation of neuron-like cells in cultured rat optic nerves: a neuron or common neuron-glia progenitor?

The optic nerve consists of axons, glia, and undifferentiated cells; neuronal cell bodies are absent. To study the developmental potential of glia and precursor cells in vitro, we devised an original, long-term culture system of optic nerve explants, called minisegments, of newborn rats; at this stage the nerves are composed of naked axons, astrocytes, and undifferentiated cells. After about 4-5 weeks in culture, neuron-like cells appeared, which showed morphological, fine structural, and immunocytochemical properties ascribed to neurons. These neuron-like cells may be derived from a common neuron-glia progenitor or from a small population of precursors, which never produce glia in situ.

Substrate-dependent migration of myelin-associated glycoprotein immunoreactive cells in cultured explants of dorsal root ganglia from chick embryos.

The aim of this study was to investigate the influence of collagen or polyornithine substrates on cell migration in explant cultures of dorsal root ganglia (DRG) by means of light microscopy and immunocytochemistry. Myelin-associated glycoprotein (MAG) immunoreactivity was used to characterize the subpopulation of small B ganglion cells, whereas neuron-specific enolase (NSE) immunoreactivity acted as a general neuronal cell marker. After a few days in culture, DRG explants grown on collagen substrate showed a flattened shape consisting of a core surrounded by a crown of neurites, which were mixed up with migrating cells of different types. These migrating cells were immunostained for both MAG and NSE and were observed after 7 days in the vicinity of the explant core, then after 14 days also at a distance from the explant core. In contrast, even after 14 days in culture, explants grown on polyornithine substrate maintained a globular shape. The MAG-positive ganglion cells were confined to the explant core and no cell migration was observed on this type of substrate. MAG immunoprecipitates located at the ganglion cell surface were observed in explants cultured on polyornithine, but rarely on collagen substrate. In conclusion, it is suggested that this pattern of intracellular distribution of MAG immunoreactive material could reflect interactions between cell surface and extracellular matrix, and could condition the migratory ability of small ganglion cells.

Filaments in astrocytes and axons of mice optic nerve. A freeze-etching study.

Fixed and nonfixed tissues from optic nerves of 20-day-old mice were examined with the electron microscope using the freeze-etching method. In this study the filaments of fibrious astrocytes are compared with those of axons. Both the astrocytic perikaron and the processes show a characteristic aspect in view of the arrangement and density of filaments. The most reliable criterion to distinguish them from nonmyelinated axons is the presence of areas with packed filaments. Furthermore, we demonstrated morphometrically that the filaments of axons and astrocytes from prefixed specimens had a statistically significant (p less than 0.001) smaller diameter (9.5 +/- 0.3 nm) than those from nonprefixed ones (10.5 +/- 0.3 nm). The diameters of filaments in axons and astrocytes are identical in fixed as well as in nonfixed material. The fine structure of filaments displays in addition to a helical form also a certain periodicity.

Myelin basic protein deficit in the PNS of mld mutant mice recovers during development.

Myelination was studied between 15 and 135 days postnatally in peripheral nerves of myelin deficient (mld) mice and in unaffected littermates. The nerve weights were not affected by the mutation and showed a 4-fold increase during the developmental period studied. The amounts of myelin present in peripheral nerves, as shown by biochemical and morphological techniques, were slightly reduced in mld in comparison to control mice. In controls, the concentration of myelin doubled during the investigation period. The increase of myelin basic protein (MBP) in total nerve homogenate paralleled the deposition of myelin, but the MBP concentration remained constant in normal myelin. In contrast, in mld myelin MBP concentrations were extremely low until 60 days of age and increased thereafter to reach almost normal values at 135 days. Similarly, the amounts of myelin isolated at 85 and 135 days were normal. 2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP; EC 3.1.4.37), the myelin-specific enzyme, showed normal specific activities in mld nerves. In mld and control myelin, CNP-specific activities decreased during development suggesting a preferential localization of CNP in Schwann cell plasma membranes. In contrast to the central nervous system, other myelin proteins were not altered in mld peripheral nervous system (PNS) and the very low MBP content had no severe repercussions on the composition and structure of the myelin sheath. Furthermore, Schwann cells appeared normal in mld PNS. Nevertheless, more subtle alterations could be detected. Slightly decreased amounts of myelin were observed in young mld mice and preliminary results indicate discrete alterations of the myelin periodicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum-free aggregate cultures of rat CNS glial cells: biochemical, immunocytochemical and morphological characterization.

Aggregate cultures of mixed glial cells, as well as of enriched astrocytes and oligodendrocytes were prepared, and maintained in serum-free medium for up to 25 days. Biochemical measurements of both neuron-specific and glia-specific enzyme activities showed that these three types of aggregate cultures were virtually devoid of neurons. Astrocyte-enriched cultures were greater than 95% pure, with oligodendrocytes as the only apparent contaminant, whereas oligodendrocyte-enriched cultures still contained a considerable proportion of astrocytes. In all these neuron-free aggregate cultures both astrocytes and oligodendrocytes attained a high degree of maturation. These findings were confirmed by morphological examinations, and by immunofluorescence studies. Furthermore, ultrastructural as well as immunocytochemical investigations using antibodies to myelin basic protein revealed that all three types of glial cell aggregate cultures contained myelin membranes, indicating that the presence of axons is not a prerequisite for the formation of myelin.