Neuronal and non-neuronal catechol-O-methyltransferase in primary cultures of rat brain cells

Previous biochemical and histochemical studies have suggested that catechol-O-methyltransferase (COMT) is a predominantly glial enzyme in the brain. The aim of this work was to study its localization and molecular forms in primary cultures, where cell types can be easily distinguished with specific markers. COMT immunoreactivity was studied in primary astrocytic cultures from newborn rat cerebral cortex, and in neuronal cultures from rat brain from 18-day-old rat embryos using antisera against rat recombinant COMT made in guinea pig. Double-staining studies with specific cell markers to distinguish astrocytes, neurons and oligodendrocytes were performed. COMT immunoreactivity colocalized with a specific oligodendrocyte marker galactocerebroside in cells displaying oligodendrocyte morphology, flat cells displaying type-1 astrocyte morphology and glial fibrillary acidic protein, in branched cells displaying type-2 astrocyte morphology and in cell bodies of neurons, the processes of which displayed neurofilament immunoreactivity. Western blots detected both soluble 24 kDa and membrane-bound 28-kDa COMT proteins in neuronal and astrocyte cultures. The results suggest that COMT is synthesized by cultured astrocytes, oligodendrocytes and neurons.     

Characterization of glial subpopulations in cultures of the ovine central nervous system

The cellular composition and in vitro development of glial cultures derived from the rat CNS has been well studied. However, less information is available on similar cultures from other species, particularly higher mammals. To study ovine glial development in vitro, cultures from 50-day fetal to adult animals were characterized with various immunocytochemical markers, which are frequently used to define neural cell subsets in rat cultures. As in rats, both A2B5+ and A2B5- astrocytes can be identified in ovine cultures. However, ovine A2B5+ and A2B5- could not be reliably differentiated by their morphology, which was more influenced by whether the cells were in serum-free or serum-containing media than by their A2B5-positive or -negative status. In addition, ovine A2B5+ astrocytes were present in cultures from early fetal brain before the development of identifiable oligodendrocytes, unlike rat type II astrocytes, which develop only after the appearance of oligodendrocytes. An A2B5+ cell, morphologically similar to the rat 02-A cell, can be found in cultures from fetal ovine cerebrum or cerebellum. A2B5+/glial fibrillary acidic protein (GFAP)- cells in cultures from 100- to 115-day ovine cerebellum appeared to differentiate into A2B5+ astrocytes in serum-containing media. However, in serum-free media, although the A2B5+ cells assumed a more "oligodendroglial-like" morphology, they did not express galactocerebroside or myelin basic protein, suggesting that these cells may not be bipotential as is the rat 02-A cell. Oligodendroglial differentiation was not induced by treatment with dibutyryl cyclic AMP or insulin-like growth factor I. Many cells in cultures from a variety of fetal ages did not label with any of the immunocytochemical markers used, suggesting the need for more cell-type-specific markers to identify neural cell subsets in higher mammals.     

Differences in vimentin distribution in glial cells in culture revealed with an antibody against a phosphorylated epitope

We have previously described that spatial and temporal changes in the organization of vimentin that are correlated with protein kinase C (PKC)-induced phosphorylation of vimentin can be detected with the mouse monoclonal antibody B3 in cultures of amoeboid microglia [Ciesielski-Treska et al. (1991) J. Neurosci. Res. 29, 362–378]. The antibodies were generated to native form of vimentin-containing filaments and antibody B3 reveals a restricted immunostaining of vimentin in glial cells from human, rat and mouse origin. In the present study we show the distribution of epitope B3 analyzed by immunofluorescence within defined populations of rat glial cells. Relatively high immunoreactivity was found in Type 1 astrocytes, Type 2 astrocytes and oligodendrocytes had low immunoreactivity. Although the results suggested that in Type 1 astrocytes the phosphorylated epitope is prominent, its phosphorylation was not found to be cell cycle-dependent, and appeared unrelated to the organizational changes of intermediate filaments associated with the morphological conversion of polygonal to stellate astrocytes. As expected, the immunofluorescence was increased by exposition of astrocyte cultures to an activator of PKC, confirming our previous conclusion that the immunoreactivity of the epitope B3 depends on PKC-mediated phosphorylation. In addition, the finding that the immunofluorescence of vimentin was more homogeneous in quiescent, serum-deprived astrocytes and also in astrocytes exposed to an inhibitor of protein synthesis, cycloheximide, may suggest that phosphorylation of the epitope B3 depends on a protein factor present in fetal calf serum. The immunofluorescence studies on cultures enriched in Type 2 astrocytes and in oligodendrocytes indicate that the epitope B3 is hypophosphorylated in glial cells of this lineage and becomes dephosphorylated after terminal differentiation of oligodendrocytes. These observations suggest that in Type 2 astrocytes and in oligodendrocytes the low level of phosphorylation of vimentin could be related to the down regulation in vimentin expression.     

