Carbonic Anhydrase, 5''-Nucleotidase, and 2'',3''-Cyclic Nucleotide-3''-Phosphodiesterase Activities in Oligodendrocytes, Astrocytes, and Neurons Isolated from the Brains of Developing Rats

The activities of three myelin-associated enzymes, carbonic anhydrase, 5'-nucleotidase, and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNP), were measured in oligodendrocytes, neurons, and astrocytes isolated from the brain of rats 10, 20, 60, and 120 days old. The carbonic anhydrase specific activity in oligodendrocytes was three- to fivefold higher than that in brain homogenates at each age, and, at all the ages, low activities of this enzyme were measured in neurons and astrocytes. The oligodendrocytes and astrocytes from the brains of rats at all ages had higher activities of the membrane-bound enzyme 5'-nucleotidase than was observed in neurons. In oligodendrocytes from 10- and 20-day-old rats, the 5'-nucleotidase activity was two-to threefold the activity in the homogenates (i.e., relative specific activity = 2.0-3.0), and the relative specific activity of this enzyme in the oligodendrocytes declined to less than 1.0 at the later ages, concomitant with the accumulation of 5'-nucleotidase in myelin. The CNP activity was always higher in oligodendrocytes than in neurons, but not appreciably different from that in astrocytes from 20 days of age onward. The relative specific activity of CNP was highest in the oligodendrocytes from 10-day-old rats but was lower, at all ages, than we had observed in bovine oligodendrocytes. These enzyme activities in oligodendroglia are quite different in amount and developmental pattern from those reported previously for myelin.     

Specific Expression of N-Acetylaspartate in Neurons, Oligodendrocyte-Type-2 Astrocyte Progenitors, and Immature Oligodendrocytes In Vitro

To test the specificity of N-acetylaspartate (NAA) as a neuronal marker for proton nuclear magnetic resonance (1H NMR) spectroscopy, purified and characterized cultured cells were analyzed for their NAA content using both 1H NMR and HPLC. Cell types studied included cerebellar granule neurons, type-1 astrocytes, meningeal cells, oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, and oligodendrocytes. A high concentration of NAA was found in extracts of cerebellar granule neurons (approximately 12 nmol/mg of protein), whereas NAA remained undetectable in purified type-1 astrocytes, meningeal cells, and mature oligodendrocytes. However, twice the neuronal level of NAA was found in O-2A progenitors grown in vitro. In addition significant levels of NAA were also detected in cultures of immature oligodendrocytes. Our data partly support previous suggestions that NAA may be a useful neuronal marker for 1H NMR spectroscopic examination of the adult brain. However, they also raise the further possibility that alterations of NAA associated with some specific brain disorders, particularly disorders seen in newborn and young children, may reflect abnormalities in the development of oligodendroglia or their precursors.     

Glutamine Synthetase in Oligodendrocytes and Astrocytes: New Biochemical and Immunocytochemical Evidence

The results of recent immunocytochemical experiments suggest that glutamine synthetase (GS) in the rat CNS may not be confined to astrocytes. In the present study, GS activity was assayed in oligodendrocytes isolated from bovine brain and in oligodendrocytes, astrocytes, and neurons isolated from rat forebrain, and the results were compared with new immunochemical data. Among the cells isolated from rat brain, astrocytes had the highest specific activities of GS, followed by oligodendrocytes. Oligodendrocytes isolated from white matter of bovine brain had GS specific activities almost fivefold higher than those in white matter homogenates. Immunocytochemical staining also showed the presence of GS in both oligodendrocytes and astrocytes in bovine forebrain, in three white-matter regions of rat brain, and in Vibratome sections as well as paraffin sections.     

