bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma membrane of cultured oligodendrocytes: I. Isolation, purification, and initial characterization
Authors: Polak Paul E. , Szuchet Sara
Published: January 2005

Oligodendrocytes generate myelin as extensions of the plasma membrane. Myelin has been well characterized, yet little is known concerning oligodendrocyte plasma membrane. We have developed a reproducible method for the isolation of an oligodendrocyte plasma membrane‐rich fraction (F2.2). F2.2 has a 25‐fold enrichment in K+‐dependent p‐nitrophenyl phosphatase, a plasma membrane marker. Impurities are composed of Golgi elements (8–12%), microsomes (4–6%), and lysosomal membranes (1–5%). Our starting material was oligodendrocytes kept in culture in a nonattached state for 3 to 5 d. After disrupting the cells and removing nuclei (P1), the supernatant (SP1) was fractionated on a self‐generating 20% Percoll gradient into three bands: F1, F2, and F3. F1 had only 3% of the applied protein and was not characterized. F2, with 11% of the protein, was fivefold enriched in plasma membrane. F3 had 27% of initial protein; it consisted of a crude mitochondrial and lysosomal fraction. F2 was further purified by first washing it hypotonically, treating it with Mg2+, and then fractionating it on a Ficoll step gradient that yielded F2.2 at the interphase. Morphologically F2.2 comprises (1) membranous sheets, often with more than one membrane in close apposition; (2) membrane vesicles of various sizes and shapes frequently filled with amorphous material; (3) Golgi elements; and (4) unrecognizable profiles. The sodium dodecyl sulfate‐polyacrylamide gel electrophoresis protein profile of F2.2 reveals CNPase as a major component in agreement with the high CNPase specific activity (3,860 μmol/mgP/h) found in F2.2 Other significant polypeptides have Mr = 170,000, 135,000, 108,000, 80,000, 53,000, 38,500, 32,000, and 22,200.
Plasma membrane of cultured oligodendrocytes: II. Possible structural and functional domains
Authors: Szuchet Sara , Polak Paul E. , Yim Sung Hye , Lange Yvonne
Published: January 2005

An oligodendrocyte plasma membrane‐rich fraction, F2.2, was resolved by equilibrium density centrifugation on a linear sucrose gradient from 0.5 M to 1.3 M into three fractions, F2.2a, F.2.2b, F2.2c, and a pellet F2.2p. F2.2a and F.2.2b were enriched 1.5‐fold relative to F2.2 in plasma membrane markers at the expense of F2.2c and F2.2p, which became correspondingly impoverished. This gave F2.2a and F2.2b a 42‐fold and 37‐fold enrichment, respectively, in plasma membrane markers relative to the initial cell homogenate. F2.2c had a sevenfold enrichment in a Golgi marker; together with F2.2p, they contained all the Golgi marker initially present in F2.2. Preliminary data indicated that the F2.2‐subfractions differed from one another in their molar ratios of cholesterol to phospholipids and protein to lipids but had similar protein profiles when examined by sodium dodecylsulfate‐polyacrylamide gel electrophoresis. Their content of fucosylated glycoproteins appeared also to be different. Morphologically, F2.2a and F2.2b were very similar: they contained large membrane vesicles, membrane sheets, and vesicles entrapped within other vesicles. Membrane‐membrane interaction was apparent in these fractions. F2.2c had many of the same elements, but most of the membrane structures contained amorphous material. F2.2p differed morphologically from the other fractions in that it had principally electron‐dense structures. It is postulated that F2.2a, F2.2b, and perhaps F2.2c represent different domains of oligodendrocyte plasma membrane. Alternatively, these fractions might correspond to the plasma membrane of oligodendrocyte subtypes.
Direct effects of carbachol on membrane potential and ion activities in leech glial cells
Authors: Ballanyi K. , Schlue W.‐R.
Published: January 2005

Ion‐selective double‐barreled microelectrodes were used to measure the activities of intracellular K+ and Na+ (a, a) and the membrane potential (Em) in neuropile glial cells as well as extracellular K+ (a) in the neuropile of segmental ganglia in the leech, Hirudo medicinalis. Bath‐application of carbachol resulted in a prominent membrane depolarization. This depolarization was accompanied by transient increases of a and a, whereas a decreased. It is suggested that the carbachol‐induced depolarization and the underlying ion activity changes are due to activation of an acetylcholine receptor‐coupled cation channel in the membrane of the neuropile glial cells.
Swelling of astrocytes causes membrane potential depolarization
Authors: Kimelberg H. K. , O'Connor E.
Published: January 2005

