BBS4 protein has basal body/ciliary localization in sensory organs but extra-ciliary localization in oligodendrocytes during human development.

Bardet-Biedl syndrome protein 4 (BBS4) localization has been studied in human embryos/fetuses from Carnegie stage 15 to 37 gestational weeks in neurosensory organs and brain, underlying the major clinical signs of BBS. We observed a correlation between the differentiation of the neurosensory cells (hair cells, photoreceptors, olfactory neurons) and the presence of a punctate BBS4 immunostaining in their apical cytoplasm. In the brain, BBS4 was localized in oligodendrocytes and myelinated tracts. In individual myelinated fibers, BBS4 immunolabelling was discontinuous, predominantly at the periphery of the myelin sheath. BBS4 immunolabelling was confirmed in postnatal developing white matter tracts in mouse as well as in mouse oligodendrocytes cultures. In neuroblasts/neurons, BBS4 was only present in reelin-expressing Cajal-Retzius cells. Our results show that BBS4, a protein of the BBSome, has both basal body/ciliary localization in neurosensory organs but extra-ciliary localization in oligodendrocytes. The presence of BBS4 in developing oligodendrocytes and myelin described in the present paper might attribute a new role to this protein, requiring further investigation in the field of myelin formation.

Oligodendrocyte gene expression is reduced by and influences effects of chronic social stress in mice.

Oligodendrocyte gene expression is downregulated in stress-related neuropsychiatric disorders, including depression. In mice, chronic social stress (CSS) leads to depression-relevant changes in brain and emotional behavior, and the present study shows the involvement of oligodendrocytes in this model. In C57BL/6 (BL/6) mice, RNA-sequencing (RNA-Seq) was conducted with prefrontal cortex, amygdala and hippocampus from CSS and controls; a gene enrichment database for neurons, astrocytes and oligodendrocytes was used to identify cell origin of deregulated genes, and cell deconvolution was applied. To assess the potential causal contribution of reduced oligodendrocyte gene expression to CSS effects, mice heterozygous for the oligodendrocyte gene cyclic nucleotide phosphodiesterase (Cnp1) on a BL/6 background were studied; a 2 genotype (wildtype, Cnp1+/- ) × 2 environment (control, CSS) design was used to investigate effects on emotional behavior and amygdala microglia. In BL/6 mice, in prefrontal cortex and amygdala tissue comprising gray and white matter, CSS downregulated expression of multiple oligodendroycte genes encoding myelin and myelin-axon-integrity proteins, and cell deconvolution identified a lower proportion of oligodendrocytes in amygdala. Quantification of oligodendrocyte proteins in amygdala gray matter did not yield evidence for reduced translation, suggesting that CSS impacts primarily on white matter oligodendrocytes or the myelin transcriptome. In Cnp1 mice, social interaction was reduced by CSS in Cnp1+/- mice specifically; using ionized calcium-binding adaptor molecule 1 (IBA1) expression, microglia activity was increased additively by Cnp1+/- and CSS in amygdala gray and white matter. This study provides back-translational evidence that oligodendrocyte changes are relevant to the pathophysiology and potentially the treatment of stress-related neuropsychiatric disorders.

The Brown Norway opticospinal model of demyelination: does it mimic multiple sclerosis or neuromyelitis optica?

Opticospinal demyelinating diseases in humans are mostly characterized by the opticospinal form of multiple sclerosis (MS) and neuromyelitis optica (NMO). Increasing attention has recently focused on astrocyte markers, aquaporin-4 (AQP4) and glial fibrillary acidic protein (GFAP) in these diseases. We induced opticospinal demyelination in Brown Norway rats with soluble recombinant rat myelin oligodendrocyte glycoprotein (1-116) and incomplete Freund's adjuvant. Clinical, MRI, neuropathological and immunological evaluations were performed, with a focus on AQP4 and GFAP. We confirmed the opticospinal phenotype, including extensive myelitis, but also showed the MRI-characterized involvement of the periventricular area. Expression levels of myelin, AQP4 and GFAP showed the early involvement of astrocytes before demyelination in the optic nerve. The overexpression of AQP4 was particularly pronounced in the spinal cord and was concomitant with demyelination and astrocyte apoptosis. The disability scores were correlated with demyelination and inflammation but not with AQP4/GFAP expression. No antibodies against the linear and conformational epitopes of AQP4 were detected. Whereas a NMO-like phenotype was observed in this model, the AQP4/GFAP expression during the disease process was more closely related to opticospinal MS than NMO. However, this model raises the question of a continuum between opticospinal MS and the seronegative NMO subtype.