Enzyme levels in cultured astrocytes,oligodendrocytes and Schwann cells,and neurons from the cerebral cortex and superior cervical ganglia of the rat

Data are presented for 16 enzymes from 8 metabolic systems in cell cultures consisting of approximately 95% astrocytes and 5% oligodendrocytes. Nine of these enzymes were also measured in cultures of oligodendrocytes, Schwann cells, and neurons prepared from both cerebral cortex and superior cervical ganglia. Activities, in mature astrocyte cultures, expressed as percentage of their activity in brain, ranged from 9% for glycerol-3-phosphate dehydrogenase to over 300% for glucose-6-phosphate dehydrogenase. Creatine phosphokinase activity in astrocytes was about the same as in brain, half as high in oligodendrocytes, but 7% or less of the brain level in Schwann cells and superior cervical ganglion neurons and only 16% of brain in cortical neurons. Three enzymes which generate NADPH, the dehydrogenases for glucose-6-phosphate and 6-phosphogluconate, and the NADP-requiring isocitrate dehydrogenase, were present in astrocytes at levels at least twice that of brain. Oligodendrocytes had enzyme levels only 30% to 70% of those of astrocytes. Schwann cells had much higher lactate dehydrogenase and 6-phosphogluconate dehydrogenase activities than oligodendrocytes, but showed a remarkable similarity in enzyme pattern to those of cortical and superior cervical ganglion neurons.Special issue dedicated to Dr. Lewis Sokoloff.     

Vitamin E, Ascorbate, Glutathione, Glutathicne Disulfide, and Enzymes of Glutathione Metabolism in Cultures of Chick Astrocytes and Neurons: Evidence that Astrocytes Play an Important Role in Antioxidative Processes in the Brain

Abstract: GSH, GSSG, vitamin E, and ascorbate were measured in 14-day cultures of chick astrocytes and neurons and compared with levels in the forebrains of chick embryos of comparable age. Activities of enzymes involved in GSH metabolism were also measured. These included -γ-glutamylcysteine synthetase, GSH synthetase, γ-glutamyl cyclotransferase, γ-glutamyltranspeptidase, glutathione transferase (GST), GSH peroxidase, and GSSG reductase. The concentration of lipid-soluble vitamin E in the cultured neurons was found to be comparable with that in the forebrain. On the other hand, the concentration of vitamin E in the astrocytes was significantly greater in the cultured astrocytes than in the neurons, suggesting that the astrocytes are able to accumulate exogenous vitamin E more extensively than neurons. The concentrations of major fatty acids were higher in the cell membranes of cultured neurons than those in the astrocytes. Ascorbate was not detected in cultured cells although the chick forebrains contained appreciable levels of this antioxidant. GSH, total glutathione (i.e., GSH and GSSG), and GST activity were much higher in cultured astrocytes than in neurons. γ-Glutamylcysteine synthetase activity was higher in the cultured astrocytes than in the cultured neurons. GSH reductase and GSH peroxidase activities were roughly comparable in cultured astrocytes and neurons. The high levels of GSH and GST in cultured astrocytes appears to reflect the situation in vivo. The data suggest that astrocytes are resistant to reactive oxygen species (and potentially toxic xenobiotics) and may play a protective role in the brain. Because enzymes of GSH metabolism are generally well represented in cultured astrocytes and neurons these cells may be ideally suited as probes for manipulating GSH levels in neural tissues in vitro. Cultured astrocytes and neurons should be amenable to the study of the effects of various metabolic insults on the GSH system. Such studies may provide insights into the design of therapeutic strategies to combat oxidative and xenobiotic stresses.     

Glutamine synthetase expression in rat oligodendrocytes in culture: regulation by hormones and growth factors.