Induced glial differentiation of fetal rat brain cells in culture: An ultrastructural study

Primary and secondary cultures of fetal rat brain cells (FBC) from 18th day of gestation have been investigated by scanning and transmission electron microscopy. Primary cultures consisted of a monolayer of flat, undifferentiated epithelioid cells, with some oligodendrocytes, astrocytes and immature neuronal cells. In secondary cultures, cells with glia morphology disappeared. Following addition of extracts from adult rat brains to secondary cultures, a dramatic of the epithelioid cells took place. They detached from the plastic surface, extruded long cytoplasmic processes with numerous microvilli and cytoplasmic blebs as well as parallel arrays of microtubules and filaments. The differentiated cells resembled astrocytes, and characteristic glia filaments were also observed. An increase of ribosomes and rough endoplasmatic reticulum suggested enhancement of protein synthesis. At the same time S-100 protein and glial fibrillary acidic protein accumulated within the cells. The morphological changes were mostly reversible within 48 h of removal of the brain extract.     

Late onset and increasing expression of the gap junction protein connexin30 in adult murine brain and long-term cultured astrocytes

In rat brain, expression of the gap junction protein connexin30 increased during the first 3 weeks after birth and reached its maximum after 4 weeks, as shown by analysis with specific connexin30 antibodies. This contrasts with the prenatal onset of connexin43 expression. On cryosections of rat brain, connexin30 immunoreactivity was found near blood vessels and in ependymal as well as in leptomeningeal cells. Expression in the neuropil was first noticed 3 weeks after birth, showing the same spatial pattern of immunoreactivity as connexin43. This late onset of connexin30 expression in astrocytes was also seen in long-term glial cell cultures, where connexin30 was coexpressed with the astrocytic marker proteins S-100β and glial fibrillary acid protein. In acute brain slices, connexin30 immunofluorescent signals were detected on processes of functionally identified astrocytes. Thus, our results show that connexin30 is expressed in three different cell types of the rodent brain. The late onset of connexin30 expression in astrocytes suggests that this gap junctional protein fulfills a role in intercellular communication among mature astrocytes. GLIA 25:111–119, 1999. © 1999 Wiley-Liss, Inc.     

Fate of developing astrocytes in the optic nerve of the myelin-deficient rat

There is considerable debate on the development of a glial cell line in the rat optic nerve, which is characterized by the specific expression of the A2B5 and HNK-1 epitopes. This cell line has been assumed to give rise to oligodendrocytes and so-called type 2 astrocytes. However, it is doubtful that the latter cell type really exists in vivo. In the present study, we have addressed this question by investigating the development of astrocytes in the myelindeficient (md) rat, which is characterized by dysmyelination and loss of oligodendrocytes. Defective oligodendrocytes were observed by the third postnatal day, well before the generation of type 2 astrocytes. Consequently, the number of type 2 astrocytes was reduced in cultures prepared from optic nerves of md rats vs. controls. This finding was not paralleled in vivo; i.e., no dying astrocytes were observed in md sections by conventional electron microscopy. However, immunoreactivity against the HNK-1 epitope was enhanced in md compared to control sections. Ultrastructurally, HNK-1 immunoreactivity was detected predominantly on the axonal surface at astroaxonal contact sites, which were found only at the nodes of Ranvier within controls but extended to the whole axonal surface in md animals. Only a minor portion of the immunoreactivity derived from glial cells, presumably from oligodendrocytes at the paranodal region in controls. Thus, the HNK-1 epitope is not a useful antigen for distinguishing astrocytes in the rat optic nerve. Accordingly, our results do not provide evidence for the existence of specialized type 2 astrocytes in vivo. In vitro, these cells are probably only oligodendrocytes that mimic some astroglial features if grown in serum-containing media. J. Comp. Neurol. 378:105–116, 1997. © 1997 Wiley-Liss, Inc.     