The myelin proteolipid protein gene modulates apoptosis in neural and non-neural tissues.

Mutations of the myelin proteolipid protein gene (Plp) are associated with excessive programmed cell death (PCD) of oligodendrocytes. We show for the first time that PLP is a molecule ubiquitously expressed in non-neural tissues during normal development, and that the level of native PLP modulates the level of PCD. We analyze three non-neural tissues, and show that native PLP is expressed in trophoblasts, spermatogonia, and cells of interdigital webbing. The non-neural cells that express high levels of native PLP also undergo PCD. The level of PLP expression modulates the level of PCD because mice that overexpress native PLP have increased PCD and mice deficient in PLP have decreased PCD. We show that overexpression of native PLP causes a dramatic acidification of extracellular fluid that, in turn, causes increased PCD. These studies show that the level of native PLP modulates the amount of PCD during normal development via a pH-dependent mechanism.

An immortalized jimpy oligodendrocyte cell line: defects in cell cycle and cAMP pathway.

Normal and jimpy oligodendrocytes in secondary cultures were transfected with plasmids containing the SV40 T-antigen gene expressed under the control of the mouse metallothionein-I promoter. Two immortalized stable cell lines, a normal (158N) and jimpy (158JP) cell line, expressed transcripts and proteins of oligodendrocyte markers, including proteolipid protein (PLP), myelin basic protein (MBP), and carbonic anhydrase II (CAII). Galactocerebroside and sulfatide were also detected with immunocytochemistry. Immunoelectron microscopy using gold particles showed that the truncated endogenous jimpy PLP was distributed throughout the cytoplasm and in association with the plasma membrane of cell bodies and processes. The length of the cell cycle in the jimpy oligodendrocytes in the absence of zinc was 31 h, about a 4-h longer cell cycle than the normal line. In the presence of 100 microM zinc, the cell cycle became 3 h shorter for both cell lines, with the jimpy cell cycle duration remaining 4 h longer than the normal line. Interestingly, the jimpy cell line showed a significant deficiency in stimulation via the cAMP pathway. While the level of oligodendrocyte markers (PLP, MBP, and CAII) were significantly increased by dibutyryl cAMP (dbcAMP) treatment in the normal cell line, no changes were observed in the jimpy cell lines. This observation, together with previous results showing jimpy oligodendrocyte's failure to respond to basic fibroblast growth factor (bFGF), suggests a role for PLP in a signal transduction pathway. Jimpy and normal oligodendrocytes transfected with the SV40T antigen gene, driven by the wild-type promoter of mouse metallothionein-I, continue to express properties of oligodendrocytes and therefore provide a powerful model to explore the function of myelin proteins and to dissect the complexity of the jimpy phenotype.

Mitochondrial carbonic anhydrase in the nervous system: expression in neuronal and glial cells.