Rat brain astrocytes growing in primary monolayer cultures were swollen by exposing them to media of decreasing osmolality caused by removal of NaCl, and the effects of this treatment on their membrane potentials were measured by intracellular recording. Depolarizations were seen that were proportional to the degree of swelling, reaching a maximum of around 60 mV when 80–100 mM NaCl was removed from the reaction media, which had an original total osmolality of 290 mosmolal. These effects were completely reversible, since restoring the cells to iso‐osmotic medium after a 2‐min exposure caused an immediate repolarization back to the original membrane potentials, and depolarizations were not seen when isotonicity was maintained by replacing NaCl with sucrose. Partial repolarization was seen during an extended period (30 min) of exposure to hypo‐osmotic medium, mirroring a regulatory volume decrease we have previously described in these cells under identical conditions. In ion‐replacement studies depolarizatins were seen when the solution was made hypo‐osmotic with the large cation N‐methyl‐D‐glucamine totally replacing Na+ in the medium. Removal of Cl− from the medium also had no effect on the initial swelling‐induced depolarization. These results show that even moderate swelling of astrocytes in primary culture results in marked depolarization of their membrane potentials; possible mechanisms for this effect and the potentially profound implications for the swelling of astrocytes seen in situ under pathological conditions are discussed.
Glial conditioned medium enables jimpy oligodendrocytes to express properties of normal oligodendrocytes: Production of myelin antigens and membranes
Authors: Bartlett William P. , Knapp Pamela E. , Skoff Robert P.
Published: January 2005

Cultures of cerebral cells from 2‐day‐old jimpy (jp) and normal animals were immunocytochemically stained with antibodies to galactocerebroside, 2′, 3′‐cyclic nucleotide 3′‐phosphohydrolase and proteolipid protein (PLP). As the normal cultures mature, oligodendrocytes express these markers in cell bodies, in processes and in expansive membrane sheets produced along the lengths of cell processes. In contrast, oligodendrocytes from jp cultures do not produce large membrane sheets and their total numbers are reduced. Only rarely do jp oligodendrocytes exhibit immunostaining for PLP. Thus, jp oligodendrocytes grown in vitro mimic deficiencies of jp CNS in situ. In order to examine the effect of environment and cell‐cell interactions on expression of the jp mutation we grew jp cerebral cells in the presence of medium conditioned for short periods by normal cerebral cells. Under these conditions jp oligodendroglia appeared nearly normal by immunostaining criteria. Their numbers were increased; they were able to produce and maintain membrane sheets; and some cells expressed PLP. These results show that jp oligodendrocytes have the capacity to express certain normal phenotypic parameters, and when given an appropriate environment they do so. The effects of normal conditioned medium may be due to a secreted factor, possibly produced by the astrocytes, which constitute the vast majority of cells in the conditioning cultures. The dramatic effect of normal glial conditioned medium on jp oligodendrocytes suggests that the steps leading to hypomyelination in jp are exceedingly complex and may involve glial‐glial interactions.
Ultracytochemical localization of adenylate cyclase and guanylate cyclase in crushed peripheral nerves
Authors: Rambotti Maria Grazia , Spreca Antonio , Rende Mario , Donato Rosario
Published: January 2005

Cellular and subcellular distribution of adenylate cyclase (AC) and guanylate cyclase (GC) activities in crushed peripheral nerves during regeneration were studied at the electron microscope level. In unlesioned nerves, no AC reaction product could be evidenced, whereas GC was detectable on the plasma membranes of Schwann cells, myelinated and nonmyelinated fibers, and within nonmyelinated axons. At 24 hours after the crush, AC reaction product was found within axonal segments proximal to the zone of the crush in association with mitochondria. At this stage, macrophage‐like cells, which probably are transformed Schwann cells, polymorphonuclear leucocytes, and endothelial cells displaying an intense AC reaction product could be detected. On the other hand, at 24 hours after the crush, GC was no longer detectable, except on occasional unlesioned nerve fibers. At 48 hours after the lesion, AC reaction product was no longer detectable within axons, and all AC positivity was associated with plasma membranes of non‐neuronal cells, including transformed Schwann cells, occasional macrophages, polymorphonuclear leucocytes, fibroblasts, and elongated cells. As to GC, images similar to those obtained at 24 hours were observed until 48 hours after the crush. From the 7th to the 28th postlesion day, AC activity was localized exclusively to the plasma membranes of fibroblasts and elongated cells. Transformed Schwann cells were no longer detectable, whereas normal Schwann cells and regenerating axons could be seen, and these showed no AC reaction product in analogy to the absence of AC reaction product of unlesioned nerves. During the same period, GC again was detectable on regenerating fibers with the same subcellular localization as that of unlesioned nerves. The present results strongly suggest that starting from the second postcrush day, cells invading the lesioned zone and transformed Schwann cells, all taking part in the formation of the new perineurial tissue, display a high AC activity, which should be taken into account when measuring cyclic adenosine monophosphate (cAMP) levels under these conditions. Also, our data suggest that GC is involved primarily in regeneration processes that occur in crushed peripheral nerves. Thus, the pattern of AC distribution in peripheral unlesioned and lesioned nerves appears to be exactly the opposite of the GC localization examined under similar experimental conditions insofar as nervous fibers are concerned.
Axon‐glia transfer of a protein and a carbohydrate
Authors: Grossfeld Robert M. , Klinge Maura A. , Lieberman Edward M. , Stewart Louise C.
Published: January 2005