Glutamine synthetase (GS, EC 6.3.1.2.) has long been considered as a protein specific for astrocytes in the brain, but recently GS immunoreactivity has been reported in oligodendrocytes both in mixed primary glial cell cultures and in vivo. We have investigated its expression and regulation in "pure" oligodendrocyte cultures. "Pure" oligodendrocyte secondary cultures were derived from newborn rat brain primary cultures enriched in oligodendrocytes as described by Besnard et al. (1987) and were grown in chemically defined medium. These cultures contain more than 90% galactocerebroside-positive oligodendrocytes and produce "myelin" membranes (Fressinaud et al., 1990) after 6-10 days in subcultures (30-35 days, total time in culture). The presence of GS in oligodendrocytes from both primary glial cell cultures and "pure" oligodendrocyte cultures was confirmed by double immunostaining with a rabbit antisheep GS and guinea pig antirat brain myelin 2', 3'-cyclic nucleotide 3'-phosphodiesterase. In "pure" oligodendrocyte cultures, about half of cells were labeled with anti-GS antibody. Furthermore, on the immunoblot performed with a rabbit antisheep GS, the GS protein in "pure" oligodendrocyte secondary cultures was visualized as a single band with an apparent molecular mass of about 43 kDa. In contrast, two protein bands for GS were observed in cultured astrocytes. On the immunoblot performed with a rabbit antichick GS, two immunopositive protein bands were observed: a major one migrating as the purified adult chick brain GS and a minor one with a lower molecular mass. Two similar immunoreactive bands were also observed in pure rat astrocyte cultures. Compared to pure rat astrocyte cultures, "pure" oligodendrocyte cultures of the same age displayed an unexpectedly high GS specific activity that could not be explained by astrocytic contamination of the cultures (less than 5%). As for cultured astrocytes, treatment of oligodendrocyte cultures with dibutyryl-adenosine 3':5'-cyclic monophosphate, triiodothyronine, or hydrocortisone increased significantly GS specific activity. Interestingly, epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor that increase the GS activity in astrocytes do not affect this activity in oligodendrocytes. Thus we confirm the finding of Warringa et al. (1988) that GS is also expressed in oligodendrocytes. We show that its activity is regulated similarly in astrocytes and oligodendrocytes by hormones, but that it is regulated differently by growth factors in these two cell types.     

Glutamine Transaminase K and ω-Amidase Activities in Primary Cultures of Astrocytes and Neurons and in Embryonic Chick Forebrain: Marked Induction of Brain Glutamine Transaminase K at Time of Hatching

Abstract: Glutamine transaminase K and ω-amidase activities are present in the chick brain and in the brains of adult mice, rats, and humans. However, the activity of gluta-mine transaminase K in adult mouse brain is relatively low. In the chick embryo, cerebral glutamine transaminase K activity is low between embryonic days 5 and 17, but by day 23 (day of hatching) activity rises dramatically (< 15-fold). Cerebral ω-amidase activity is relatively high at embryonic day 5 but lower between days 5 and 17; at embryonic day 23 the activity rises to a maximum. Both glutamine transaminase K and ω-amidase are present in cultured chick, rat, and mouse astrocytes and neurons. For each species, the activity of glutamine transaminase K is higher in the astrocytes than in the neurons. The activity of ω-amidase is about the same in the cultured chick astrocytes and neurons but significantly higher in rat astrocytes than in rat neurons. The data suggest that the rise in brain glutamine transaminase K activity in the chick embryo at hatching correlates with maturation of astrocytes. Glutamine transaminase K may be involved in glutamine cycling in astrocytes. Glutamine transaminase K appears to be a major cysteine S-conjugate β-lyase of the brain and may play a role in the neurotoxicity associated with exposure to dichloroacetylene and perhaps to other toxins.     

Cytosolic and mitochondrial isoforms of NADP+-dependent isocitrate dehydrogenases are expressed in cultured rat neurons,astrocytes, oligodendrocytes and microglial cells

NADP+-dependent isocitrate dehydrogenases (ICDHs) are enzymes that reduce NADP+ to NADPH using isocitrate as electron donor. Cytosolic and mitochondrial isoforms of ICDH have been described. Little is known on the expression of ICDHs in brain cells. We have cloned the rat mitochondrial ICDH (mICDH) in order to obtain the sequence information necessary to study the expression of ICDHs in brain cells by RT-PCR. The cDNA sequence of rat mICDH was highly homologous to that of mICDH cDNAs from other species. By RT-PCR the presence of mRNAs for both the cytosolic and the mitochondrial ICDHs was demonstrated for cultured rat neurons, astrocytes, oligodendrocytes and microglia. The expression of both ICDH isoenzymes was confirmed by western blot analysis using ICDH-isoenzyme specific antibodies as well as by determination of ICDH activities in cytosolic and mitochondrial fractions of the neural cell cultures. In astroglial and microglial cultures, the total ICDH activity was almost equally distributed between cytosolic and mitochondrial fractions. In contrast, in cultures of neurons and oligodendrocytes about 75% of total ICDH activity was present in the cytosolic fractions. Putative functions of ICDHs in cytosol and mitochondria of brain cells are discussed.     