Carbonic anhydrase (CA) V is a mitochondrial enzyme that has been reported in several tissues of the gastrointestinal tract. In liver, it participates in ureagenesis and gluconeogenesis by providing bicarbonate ions for two other mitochondrial enzymes: carbamyl phosphate synthetase I and pyruvate carboxylase. This study presents evidence of immunohistochemical localization of CA V in the rodent nervous tissue. Polyclonal rabbit antisera against a polypeptide of 17 C-terminal amino acids of rat CA V and against purified recombinant mouse isozyme were used in western blotting and immunoperoxidase stainings. Immunohistochemistry showed that CA V is expressed in astrocytes and neurons but not in oligodendrocytes, which are rich in CA II, or capillary endothelial cells, which express CA IV on their plasma face. The specificity of the immunohistochemical results was confirmed by western blotting, which identified a major 30-kDa polypeptide band of CA V in mouse cerebral cortex, hippocampus, cerebellum, spinal cord, and sciatic nerve. The expression of CA V in astrocytes and neurons suggests that this isozyme has a cell-specific, physiological role in the nervous system. In astrocytes, CA V may play an important role in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase. The neuronal CA V could be involved in the regulation of the intramitochondrial calcium level, thus contributing to the stability of the intracellular calcium concentration. CA V may also participate in bicarbonate ion-induced GABA responses by regulating the bicarbonate homeostasis in neurons, and its inhibition could be the basis of some neurotropic effects of carbonic anhydrase inhibitors.

Abnormal Ca(2+) regulation in oligodendrocytes from the dysmyelinating jimpy mouse.

Jimpy (jp) is a point mutation in the gene on the X chromosome which codes for the major myelin proteolipid protein. Most oligodendrocytes (OLs) in the jp mouse undergo cell death at the time when they should be actively myelinating. Loss of mature OLs results in severe CNS dysmyelination. Dying jp OLs have the morphology of apoptotic cells but it is not clear how the mutation activates biochemical pathways which lead to programmed death of OLs in jp CNS. There is compelling evidence from a number of systems that high levels of intracellular Ca(2+) ([Ca2+]i) can activate downstream processes which result in both apoptotic and necrotic cell death. To determine whether [Ca2+](i) dysregulation might be involved in the death of jp OLs, we used ratiometric imaging to determine levels of [Ca2+](i) in OLs cultured from jp and normal CNS and in immortalized cell lines derived from jp and normal OLs. Immortalized jp OLs and OLs isolated directly from jp brain both showed a similar elevation in [Ca2+](i) ranging from 60% to 150% over control values. A higher baseline [Ca2+](i) in jp OLs might increase their vulnerability to other insults due to abnormal protein processing or changes in signaling pathways which act as a final trigger for cell death.

Oligodendrocytes express different isoforms of beta-amyloid precursor protein in chemically defined cell culture conditions: in situ hybridization and immunocytochemical detection.

The expression of beta-amyloid precursor protein (betaAPP) by astrocytes is well documented; however, data concerning oligodendrocytes remain controversial. The main goal of the present study was to determine whether or not oligodendrocytes in culture constitutively express the different betaAPP isoforms. Oligodendrocytes were cultured in a chemically defined medium that avoids putative effects of unknown serum factors on oligodendrocyte development. We have employed immunocytochemistry and in situ hybridization with antibodies and synthetic oligonucleotides recognizing, respectively, specific protein epitopes and mRNA transcripts of rat betaAPP isoforms. Oligodendrocytes, in both mixed primary cultures in the presence of serum or in secondary cultures in defined medium, were clearly labeled by antibodies directed to different betaAPP sequences. Antibodies against the serine protease inhibitor domain of betaAPP, also strongly labelled oligodendrocytes. Immunohistochemistry and in situ hybridization were combined to determine precisely the expression of different isoforms of betaAPP. In situ hybridization revealed the presence in oligodendrocytes of mRNA transcripts coding not only for betaAPP695 but also for betaAPP770 and betaAPP751. This indicates that betaAPP immunoreactivity found in oligodendrocytes corresponds to constitutive expression of betaAPP. Oligodendrocyte cultured in chemically defined medium are able to express not only betaAPP695 but also betaAPP770, betaAPP751 isoforms containing the Kunitz protease inhibitor domain. Although the role of betaAPP in the pathological processes of Alzheimer's disease (AD) remains unknown, possible disturbances of betaAPP processing and/or synthesis in oligodendrocytes may account for some myelin disorders observed in AD and other senile dementias.

Effects of an 11-day spaceflight on the choroid plexus of developing rats.