We have investigated the transfer of a fluorescent protein, the fluorescein isothiocyanate derivative of bovine serum albumin (FITC‐BSA), and a fluorescent carbohydrate, FITC‐dextran, from the crayflsh medial giant axon (MGA) to the periaxonal glial cells. The dialyzed tracer was injected into one of the two MGAs, and, after a transfer period of 15–60 min, the tissue was fixed for histological examination of fluorescence distribution. With each tracer, the periaxonal sheath of the injected MGA was specifically labeled. Similar results were obtained with several different fixatives. During the transfer period, there was no appreciable change in the resting potential or conducted action potential of the MGA or in the resting potentials of the adaxonal glial cells. Polyacrylamide gel electrophoresis indicated that the axoplasmic and sheath fluorescence was produced by the intact tracers. These results suggest that “foreign” macromolecules can be exchanged from crayfish axons to glia under physiological conditions. Such transfers may indicate a substantial intercellular traffic of molecules or a means whereby neurons can eliminate waste materials.
Effect of thyroid deficiency on the development of glia in the hippocampal formation of the rat: An immunocytochemical study
Authors: Rami Abdelhaq , Rabíe Alain
Published: January 2005

The development of glia in the hippocampal formation of normal and hypothyroid rats was studied using immunocytochemical staining for either glial fibrillary acidic protein (GFAP) or vimentin. Light microscopy showed lower GFAP immunoreactivity in the radial glial processes of young hypothyroid rats compared to normal animals. These processes followed the known path of neuroblast migration toward the proliferative zone of the dentate gyrus until the end of the 1st postnatal week. Vimentin immunoreactivity showed that the glial processes were present and therefore immature at least with respect to their cytoskeletal composition. We propose that this early defect in the maturation of the radial glial fibers accounts for the final deficit in the granule cells of the dentate gyrus. Later in development, thyroid deficiency also reduced the density and number of GFAP‐labeled astrocytes and the growth of their processes. This observation is in complete disagreement with the glial hypertrophy induced by thyroid deficiency in the cerebellum. The considerably increased histogenetic cell death observed in the cerebellum of young hypothyroid rats could in turn induce glial hypetrophy, whereas the hippocampal formation, where a normal low number of cell deaths is observed, is only subjected to the general depressive effect of thyroid deficiency on cell maturation.
Glutamate and kainate increase intracellular sodium activity in leech neuropile glial cells
Authors: Ballanyi K. , Dörner R. , Schlue W.‐R.
Published: January 2005

Na+‐selective, double‐barrelled microelectrodes were used to measure intracellular Na+ activity (a) and membrane potential (Em) in neuropile glial cells of isolated segmental ganglia in the leech Hirudo medicinalis. Bath application of glutamate (10−3 M) resulted in membrane depolarizations of about 5 mV and a concomitant increase of a by between 2 and 10 mM. Kainate (10−4 M) elicited depolarizations of up to 40 mV amplitude followed by a prominent afterhyperpolarization. During kainate, a increased by 7 to 25 mM. In contrast to glutamate, an initial decrease of a was detected during the action of kainate. N‐methyl‐D‐aspartate (NMDA, 10−5–10−3M) had no effect of Em and a. The results indicate that leech glial cells have a kainate‐preferring non‐NMDA glutamate receptor.
Glia:Neuron index: Review and hypothesis to account for different values in various mammals
Authors: Reichenbach Andreas
Published: January 2005