Appearance of neuronal S-100β during development of the rat brain

In addition to being an astroglial protein, S-100β is localised in distinct populations of neurons in the adult rat hindbrain. We report, here, the expression of S-100β in both neurons and glia of the rat brain during development. Prenatally, S-100β immunoreactivity was confined to glial cells close to the germinal zone. After birth, S-100β positive glial cells were seen mainly in the brainstem and cerebellum, while only a few were detected in cerebral cortex and hippocampus. The number of S-100β containing glial cells increased steadily during the first 2 postnatal weeks after which the adult pattern was attained. No S-100β containing neurons were present prenatally. The first S-100β containing neurons were seen in the mesencephalic trigeminal nucleus at postnatal day 1 (P1), and in the motor trigeminal nucleus at P3. Neuronal S-100β immunoreactivity in other nuclei was mostly attained from the 10th to the 21st postnatal day. The neuronal S-100β immunoreactivity was first detected in the cell nuclei during development, then increased in the cytoplasm with ages. A nuclear staining in many immunoreactive neurons persisted until the adult. It usually took 1 to 2 weeks for neuronal S-100β to attain the adult staining pattern, i.e., heavy staining of the cytoplasm and processes, after its appearance. The forebrain never contained S-100β positive neurons. The S-100β is first expressed in glial cells, suggesting it is primarily of the glial origin. Coupled with neurotrophic effects of the protein, the time course of neuronal S-100β expression during the critical period of neuronal development implies that it may be involved in neuronal differentiation and maturation.     

Expression of heat shock protein-65 by oligodendrocytes in vivo and in vitro: implications for multiple sclerosis.

We studied immunoreactivity for heat shock proteins (HSPs) in multiple sclerosis (MS) brain tissue and detected HSP-65 in chronic MS plaques at the edge of thinly myelinated (or remyelinated) areas. Serial-section immunocytochemistry and double staining revealed that HSP-65+ cells represented reactive, immature oligodendrocytes with strong reactivity for myelin basic protein and weak reactivity for galactocerebroside. Mature oligodendrocytes outside MS plaques did not stain for HSP-65. Control brain sections showed no HSP-65 reactivity. Oligodendrocytes expressed HSP-65 in mixed glial cell cultures. In this system in vitro, oligodendrocytes, but not astrocytes, showed constitutive expression of HSP-65. There was immunoreactivity for HSP-72 in astrocytes in MS and non-MS brains to a similar extent but no association between HSP-72 reactivity and MS plaques. Interestingly, HSP-65+ oligodendrocytes colocalized with T lymphocytes expressing the gamma delta T-cell receptor (TcR). Since HSP-65 has been implicated as a major antigen recognized by TcR gamma delta lymphocytes, our findings might represent a new immunologic interaction within MS plaques that leads to the destruction of immature oligodendrocytes involved in remyelination.     

Differential distribution of cellular forms of beta-amyloid precursor protein in murine glial cell cultures.

The production and localization of cell-associated forms of beta-amyloid precursor protein (APP) of Alzheimer's disease was investigated in primary cultures of mouse glial cells. In both oligodendrocytes and astrocytes, immunofluorescence staining with an antibody against the carboxy terminus of APP revealed an intense cytoplasmic immunoreactivity. Immunoblotting of the cell extracts detected differences in the composition of APP between oligodendrocytes and astrocytes, notably the abundance of 107 kDa subtype in oligodendrocytes. Differences in immunoblot patterns were also noted between two buffer-insoluble, membrane-rich subcellular fractions of the glial cells, nuclear-mitochondrial and microsomal; the 119 kDa APP was enriched in the former, whereas the 73 and 115 kDa APPs in the latter. The results suggest that each APP subspecies may play a distinct functional role in different cell types and subcellular fractions.     