Differential expression of isoforms of the regulatory subunit of type II cAMP-dependent protein kinase in rat neurons, astrocytes, and oligodendrocytes

The RII-B isoform of the regulatory subunit (R) of cAMP-dependent protein kinase II is abundantly and selectively expressed in cerebral cortex (Erlichman, J., Sarkar, D., Fleischer, N., and Rubin, C. S. (1980) J. Biol. Chem. 255, 8179-8184). In contrast to the cytosolic RII-H isoform from heart and other non-neural tissues, a substantial fraction of cerebral cortex RII-B is tightly associated with cell organelles. In order to study the cellular basis for the localization and abundance of RII-B in this complex and heterogeneous tissue, rat cerebral cortex was fractionated into highly purified populations of neurons, astrocytes, and oligodendrocytes. In neurons and astrocytes more than 80% of the total cAMP-binding activity is contributed by RII subunits, whereas the myelin-producing oligodendrocytes contain nearly equal proportions of RI (from protein kinase I) and RII. Approximately 70% of RII and RI subunits are associated with the particulate fraction in each of the three types of brain cells. The nature of the RII isoforms expressed in the cytosolic and particulate fractions of the purified brain cells was established by performing Western immunoblot and indirect immunoprecipitation analyses with selective and sensitive polyclonal antibodies directed against RII-B. Astrocytes and neurons exhibit high levels of RII-B, whereas oligodendrocytes contain the RII-H isoform. Thus, the expression of RII isoforms is not uniform among brain cells that are anatomically and developmentally related. Rather, it appears that RII-B and RII-H are expressed in a cell-specific fashion within cerebral cortex and this might reflect an RII-mediated adaptation of protein kinase II to the specialized metabolic and functional roles of neurons, astrocytes, and oligodendrocytes.     

Differences in nuclear proteins of neurons, astrocytes and C-6 glioma cells

In an effort to identify cell type specific proteins from brain, we have compared proteins of the cell nucleus from two brain cell types. Using a bulk isolation procedure, we fractionated neurons and astrocytes from adult rat brain. In addition, primary cultures of astrocytes were prepared from one-day old rats. Nuclei from these cells and C-6 glioma cell cultures were isolated and the resulting proteins subjected to two-dimensional gel electrophoresis. Several proteins specific for each cell type were found. While many similarities between bulk brain astrocyte preparations and cultured astrocytes were found, less than pure bulk astrocytes from brain were found to be most similar to those of neurons and not to those from primary cell culture.

The nuclear protein profile of cultured astrocytes differed significantly from that of C-6 cells, indicating the utility of two-dimensional gel analysis for detecting major cell type differences in uniform populations of cells.     

Cellular Location of Glutamine Synthetase and Lactate Dehydrogenase in Oligodendrocyte-Enriched Cultures from Rat Brain

Glial cells were isolated from 1-week-old rat brain and cultured in a serum-free medium supplemented with the hormones insulin, hydrocortisone, and triiodothyronine. After 1 week in culture the cell population consisted mainly of galactocerebroside-positive cells (GC+; oligodendrocytes), the remainder of the cells being positive for glial fibrillary acidic protein (GFAP+; astrocytes). Oligodendrocytes were selectively removed from the cultures by complement-mediated cytolysis. The activities of glutamine synthetase and of various marker enzymes were measured in the nonlysed cells remaining after complement treatment of the cultures and in the culture medium containing proteins of the lysed cells. We found that the cellular activity of glutamine synthetase decreased in parallel with the lysis of GC+ cells and that the activity of glutamine synthetase in the supernatant increased. The activity of glycerol-3-phosphate dehydrogenase, a marker enzyme for oligodendrocytes, was no longer detectable in complement-treated cultures and the activity of glutamine synthetase was markedly lowered, whereas the activity of lactate dehydrogenase was as high as in untreated cultures. The location of glutamine synthetase both in oligodendrocytes and in astrocytes was confirmed by double-label immunocytochemistry with antisera against glutamine synthetase, GC, and GFAP. We conclude that in this culture system glutamine synthetase is expressed in both types of glial cells and that the activity of lactate dehydrogenase is at least one order of magnitude higher in astrocytes than in oligodendrocytes.     