The present paper proposes a hypothesis to account for different values of the glia:neuron index in comparable central nervous system tissues of various mammals. This hypothesis assumes that K+ ions released by active neurons are a mitogenic signal for glial cells. The thicker the tissue (for example, the brain wall), the more difficult is efficient K+ clearance, and more perinatal glial cell proliferation should occur. Thus, this hypothesis accounts for higher glia:neuron indices in mammals with thicker brain walls.
Pharmacological identification of the α‐adrenergic receptor type which inhibits the β‐adrenergic activated adenylate cyclase system in cultured astrocytes
Authors: Northam William J. , Bedoy Cynthia A. , Mobley Philip L.
Published: January 2005

The adrenergic agonist norepinephrine can exert its influence on cell function by activating both α‐ and β‐adrenergic receptors. In astrocytes, the α‐adrenergic receptor activity of norepinephrine is known to inhibit the cyclic AMP response elicited by its action at β‐adrenergic receptors. Pharmacological studies were conducted to identify the subtype of α‐adrenergic receptor which mediates this inhibitory action. The α2‐adrenergic antagonist yohimbine potentiated the cyclic AMP response elicited by norepinephrine, whereas the α1‐adrenergic antagonist prazosin did not affect the response. The α2‐adrenergic agonist clonidine inhibited the cyclic AMP response elicited by the β‐adrenergic agonist isoproterenol and this inhibition could be blocked by yohimbine but not by prazosin. In contrast, the α1‐adrenergic agonist phenylephrine did not inhibit the cyclic AMP response to isoproterenol. These studies indicate that the inhibitory action of norepinephrine is mediated by its action at α2‐adrenergic receptors.
Astrocytes in cell culture incorporate GM1 ganglioside
Authors: Masco Daniel , Flott Berenike , Seifert Wilfried
Published: January 2005

Ganglioside GM1 3H‐labelled at the terminal galactose was added to astrocyte cell cultures. GM1 incorporation was studied in the two typical forms of astrocytes in cell culture of flat and stellate morphology. There was a strong time‐ and concentration‐dependent increase in GM1 incorporation for both cell types of astrocytes. The incorporation of GM1 into the stellate form increased continuously up to 48 h (maximum time studied), while the incorporation into the flat form reached a plateau at the same time. After 2 h of GM1 incubation additional gangliosides appeared; the latter resulted from the metabolism of the GM1 incorporated, indicating that astrocytes in cell culture can biosynthesize more complex gangliosides. To confirm that GM1 was indeed incorporated into astrocytes, two other different approaches were used. Astrocyte cells treated with 3H‐GM1 were visualized using autoradiography. The specific marker for GM1, rhodamine‐labelled choleratoxin, was used to detect the incorporated GM1 using fluorescence microscopy. In both cases GM1 treated cells were intensely labelled. These observations indicate that exogenous GM1 ganglioside can also be integrated into the astrocyte membranes as occurs in other types of cells and membranes.
Patterns of gliosis in alzheimer's disease and aging cerebrum
Authors: Beach T.G. , Walker R. , McGeer E.G.
Published: January 2005

The distribution of astrocytic gliosis in Alzheimer's disease (AD) and aging cerebrum, as marked by immunoperoxidase staining for glial fibrillary acidic protein (GFAP), was examined in whole‐hemisphere coronal sections. Cortical gliosis in AD had an obvious laminar pattern. There were two heavy bands of staining, one in layers II–III and another in layer V. Normal aging cases sometimes displayed considerable cortical gliosis, but no specific patterns were apparent. Most AD cases, and some normal aging cases, displayed hypertrophy of immunoreactive astrocytes at grey matter‐white matter interfaces, especially the cortico‐medullary junction. Subcortical grey matter gliosis was common in both normal aging and AD, but there was no consistent pattern in either group. The deep cerebral white matter, which is stained evenly and heavily in young, healthy individuals, showed uneven staining in both normal elderly and AD brains. In both AD and aging, perivascular gliosis was prominent throughout the cerebrum and especially in the putamen. In conclusion, both AD and aging cerebri show extensive gliosis: AD cortical gliosis has a specific laminar pattern, but there does not appear to be an AD‐specific pattern of subcortical gliosis.
Cell cultures enriched in oligodendrocytes from the central nervous system of trout in terms of phenotypic expression exhibit parallels with cultured rat schwann cells
Authors: Jeserich Gunnar , Rauen Thomas
Published: January 2005