Immunocytochemical study of S-100 positive glial cells in the brainstem and spinal cord of the rat embryo

A light and electronmicroscopic immunocytochemical study of the glial cells in the brainstem and spinal cord of the 18th day rat embryo was performed using an anti-S-100 protein antiserum. Only the radial glia and the free immature glial cells are S-100 immunoreactive. Neurons are devoid of S-100 immunoreactivity. The radial glia form two paramedial plates and a great number of lateral plates, uniformly spaced along the ventral portion of the brainstem from the mesencephalon to the medulla. The S-100 protein was also detected in the perivascular membranes and glial limitans. Embryonic glia adopt a highly organized spatial pattern in the brainstem that could set the structural basis for an organized assembly of the developing nervous tissue. The use of the S-100 protein as a glial marker in the embryonic rat brain proved to be of great value. Antibodies to S-100 protein allow the demonstration of immature glial cells and a highly organized spatial pattern in the brainstem and spinal cord of the rat embryo.     

Pure astrocyte cultures derived from cells isolated from mature brain

Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30-day-old rat brain, eventually yield cultures in MEM-15% calf serum that contain, in addition to oligodendrocytes, proliferating astrocytes and variable numbers of fibroblast-like cells. If these cultures are switched to a serum-free defined medium during the 1st week, mixed cultures containing only oligodendrocytes and astrocytes are obtained. Bovine cultures can be replated and purified by selective adhesion to yield cultures that are greater than 99% astrocytes; similar procedures were not successful with rat cultures. Cytoskeletal preparations of the purified astrocyte cultures from mature bovine brain contain both vimentin and glial fibrillary acidic protein (GFAP), but vimentin is by far the major intermediate filament protein. Thus, the intermediate filament composition of these astrocytes is similar to that of astrocytes in primary cultures obtained from neonatal rat brain. Immunofluorescent studies of these cultures at 24 hr in vitro show that there are no GFAP+ cells in cultures of either species; the bovine cultures contain greater than 95% GC+ cells; and the rat cultures contain 90% GC+ cells. After a few days in vitro flat cells appear that are vimentin+/GFAP-/GC-. In serum-free medium these cells eventually become vimentin+/GFAP+. We propose that the astrocytes that grow in these cultures arise from a population of glial precursor cells, which are present even in adult brain and are isolated together with oligodendroglia, and that they do not derive from contaminating mature astrocytes. Thus, the astrocytes in our cultures may have the same origin as astrocytes grown in culture from dissociated neonatal brain.     

Differential distribution of cellular forms of β-amyloid precursor protein in murine glial cell cultures

The production and localization of cell-associated forms of β-amyloid precursor protein (APP) of Alzheimer's disease was investigated in primary cultures of mouse glial cells. In both oligodendrocytes and astrocytes, immunofluorescence staining with an antibody against the car☐y terminus of APP revealed an intense cytoplasmic immunoreactivity. Immunoblotting of the cell extracts detected differences in the composition of APP between oligodendrocytes and astrocytes, notably the abundance of 107 kDa subtype in oligodendrocytes. Differences in immunoblot patterns were also noted between two buffer-insoluble, membrane-rich subcellular fractions of the glial cells, nuclear-mitochondrial and microsomal; the 119 kDa APP was enriched in the former, whereas the 73 and 115 kDa APPs in the latter. The results suggest that each APP subspecies may play a distinct functional role in different cell types and subcellular fractions.     

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.     

Ca2+ Mobilization in Cultured Rat Cerebellar Cells: Astrocytes are Activated by t-ACPD

Using primary rat cerebellar cell cultures we observed that trans-1-amino-cyclopentyl-1,3-dicarboxylic acid (t-ACPD) was able to induce an increase in intracellular [Ca2+] in different cell types. This response was not abolished by external Ca2+ withdrawal, indicating that t-ACPD triggered the release of intracellularly stored Ca2+. In neurons the t-ACPD response was monophasic and inhibited by l-2-amino-4-phosphonobutyrate (APB). In astrocytes, characterized by their immunoreactivity to antisera to glial fibrillary acidic protein and S-100 protein, the response was oscillatory and resistant to APB application. These results suggest the presence of glutamate metabotropic receptor subtypes in the mammalian brain.     