Interleukin-2-like activity in injured rat brain

The lymphokine interleukin-2 (IL-2) promotes division and maturation of oligodendrocytes in culture (1). We now report that a IL-2-like activity was present in injured rat brain. The ion-exchange properties of this activity were similar to those of splenocyte IL-2 but its apparent molecular weight was higher. Brain IL-2-like activity was highest in the tissue immediately adjacent to the injury, reaching a maximal activity of about 8000 U/g tissue after 10 days postlesion. The mitogenic activity of injured-brain extracts on astrocytes and CTLL thymocytes was partially inhibited by monoclonal antibodies to murine IL-2 receptor. However, pure human IL-2 did not have mitogenic activity for cultured rat astrocytes. Purified astrocytes, alone or stimulated in a variety or ways, did not produce IL-2-like activity.     

Aralar mRNA and protein levels in neurons and astrocytes freshly isolated from young and adult mouse brain and in maturing cultured astrocytes

Intense glucose-based energy metabolism and glutamate synthesis by astrocytes require malate–aspartate-shuttle (MAS) activity to regenerate NAD⁺ from NADH formed during glycolysis, since brain lacks significant glycerophosphate shuttle activity. Aralar is a necessary aspartate/glutamate exchanger for MAS function in brain. Based on cytochemical immunoassays the absence of aralar in adult astrocytes was repeatedly reported. This would mean that adult astrocytes must regenerate NAD⁺ by producing lactate from pyruvate, eliminating its use by oxidative and biosynthetic pathways. We alternatively used astrocytes and neurons from adult brain, freshly isolated by fluorescence-activated cell sorting, to determine aralar protein by a specific antibody and its mRNA by real-time PCR. Both protein and mRNA expressions were identical in adult neurons and astrocytes and similar to whole brain levels. The same level of aralar expression was reached in well-differentiated astrocyte cultures, but not until late development, coinciding with the late-maturing brain capability for glutamate formation and degradation.     

Glycerol Phosphate Dehydrogenase Activity of Oligodendrocytes Isolated from Adult Pig Brain: Its Inducibility by Hydrocortisone

Developing oligodendrocytes cultured in vitro express glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) and are known to respond to glucocorticoid treatment by increased activity of GPDH. We present evidence that GPDH is enriched in white matter and oligodendrocytes of adult pig brain. Bulk-isolated oligodendrocytes maintained in culture for several weeks exhibit an almost constant level of GPDH activity. Furthermore, a 4-day stimulation with hydrocortisone induces GPDH specific activity of long-term cultured oligodendrocytes from adult pig brain.     

Neuromodulin (GAP43): a neuronal protein kinase C substrate is also present in 0-2A glial cell lineage. Characterization of neuromodulin in secondary cultures of oligodendrocytes and comparison with the neuronal antigen

Neuromodulin (also called GAP43, G50, F1, pp46), a neural-specific calmodulin binding protein, is a major protein kinase C substrate found in developing and regenerating neurons. Here, we report the immunocytochemical characterization of neuromodulin in cultured 0-2A bipotential glial precursor cells obtained from newborn rat brain. Neuromodulin is also present in oligodendrocytes and type 2 astrocytes (stellate-shaped astrocytes), which are both derived from the bipotential glial 0-2A progenitor cells, but is absent of type 1 astrocytes (flat protoplasmic astrocytes). These results support the hypothesis of a common cell lineage for neurons and bipotential 0-2A progenitor cells and suggest that neuromodulin plays a more general role in plasticity during development of the central nervous system. The expression of neuromodulin in secondary cultures of newborn rat oligodendrocytes and its absence in type 1 astrocytes was confirmed by Northern blot analysis of isolated total RNA from these different types of cells using a cDNA probe for the neuromodulin mRNA and by Western blot analysis of the cell extracts using polyclonal antibodies against neuromodulin. The properties of the neuromodulin protein in cultured oligodendrocytes and neuronal cells have been compared. Although neuromodulin in oligodendrocytes is soluble in 2.5% perchloric acid like the neuronal counterpart it migrates essentially as a single protein spot on two-dimensional gel electrophoresis whereas the neuronal antigen can be resolved into at least three distinct protein spots. To obtain precise alignments of the different neuromodulin spots from these two cell types, oligodendrocyte and neuronal cell extracts were mixed together and run on the same two-dimensional gel electrophoresis system. Oligodendroglial neuromodulin migrates with a pI identical to the basic forms of the neuronal protein in isoelectric focusing gel. However, the glial neuromodulin shows a slightly lower mobility in the second dimensional lithium dodecyl sulfate-PAGE than its neuronal counterpart. As measured by 32Pi incorporation, neuromodulin phosphorylation in oligodendrocytes is dramatically increased after short-term phorbol ester treatments, which activate protein kinase C, and is totally inhibited by long-term phorbol ester treatments, which downregulates protein kinase C, thus confirming its probable specific in vivo phosphorylation by protein kinase C. In primary cultures of neuronal cells, two of the three neuromodulin spots were observed to be phosphorylated with an apparent preferential phosphorylation of the more acid forms.     