Oligodendrocytes were isolated from the white matter of young trout by Percoll density centrifugation of enzymatically dissociated tissue and cultured on poly‐D‐lysine–coated petri dishes. Using antisera recognizing myelin‐specific compounds of fish CNS (36K, IP2) up to 72% of the isolated cells could be identified as oligodendrocytes with an average yield of 4 X 106 cells per gram of wet tissue. Taken in culture, the cells rapidly regenerated their processes and soon acquired a morphology closely resembling mammalian oligodendrocytes in vitro. On the other hand, in terms of phenotypic expression, interesting parallels were revealed with the known in vitro behavior of Schwann cells: Galactocerebroside, which in mammalian oligodendrocytes is persistently expressed over longer periods of time in vitro, rapidly disappeared from the surface of cultured trout oligodendrocytes. In contrast, the fish CNS myelin glycoprotein IP2, which like IP1 is immunologically related to the major myelin product of Schwann cells, Po, was continuously expressed over several weeks in culture. Two other myelin protein constituents, 36K and IP1, transiently declined in vitro, but later on fully reappeared in the glial cells of trout. The present cell culture system offers an experimental model for studying in vitro the factors underlying oligodendroglial regeneration and remyelination in the fish CNS.
Immunohistochemical localization of glutamine synthetase in mesencephalon and telencephalon of the lizard Gallotia galloti during ontogeny
Authors: Monzon‐Mayor M. , Yanes C. , Tholey G. , De Barry J. , Gombos G.
Published: January 2005

The immunohistochemical localization of glutamine synthetase, an astrocyte marker in mammals, was determined in the telencephalon and mesencephalon of the lizard Gallotia galloti during development by using an antiserum raised against chicken brain glutamine synthetase. Ependymal glial cells and their radial processes were glutamine synthetase immunoreactive, and they were present also in the adult. Immunoreactivity was also detected in two populations of scattered cell bodies, each preferentially localized in different zones: star‐shaped cells morphologically similar to mammalian astrocytes, and ovoid or pear‐shaped cell bodies, the processes of which were aligned with radial fibers and formed perivascular end‐feet. Both populations displayed ultrastructural characteristics of astrocytes even though a comparison with our previous results (Monzon‐Mayor et al., 1989; Yanes et al., 1989) indicated that many of these cells did not react with antibodies directed against the astrocyte‐specific glial fibrillary acidic protein.

During ontogeny, glutamine synthetase immunoreactivity appeared in radial glial processes and in ependymal glial cells of midbrain at embryonic stage 35 (E35) and of telencephalon at E37; in both regions, immunoreactivity in the radial glia increased until hatching and then decreased until adulthood, but it did not disappear. Labelled scattered cells became progressively more numerous and more immunoreactive.

A comparative analysis of the distribution of these cells at different ages tends to suggest that some of the “ovoid” astrocytes originate in, and migrate out from, the proliferative zone of the different sulci, whereas the star‐shaped cells appear directly in situ, probably because they begin to express glutamine synthetase after they have reached their final location.
Short‐term post‐grafting morphological alterations of glia from an adult brain transplant
Authors: Ignacio V. , Gansmuller A. , Collins V. P. , Suard I. , Jacque C.
Published: January 2005

Fragments of corpus callosum from adult rabbit have been implanted into the brain of newborn mice. Previous studies had shown that under such conditions transplant‐derived astroglial cells differentiate in the host and survive for at least 2 months. The present study was devised to clarify the fate of the differentiated astrocytes present in the adult transplant by using combined ultrastructural and immunohistochemical approaches. These mature cells are shown to degenerate and die within 2 days after the implantation. Therefore, we suggest that stem cells present in adult tissue would account for the surviving population of transplant‐derived glial cells.
Enhanced release of plasminogen activator inhibitor(s) but not of plasminogen activators by cultured rat glial cells treated with interleukin‐1
Authors: Rogister B. , Leprince P. , Delree P. , Van Damme J. , Billiau A. , Moonen G.
Published: January 2005

Astroglial cells are known to proliferate during development of the nervous system, as well as during post‐traumatic gliosis. We have previously shown that the proliferation of cultured astrocytes can be stimulated by the urokinase‐type (uPA) of plasminogen activator (PA) and that astrocytes are able to release such uPA upon stimulation with basic fibroblast growth factor, which is known to act as a mitogen for these cells.