Cellular distributions of ezrin, a cytoskeletal protein involved in apical cell differentiation in choroid plexus, and carbonic anhydrase II, which is partly involved in the cerebrospinal fluid production, were studied by immunocytochemistry, at the level of choroidal epithelial cells from the lateral, third and fourth ventricles in normal or experimental fetuses, in parallel with the ultrastructure of apical microvilli, observed by transmission electron microscopy. We compared choroid plexuses from developing normal rats (gestational day 15 to birth) with choroid plexuses from 20-day-old rat fetuses, developed for 11 days in space, aboard a space shuttle (NASA STS-66 mission, NIH-R1 experiments), from gestational day 9 to day 20. The main changes observed in fetuses developed in space were demonstrated by immunocytochemistry and concerned the distribution of ezrin and carbonic anhydrase II. Thus, in fetuses developing in space, ezrin was strongly detected in the choroidal cytoplasm and weakly associated to the membrane in the apical domain of the choroid plexus from the fourth ventricle. Such alterations suggested that choroid plexus from rat fetal brain displays a delayed maturation under a micro-gravitational environment. In contrast, intense immunoreactions to anti-carbonic anhydrase II antibodies showed that this enzyme is very abundant in rats developed in space, compared to ground control fetuses.

Isolation and characterization of defective jimpy oligodendrocytes in culture.

This study characterizes jimpy oligodendrocyte-enriched secondary cultures isolated from 10-12 days in vitro primary glial cell cultures derived from 1-2-day-old jimpy mouse brains. Proliferation of defective oligodendrocytes was carefully investigated with regard to the expression of myelin basic protein and proteolipid protein and their respective mRNAs. Less than 5% of contaminating astrocytes (GFAP+ cells) were usually present. The identity of jimpy oligodendrocytes was confirmed using an antibody directed against a peptide from the wild type proteolipid protein C-terminal sequence for immunocytochemistry and an oligonucleotide complementary to mRNA derived from exon 5 of the proteolipid protein gene for in situ hybridization. Both the antibody and the probe recognize only normal oligondedrocytes while jimpy oligodendrocytes always remain unstained. Proteolipid protein in normal and jimpy oligodendrocytes was detected with antibody recognizing normal and mutated forms. Between 80 and 95% of the cells in normal and jimpy cultures at 2 and 4 days in vitro in secondary cultures express myelin basic protein and proteolipid protein and their respective mRNAs. The percentage of oligodendrocytes (PLP+ or MBP+) in S phase of the cell cycle was 7-10% for both normal and jimpy oligodendrocytes. This contrasts with the in vivo situation where the proliferation rate of oligodendrocytes in jimpy brains is higher than in normal brains. In addition, jimpy oligodendrocytes remain unresponsive to basic fibroblast growth factor treatment while a similar treatment stimulates the proliferation of normal oligodendrocytes.

Oligodendrocytes in female carriers of the jimpy gene make more myelin than normal oligodendrocytes.

The female carrier of the jimpy (jp) gene is a model system to study the plasticity of neuroglial cells and the mechanisms they use to compensate for a temporary deficit in myelin. Myelin in the female carriers is reduced 30-40% during the first postnatal month but is normal in adults. We hypothesized that the number of oligodendrocytes (OLs) in the female carriers is increased, based upon previous data showing OL proliferation is increased but the number of dying OLs is only slightly elevated in development. To test this hypothesis, antibodies to carbonic anhydrase (CA)II, an OL-specific marker, were used to quantify the number of OLs in the spinal cords of 1-month-old and adult female carriers. Contrary to expectations, the number of OLs is significantly reduced in the dorsal funiculus and grey matter by 21% in adult female carriers compared to controls. A reduction of lesser magnitude is present in the 1-month-old animals. Electron microscopic montages prepared from normal and carrier dorsal funiculus were used to count total numbers of glia. Ultrastructural quantification shows a similar reduction in the number of OLs and confirms the validity of the CAII immunostaining as a means to quantify OLs. These data show that there are 21% fewer OLs in the central nervous system (CNS) of adult female carriers but normal amounts of myelin. Presumably, some OLs in the carrier CNS are maintaining more myelin than their counterparts in normal CNS would. These findings demonstrate that (1) a reduction in number of OLs does not necessarily involve a reduction in the amount of myelin, and (2) OLs have considerable flexibility in the amount of myelin they can make.