Aralar mRNA and protein levels in neurons and astrocytes freshly isolated from young and adult mouse brain and in maturing cultured astrocytes

Intense glucose-based energy metabolism and glutamate synthesis by astrocytes require malate–aspartate-shuttle (MAS) activity to regenerate NAD⁺ from NADH formed during glycolysis, since brain lacks significant glycerophosphate shuttle activity. Aralar is a necessary aspartate/glutamate exchanger for MAS function in brain. Based on cytochemical immunoassays the absence of aralar in adult astrocytes was repeatedly reported. This would mean that adult astrocytes must regenerate NAD⁺ by producing lactate from pyruvate, eliminating its use by oxidative and biosynthetic pathways. We alternatively used astrocytes and neurons from adult brain, freshly isolated by fluorescence-activated cell sorting, to determine aralar protein by a specific antibody and its mRNA by real-time PCR. Both protein and mRNA expressions were identical in adult neurons and astrocytes and similar to whole brain levels. The same level of aralar expression was reached in well-differentiated astrocyte cultures, but not until late development, coinciding with the late-maturing brain capability for glutamate formation and degradation.     

Uptake, Release, and Metabolism of Citrate in Neurons and Astrocytes in Primary Cultures

Abstract: Synthesis, uptake, release, and oxidative metabolism of citrate were investigated in neurons and astrocytes cultured from cerebral cortex or cerebellum. In addition, the possible role of citrate as a donor of the carbon skeleton for biosynthesis of neurotransmitter glutamate was studied. All cell types expressed the enzyme citrate synthase at a high activity, the cerebellar granule neurons containing the enzyme at a higher activity than that found in the astrocytes from the two brain regions or the cortical neurons. Saturable citrate uptake could not be detected in any of the cell types, but the astrocytes, and, in particular, those of cerebellar origin, had a very active de novo synthesis and release of citrate (～70 nmol × h^?1× mg of protein^?1). The rate of release of citrate from neurons was <5% of this value. Using [¹⁴C]citrate it could be shown that citrate was oxidatively metabolized to ¹⁴CO₂ at a modest rate (～1 nmol × n^?1× mg^?1 of protein) with slightly higher rates in astrocytes compared with neurons. Experiments designed to investigate the ability of exogenously supplied citrate to serve as a precursor for synthesis of transmitter glutamate in cerebellar granule neurons failed to demonstrate this. Rather than citrate serving this purpose it may be suggested that astrocytically released citrate may regulate the extracellular concentration of Ca²⁺ and Mg²⁺ by chelation, thereby modulating neuronal excitability.     

Effects of Arachidonic Acid on Glutamate and γ-Aminobutyric Acid Uptake in Primary Cultures of Rat Cerebral Cortical Astrocytes and Neurons