Here we report studies on the effects of human interleukin‐1 (IL‐1) on the release of PA activity by cultured newborn rat astroglial cells. Whereas there is controversy in the literature as to whether IL‐1 stimulates multiplication of astroglial cells, we failed to observe such an effect in our system. We did observe, however, a dose‐dependent decrease in PA activity in the supernatant of the IL‐1 treated cultures. Further analysis revealed that this apparent decrease in PA release was in fact due to an increased release of plasminogen activator inhibitor (PAI). A similar IL‐1 induced increase in PAI release was also found to occur in cultures of transformed astrocytes (human glioma LN18) and in cultured Schwann cells, but not in cultures of neurons or neuronal tumour cells.

Since protease inhibitors are known to possess neuritogenic properties, our results suggest that IL‐1, by its capacity to induce PAI, may promote neuritogenesis.
Increased glutamate uptake and glutamine synthetase activity in neuronal cell cultures surviving chronic hypoxia
Authors: Sher Phyllis K. , Hu Shuxian
Published: January 2005

To examine the neurochemical effects of chronic hypoxia on immature nervous tissue in vitro, mixed neuronal–glial cell cultures derived from fetal mice were exposed to 5% O2 for 24 or 48 h. Those cultures subjected to longer hypoxia manifested improved neuronal survival compared to those with the shorter insult, both with respect to neuronal morphology and also cell counts. Neurochemical assays were performed on living cells in situ to determine the possible basis for differential cell survival. After both exposure conditions, Ro5–4864‐displaceable benzodiazepine (BDZ) binding, reflecting nonneuronal BDZ binding sites, was either not reduced or was elevated. Although initially reduced, binding of the excitatory amino acid (EAA) glutamate was progressively increased after both insults and, within 2 days after return to normoxia, was increased relative to control values (121 and 128% of controls, P < 0.05). The most impressive neurochemical differences between the two conditions related to changes in the predominantly or exclusively glial functions of glutamate uptake and glutamine synthetase activity. In those cultures with relatively preserved neuronal morphology: 1) high affinity uptake of glutamate was elevated compared to the shorter hypoxic insult by 3 days of recovery (104 vs 70%, P < 0.001) and 2) glutamine synthetase, an enzyme localized primarily within astrocytes, was significantly elevated even when compared to absolute control values (148%, P < 0.001). These data suggest that longer periods of hypoxia may be less deleterious to neurons than shorter hypoxic events because of a time‐dependent stimulation of specific glial cell functions which relate to increased metabolism of potentially neurotoxic EAAs such as glutamate.
Patchy myelination pattern in the jimpy mouse brain: Immunohistochemical study
Authors: Lachapelle F. , Lapie P. , Gansmuller A. , Villarroya H. , Gumpel M.
Published: January 2005

The jimpy (jp) mutation of the mouse leads to a dramatic decrease of myelination in the hemizygous mutant central nervous system (CNS). Several descriptions based on classical histology, immunohistochemistry, and electron microscopy (EM) have demonstrated the scarcity of myelin formation in the different parts of the CNS. The immunohistochemical study presented here showed a very singular patchy pattern of myelin distribution in the different areas of the whole mutant brain. The myelin patches are randomly dispersed without bilateral symmetry, and their density and location vary from one animal to another. No reproducible pattern of myelination could be found among the population observed. This distribution has been compared with observations on young heterozygotes and wild‐type homozygotes from the same strain. A similar patchy and random distribution of myelin could be observed in heterozygotes, which present an intermediate level of myelination. This strongly suggests that a migration of precursors or immature oligodendrocytes (ODCs) from the periventricular zone followed by local multiplication of colonies of ODCs before myelination is a general feature in normal as well as pathological conditions.
Channel‐mediated and carrier‐mediated uptake of K+ into cultured ovine oligodendrocytes
Authors: Hertz L. , Soliven B. , Hertz E. , Szuchet S. , Nelson D. J.
Published: January 2005