Postmitotic oligodendrocytes generated during postnatal cerebral development are derived from proliferation of immature oligodendrocytes.

The phenotype of proliferating glia is examined during postnatal rodent development by combining immunocytochemistry (ICC) with 3H-thymidine autoradiography (ARG) to identify cells in the S phase of the cell cycle. Antibodies (ABs) which are specific for cells in the oligodendrocyte (OL) lineage were utilized, with emphasis placed upon the proliferation of OLs as it remains unclear whether this cell type divides in situ. The results show that proliferating cells stain with ABs which are specific for OLs and myelin glycolipids. The proliferating OLs (oligodendroblasts), although they do not appear to have formed myelin sheaths, have quite elaborate and distinctive morphologies. These oligodendroblasts give rise to very long, thin processes which in turn have additional branches. Their cytoarchitecture corresponds closely to cells described as oligodendroblasts with electron microscopy and whose processes often appear to be in the initial phase of myelination (Skoff et al: J. Comp. Neurol. 169:291-312, 1976a). These proliferating OLs are still quite immature because the expression of myelin specific proteins is only occasionally observed in 3H-thymidine labeled cells. The phenotype of the oligodendroblasts is quite different from that of proliferating astrocytes (astroblasts). As shown in previous studies (Skoff; Dev. Biol. 139:149-163, 1990), the astroblasts, which are identified by the presence of glial fibrillary acidic protein (GFAP), usually have thick, stubby processes, and both their nucleus and cytoplasm are larger and of lighter density than those found in oligodendroblasts. In early myelinating regions of the cerebrum, glycolipid positive cells account for the majority of the 3H-thymidine labeled cells. This data, when combined with the quantification of proliferating astrocytes (ASs) from previous immunocytochemical and electron microscopic studies, indicate that oligodendroblasts and astroblasts constitute the vast majority of the proliferating glia in the brain and in optic nerve at times when ASs and OLs are being generated. In normal postnatal cerebral development, the immature ASs and OLs which proliferate are the direct, immediate precursors for most postmitotic ASs and OLs.

Carbonic anhydrase IV on brain capillary endothelial cells: a marker associated with the blood-brain barrier.

Carbonic anhydrase (CA) activity plays an important role in controlling cerebrospinal fluid production and also influences neuroexcitation and susceptibility to seizures. Until recently, CA II was the only CA demonstrated in brain. Its distribution is limited to the epithelial cells of the choroid plexus and to the myelin-forming cells, the oligodendrocytes. In this report, we present immunoblots, using an antibody raised to CA IV from rat lung, that show that CA IV is also present in rat and mouse brain. Results of immunohistochemistry and immunoelectron microscopy on sections from rat and mouse brain are presented that show the distribution of CA IV to be quite distinct from that of CA II. CA IV is expressed on and is limited to the luminal surface of endothelial cells of cerebral capillaries. These results establish CA IV as a cytochemical marker associated with the blood-brain barrier and suggest an important role for CA IV in CO2 and HCO3- homeostasis in brain.

Double-labeling in situ hybridization analysis of mRNAs for carbonic anhydrase II and myelin basic protein: expression in developing cultured glial cells.