The effects of arachidonic acid on glutamate and gamma-aminobutyric acid (GABA) uptake were studied in primary cultures of astrocytes and neurons prepared from rat cerebral cortex. The uptake rates of glutamate and GABA in astrocytic cultures were 10.4 nmol/mg protein/min and 0.125 nmol/mg protein/min, respectively. The uptake rates of glutamate and GABA in neuronal cultures were 3.37 nmol/mg protein/min and 1.53 nmol/mg protein/min. Arachidonic acid inhibited glutamate uptake in both astrocytes and neurons. The inhibitory effect was observed within 10 min of incubation with arachidonic acid and reached approximately 80% within 120 min in both types of culture. The arachidonic acid effect was not only time-dependent, but also dose-related. Arachidonic acid, at concentrations of 0.015 and 0.03 mumol/mg protein, significantly inhibited glutamate uptake in neurons, whereas 20 times higher concentrations were required for astrocytes. The effects of arachidonic acid were not as deleterious on GABA uptake as on glutamate uptake in both astrocytes and neurons. In astrocytes, GABA uptake was not affected by any of the doses of arachidonic acid studied (0.015-0.6 mumol/mg protein). In neuronal cultures, GABA uptake was inhibited, but not to the same degree observed with glutamate uptake. Lower doses of arachidonic acid (0.03 and 0.015 mumol/mg protein) did not affect neuronal GABA uptake. Other polyunsaturated fatty acids, such as docosahexaenoic acid, affected amino acid uptake in a manner similar to arachidonic acid in both astrocytes and neurons. However, saturated fatty acids, such as palmitic acid, exerted no such effect. The significance of the arachidonic acid-induced inhibition of neurotransmitter uptake in cultured brain cells in various pathological states is discussed.     

Selective tropism of a neurotropic coronavirus for ependymal cells, neurons, and meningeal cells.

The ability of a neurotropic virus, mouse hepatitis virus type 3 (MHV3), to invade the central nervous system (CNS) and to recognize cells selectively within the brain was investigated in vivo and in vitro. In vivo, MHV3 induced in C3H mice a genetically controlled infection of meningeal cells, ependymal cells, and neurons. In vitro, purified MHV3 bound to the surface of isolated ependymal cells and cultured cortical neurons but not to oligodendrocytes or cultured astrocytes. MHV3 replicated within cultured cortical neurons and neuroblastoma cells (NIE 115); infected cultured neurons nonetheless survived and matured normally for a 7-day period postinfection. On the other hand, MHV3 had a low affinity for cortical glial cells or glioma cells (C6 line), both of which appear to be morphologically unaltered by viral infection. Finally, MHV3 infected and disrupted cultured meningeal cells. This suggests that differences in the affinity of cells for MHV3 are determinants of the selective vulnerability of cellular subpopulations within the CNS. In vivo, a higher titer of virus was needed for CNS penetration in the genetically resistant (A/Jx) mice than in the susceptible (C57/BL6) mouse strain. However, in spite of viral invasion, no neuropathological lesions developed. In vitro viral binding to adult ependymal cells of susceptible and resistant strains of mice was identical. Genetic resistance to MHV3-CNS infection appeared to be mediated both by a peripheral mechanism limiting viral penetration into the CNS and by intra-CNS mechanisms, presumably at a stage after viral attachment to target cells.     

Protein synthesis by astrocytes in primary cultures

Protein synthesis, measured as leucine incorporation into acid-precipitable proteins, was determined in astrocytes in primary cultures obtained from the cerebral hemispheres of newborn mice. As can be expected for eucaryotic, ribosomal protein synthesis, the incorporation was almost completely inhibited by cycloheximide (0.01 mM), but unaffected by chloramphenicol (0.03 mM). The rate of synthesis, measured during exposure to a high (0.8 mM) concentration of leucine was 5.4 nmol/hr/mg protein in mature (i.e., at least 4-week-old) cultures. This value is at least twice as high as the protein synthesis rates reported for the adult brain in vivo, suggesting that a very considerable part of the protein synthesis in the adult brain may take place in astrocytes. The molecular weight distribution of the synthesized proteins was determined by polyacrylamide gel electrophoresis, demonstrating synthesis of at least 50 different polypeptides, ranging in molecular weight between 190,000 and 27,000 daltons. The pattern of the synthesized proteins underwent considerable alteration with age in young cultures in which the total content of protein was still increasing, but it was remarkably stable after the age of two weeks. Exposure to dibutyryl cyclic AMP, which is known to alter morphology, content of glial fibrillary acidic protein (GFA), and activities of certain enzymes in the cultured astrocytes, caused marked alterations in the pattern of the synthesized proteins.