Uptake of radioactive K+ by mature ovine oligodendrocytes (OLGs) maintained in primary culture was measured under steady‐state conditions, i.e., in cells maintained in a normal tissue culture medium (5.4 mM K+), and in cells after depletion of intracellular K+ to less than 15% of its normal value by pre‐incubation in K+‐free medium. The latter value is dominated by an active, carrier‐mediated uptake (although it may include some diffusional uptake), wheras the former, in addition to active uptake, also reflects passive K+ diffusion through ion selective channels and possible selfexchange between extracellular and intracellular K+, which may be carrier‐mediated. The total uptake rate was 144 ± 10 nmol/min/mg protein, and the uptake after K+ depletion was 60 ± 2 nmol/min/mg protein, much lower rates than previously observed in astrocytes. The uptake into K+‐depleted cells was inhibited by about 80% in the presence of ouabain (1 mM) and about 30% in the presence of furosemide (2 mM). Activators of protein kinase C (phorbol esters) and cAMP‐dependent protein kinase (forskolin) have been shown to alter the myelinogenic metabolism as well as outward K+ current in cultured OLGs. The present study demonstrates that K+ homeostasis in OLGs is modulated through similar second messenger pathways. Active uptake was inhibited by about 60% in the presence of active phorbol esters (100 nM) but was not affected by forskolin (100 nM). Forskolin likewise had no effect on total uptake, whereas phorbol esters caused a much larger inhibition than expected from their effect on carrier‐mediated uptake alone, suggesting that channel‐mediated uptake was also reduced. The relatively small difference between the carrier‐mediated and the total uptake made it difficult to assess accurately the effects of these agents on channel‐mediated uptake. Electrophysiological experiments were carried out to assess directly the inhibitory effects of these compounds on inward rectifier K+ channel (Kir+) activation. The phorbol ester, phorbol 12‐myristate 13‐acetate (1.5 to 100 nM), decreased the amplitude of the steady‐state Kir+ current at ‐120 mV by approximately 40%; however, neither the inactive analog 4‐α phorbol‐12–13 didecanoate nor forskolin (0.1 to 1 μM) decreased current amplitude when extracellular solutions contained normal K+ concentrations. It can be concluded that both the carrier‐mediated K+ uptake and passive K+ influx through inwardly rectifying K+ channels in OLGs are modulated by protein kinase C but not by cAMP‐dependent protein kinase activation. Furthermore, both carrier‐mediated and total K+ uptake in oligodendrocytes are significantly lower than that measured in astrocytes, implying differential physiological roles for astrocytes and oligodendrocytes in K+ homeostasis.
Glial cell lineage in the cerebral cortex: A review and synthesis
Authors: Cameron Richard S. , Rakic Pasko
Published: January 2005

The present review is focused on the cell lineage relationships underlying gliogenesis in the cerebral cortex. Studies conducted both in vivo and in vitro suggest that the process of cortical gliogenesis involves a hierarchy of progressively restricted progenitor cell pools. In the cerebral cortex, as well as other areas of the central nervous system, glial cells differentiate from one another through a series of steps that can be defined at molecular, structural, and functional levels. Although the precise timing, sequence, and diversity of the steps involved in cortical gliogenesis are still not fully defined, the emerging picture suggests that both cell lineage and cell‐cell interactions play a synergetic role in the determination and maintenance of the proper blend of glial cells in the cerebrum.
The diversity and pattern of glia during axon pathway formation in the drosophila embryo
Authors: Klämbt Christian , Goodman Corey S.
Published: January 2005

Enhancer trap lines have been used to generate a collection of molecular lineage markers specific for different subsets of glia in the Drosophila embryo. Using these markers, we have been able to describe the diversity and pattern of glia along the major axon pathways in the embraynic central and peripheral nervous system. Just as these and other studies show the great diversity of embryonic glia, so too the enhancer trap lines described here point to a remarkable degree of molecular heterogeneity, and probably a concomitant functional specificity, of the embryonic glia.
Diversity amongst the microglia in growing and regenerating fish CNS: Immunohistochemical characterization using FL. 1, an anti‐macrophage monoclonal antibody
Authors: Dowding Alan J. , Maggs Alison , Scholes John
Published: January 2005

We have immunohistochemically characterized the forms and distribution of microglia—the macrophages of the CNS—in fish, using a new monoclonal antibody (mAb), FL.1. This mAb specifically reacts with resident macrophages throughout the body in Oreochromine fish, including Kupffer cells, gut‐associated myeloid cells, and peritoneal macrophages, as well as with microglia, but circulating monocytes are not labelled with FL.1. The FL.1‐epitope, which is lost following treatment with reducing agents, has an extracellular location and is associated with three integral membrane glycoprotein variants.

FL.1‐staining shows that microglia are extremely abundant throughout the fish CNS. For example, they comprise a third of the glia in the optic nerve, and 30% of all cells, including neurons, in the spinal cord, i.e., fish have about tenfold more microglia than mammals. Two forms of FL.1‐positive microglia are predominant in fish, one resembling their mammalian counterparts, but less ramified, and the other comprising smaller rounded cells with very little cytoplasm, which are most numerous in the ependymal region of the optic tectum.