We applied in situ hybridization to analyze the location and the developmental changes in the distribution of the transcripts for carbonic anhydrase II (CAII) and myelin basic protein (MBP) in mouse primary cultured glial cells. Both mRNAs were localized to the oligodendrocyte using double-labeling in situ hybridization. No evidence for CAII transcripts in astrocytes was obtained, indicating that CAII is expressed only by oligodendrocytes in normal rodent glia. As early as 48 h after plating, CAII and MBP mRNAs are present in a few, small round cells. Message is present 2-4 days before levels of these proteins can be detected in similar primary glial cultures. The intensity of labeling for MBP and CAII mRNA positive cells increases significantly during the second week but then decreases after the end of the third week. Only the oligodendrocyte perikaryon and a few processes are positive during the first week. In contrast, at 14 days, a large number of cell processes in addition to the cell bodies are heavily stained for both mRNAs. Both mRNAs could be detected far away from the cell body, up to 250 microns in some cell processes. Some segments on a cell process accumulate higher levels of mRNA than other areas. These areas may correspond to the accumulation of free ribosomes and to starting points for the membrane sheets elaborated by cultured oligodendrocytes. The developmental profile for timing and distribution of these two messages mimics closely their in situ pattern.

Oligodendrocyte cell surface recognized by a novel monoclonal antibody specific to sulfatide.

A rat monoclonal antibody (OL-1) was obtained by in-vitro immunization of rat spleenocytes with paraformaldehyde-fixed primary cultured glial cells derived from newborn rat brain and subsequent fusion with a rat myeloma cell line. The antibody secreted by the hybridoma immunostains live rat and mouse oligodendrocytes in primary and secondary cultures. The antibody binds specifically to oligodendrocytes and myelin structures in-situ. Radioimmunolabelling assays with a number of purified glycolipids offer thin layer chromatography separation show that OL-1 antibody binds strongly to sulfatide and to a lesser extent to galactosyl diglyceride.

Radial glia and astrocytes in developing and adult telencephalon of the lizard Gallotia galloti as revealed by immunohistochemistry with anti-GFAP and anti-vimentin antibodies.

The development of radial glia and astrocytes in the telencephalon of the lizard Gallotia galloti was studied by immunohistochemistry with anti-vimentin and anti-GFAP antibodies. Vimentin appears at embryonic stage 32 (E32) in the proliferative zone of the lateral ventricle and subpial end-feet in the marginal zone. At E34-35 the staining intensity for vimentin in all radial glia is maximal. It then decreases and disappears in most structures in adult animals. GFAP appears at E35 in the end-feet in the marginal zone and its intensity increases until adulthood, particularly in radial and sinuous fibers and in fibers that originate from the sulci and invade the ventral striatum and the septum. In contrast, the reaction is weak in the cortex, in the anterior dorso-ventricular ridge, and in the amygdala nuclei. Radial glia is still present in the adult, and the composition of its intermediate filaments changes during development from vimentin to GFAP. No GFA-positive cell bodies except those of ependymal glia were detected in telencephalon.

Glial fibrillary acidic protein and vimentin immunohistochemistry in the developing and adult midbrain of the lizard Gallotia galloti.

The distribution of glial fibrillary acidic protein (GFAP)- and vimentin-containing cells was studied by immunohistochemistry in the midbrain of the lizard Gallotia galloti. At embryonic stage 32 (E32), vimentin immunoreactivity appeared first in cell bodies located in the ventricular walls, in radial fibers, and subpial end-feet and increased in these structures until E34/E35. Faint GFAP immunoreactivity gradually appeared in the same structures between E34 and E37, and this increased until adulthood, whereas vimentin immunoreactivity decreased after E35, becoming limited to a few end-feet and fibers in the adult, mainly in the tegmentum. Thus, in developing Gallotia midbrain a shift from vimentin-containing to GFAP-containing intermediate filaments begins around E36 or E37. At E40, in addition to the cell bodies in the ependymal area, dispersed GFAP-positive cells, possibly immature astrocytes appeared. These cells showed the same shift. In the adult lizard, GFAP-positive radial glia are still present and coexist with GFAP-positive astrocytes, which are prefentially located in the marginal optic tract and the oculomotor nuclei, but are absent in the fasciculus longitudinalis medialis. Optic tectum, pretectum, tegmentum, and isthmic nuclei are the areas richest in GFAP-positive radial fibers: these were much less abundant in the deep mesencephalic nuclei. Thus, in this lizard, GFAP-positive astrocytes display a clear cut regional distribution: they are present in mesencephalon, whereas they are absent in telencephalon.