Vulnerability of rat and mouse brain cells to murine hepatitis virus (JHM-strain): studies in vivo and in vitro

The pathogenicity and cell tropism of mouse hepatitis virus (MHV-JHM-strain) in the developing mouse (Balb/c) and rat (Wistar and Lewis) brain were analysed. Intracranial infection of Balb/c mice at postnatal day 5 induced a lethal encephalitis in all animals. Of Wistar rats infected at day 2 or 5 after birth, 30 to 70%, respectively, survived. The distribution of viral antigen was studied in frozen brain sections of animals that died after infection; astrocytes were found to be the major virus-infected cell type throughout the central nervous system. More than 75% of the surviving rat pups developed paralysis, but viral antigen was detected in only few brain cells and not in astrocytes. The cell tropism of MHV-JHM was examined further in virus-infected glial cell cultures derived from brains of rats or mice. In the glial cultures derived from Wistar rats, only oligodendrocytes were infected, whereas in cultures derived from mouse or Lewis rat brain viral antigen was detected in both astrocytes and oligodendrocytes. Infection of astrocytes led to the formation of syncytia and degradation of the cytoskeleton. Infected rat oligodendrocytes gradually disappeared from the cultures because of cell death. These phenomena indicate that, besides an indirect autoimmune response triggered by infected astrocytes, direct virus-induced injury to astrocytes or to oligodendrocytes can have a dominant role in the neuropathogenicity of mouse hepatitis virus. The present results underscore the importance of species and developmental stage of experimental animals in the neurotropism and pathogenicity of MHV-JHM.     

Cellular and subcellular distribution of the S-100 protein in rabbit and rat central nervous system

The cellular and subcellular distribution of the S-100 protein in rabbit and rat central nervous system was studied both quantitatively and qualitatively. Microcomplement fixation estimations on bulk-prepared neuronal and glial cells showed at least five to six times higher amounts of water-soluble S-100 protein. High levels of S-100 protein were found in mitochondria and soluble protein fractions. Immunoelectron microscope investigations demonstrated S-100 protein in neuronal structures, such as the postsynaptic membrane and part of the plasma membrane. Among glial cells the astrocytic filaments contained high levels of S-100 protein. S-100 was also found in most subcellular membranes of astrocytes and oligodendrocytes.     

Effects of lipopolysaccharide on oligodendrocyte progenitor cells are mediated by astrocytes and microglia

Oligodendrocytes are the primary cells injured in periventricular leukomalacia (PVL), a predominant form of brain white matter lesion in preterm infants. To explore the possible linkage between white matter injury and maternal infection, purified rat O-2A progenitor (Oligodendrocyte-type 2 astrocyte progenitor) cell cultures were used as a model in studying the effects of lipopolysaccharide (LPS), an endotoxin, on survival and differentiation of oligodendrocytes and the involvement of other glial cells in the effects of LPS. O-2A progenitor cells were cultured from optic nerves of 7-day-old rat pups in a chemically defined medium (CDM). Astrocyte and microglia cell cultures were prepared from the cortex of 1-day-old rat brains in the CDM. Direct treatment of LPS (1 microg/ml) to O-2A cells had no effect on viability or differentiation of these cells. When O-2A progenitor cells were cultured in the conditioned medium obtained from either astrocyte or microglial cell cultures for 48 hr, survival rate and differentiation of O-2A cells into mature oligodendrocytes were greatly enhanced as measured by the MTT assay and immunocytochemistry. The conditioned medium obtained from astrocytes or microglia treated with LPS for 48 hr, however, failed to show such a promotional effect on viability and differentiation of O-2A cells. When 5 microg/ml LPS was used to stimulate astrocytes or microglia, the conditioned medium from these glial cell cultures caused O-2A cell injury. The overall results indicate that astrocytes and microglia may promote viability and differentiation of O-2A progenitor cells under physiological conditions, but they may also mediate cytotoxic effects of LPS on oligodendrocytes under an infectious disease biochemical environment.