Oligodendrocytes express a normal phenotype in carbonic anhydrase II-deficient mice.

The nervous system of a mouse mutant characterized by a carbonic anhydrase II (CA II) deficiency was examined with light and electron microscopy and with immunocytochemistry using different glial cell markers. No major morphologic abnormalities at either the cellular or subcellular level are detectable in the brains of CAII-deficient mice, even though CAII is the main isozyme of CA in the brain. The oligodendrocytes, which characteristically express high levels of CA II, do not exhibit signs of degeneration or abnormalities even in 1-year-old CA II-deficient mice. Similarly, neurons and astrocytes have a normal structure and distribution. Oligodendrocytes show a normal staining pattern and distribution for galactocerebroside (GC), 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), and myelin basic protein (MBP). Astrocytes have a normal morphology and distribution when stained for GFAP and S100 protein. The lack of major degeneration in the brain due to a CA II deficiency suggests these mice utilize other enzymatic or physiological pathways to compensate for the enzyme absence.

Expression of galactocerebroside in developing normal and jimpy oligodendrocytes in situ.

Intense and specific immunostaining of oligodendrocytes in vivo has been obtained for the first time using antibodies to galactocerebroside. We have examined the differentiation of oligodendrocytes in normal mice and then compared their differentiation to the myelin-deficient mouse jimpy, using immunoperoxidase, immunogold and immunofluorescence labelling techniques. We also compared staining for galactocerebroside with staining obtained using antibodies to myelin basic protein, carbonic anhydrase II, 2', 3'-cyclic nucleotide 3'-phosphohydrolase and proteolipid protein. The results of this comparative study confirm previous tissue culture studies and show that galactocerebroside is specific for oligodendrocytes in situ. As in tissue culture, galactocerebroside is one of the earliest oligodendrocyte markers to be expressed, making it an important marker for studying the differentiation of this cell type. The shape of oligodendrocytes in situ changes distinctly with time, shifting from an early stellate form with numerous spidery processes to a cell with a few processes radiating from the perikaryon. These morphological changes are observed for both normal and jimpy mice and they parallel those described in vitro. Oligodendrocytes in jimpy mice express most myelin markers, but the staining within the cells is generally less intense than in normal oligodendrocytes and the antigens are restricted to the cell body and processes without being incorporated into myelin sheaths. Quantification of the number of oligodendrocytes stained for galactocerebroside in normal and jimpy mice show that their number is not reduced in the corpus callosum and cerebellum during the first 2 weeks postnatal. This finding shows that many cells in jimpy mice which were considered to be unclassifiable by the application of morphological criteria have, in fact, differentiated to the stage where they are galactocerebroside-positive.

Characterization of ependymal cells in hypothalamic and choroidal primary cultures.

Long-term primary cultures derived from fetal mouse or rat hypothalamus and choroid plexus were obtained in serum-supplemented and chemically defined media. In order to identify and characterize cell types growing in our cultures, we used morphological features provided by phase-contrast, scanning and transmission electron microscopy. Immunological criteria were recognized, using antibodies against intermediate filament proteins (vimentin, gliofibrillar acid protein, cytokeratin, desmin, neurofilament proteins), actin, myosin, ciliary rootlets, laminin and fibronectin in single or double immunostaining, and monoclonal antibodies known to detect epitopes of ependymal or endothelial cells. Minor cell types such as astrocytes, fibroblasts and endothelial cells were distinguished. Ependymal cells, which exceeded 75% of the cultured cells, were identified by their cell shape and epithelial organization revealed by phase-contrast and transmission electron microscopy, by their apical differentiation evidenced by scanning and transmission electron microscopy, and by certain molecular markers (e.g. gliofibrillar acid or ciliary rootlet proteins) detected by immunofluorescence. Four ependymal cell types were recognized: choroidal ependymocytes, ciliated and unciliated ependymal cells, and tanycytes. All these cultured ependymal cell types showed a remarkable resemblance to in vivo ependymocytes, in terms of marker expression and ultrastructural features.