Apart from the conventional microglia, the optic nerves also contain large lipid‐laden macrophages which comprise a third form of FL.1‐positive cell in the CNS. Fish optic nerves contain astrocytes of a distinct type which form reticular networks, but lack connections to capillaries (Maggs and Scholes, J. Neurosci. 1990;10:1600–1614). The co‐distribution of foamy macrophages may have a metabolic role that is performed by ordinary astrocytes elsewhere in the CNS. An antiserum against the β2 subunit of the human leukocyte integrins (Kishimoto et al., Cell 1987a; 50:193–202) was found selectively to recognize the foamy macrophages in Oreochromis.

Following lesion to the optic nerve, FL.1‐labelling shows that microglia proliferate throughout the visual pathway. In the optic tectum, the additional FL.1‐positive cells are concentrated in the vicinity of degenerating retinal axons and their terminals. Most of the microglia in the injured optic nerve have amoeboid morphologies, and the foamy macrophages become depleted.
Further studies of electrogenic Na+/HCO cotransport in glial cells of necturus optic nerve: Regulation of pHi
Authors: Astion M. L. , Chvatal A. , Orkand R. K.
Published: January 2005

In the presence of Ba++, an increase in the bath HCO at constant CO2 (i.e., Variable bath pH) produced a hyperpolarization. The hyperpolarizing effect of adding HCO3−/CO2 at constant bath pH was not significantly affected by the presence of 50 μmol/l strophanthidin. In the absence of Ba++, addition of HCO3−/CO2 at constant bath pH produced a Na+‐dependent hyperpolarization. Therefore, CO2 movements, electrogenic Na+/K+ pump activity and changes in Ba++ binding do not contribute significantly to the hyperpolarization induced by HCO3−. These results along with the results of previous studies (Astion et al: J Gen Physiol 93:731, 1989) strongly suggest that the hyperpolarization induced by the addition of HCO3− is due to an electrogenic Na+/HCO3− cotransporter, which transports Na+, HCO3− (or its equivalent), and net negative charge across the glial membrane.

To study the role of electrogenic Na+/HCO cotransport in the regulation of pHi in glial cells, we used intracellular double‐barreled, pH‐sensitive microelectrodes. At a bath pH of 7.5, the mean initial intracellular pH (pHi) was 7.32 (SD 0.03, n = 6) in HEPES‐buffered Ringer's solution and 7.39 (SD 0.1, n = 6) in HCO3−/CO2 buffered solution. These values for pHi are more than 1.2 pH units alkaline to the pHi predicted from a passive distribution of protons; thus, these cells actively regulated pHi. Superfusion and with‐drawal of 15 mmol/l NH4+ induced an acidification of 0.2 to 0.3 pH units, which recovered toward the original steady‐steady‐state pHi. Recovery from acidification was stimulated by adding HCO3−/CO2 at constant pH. In HCO3−/CO2‐buffered solutions, the recovery was Na+‐dependent, inhibited by 4‐acetamido‐4′‐isothiocyanato‐stilbene‐2,2′‐disulfonic acid (SITS), and associated with a hyperpolarization of the membrane. Thus it appears that the electrogenic Na+/HCO3− cotransporter helps maintain the relatively alkaline pHi of glial cells and also contributes to the ability of glial cells to buffer changes in pH in the neuronal microenvironment.
A study of in vitro and in vivo morphological changes of ependymal cells induced by galactocerebrosides
Authors: Laabich Aicha , Graff Marie‐Noëlle , Dunel‐Erb Suzanne , Sensenbrenner Monique , Delaunoy Jean‐Pierre
Published: January 2005

Ependymal cells in culture and in vivo were treated with mixture of galactocerebrosides. Galactocerebroside is the major glycolipid of myelin and in demyelinating diseases is found in cerebrospinal fluid. Morphological changes induced by this treatment were examined by microscopy at both optical and ultrastructural levels. In vitro, cilia, microvilli, and junctions between the cells disappeared, processes containing intermediate filaments developed, and the cells lost characteristics typical of ependymal cells and became more astrocyte‐like. As shown by vital staining with a fluorescent compound and by nuclear incorporation of bromodeoxyuridine, cells did not proliferate during the period of galactocerebroside treatment and the morphological transformation was restricted to the ependymal cells. In contrast, asialoganglioside‐GM1 and sulfatides had no effect on ependymal cell morphology. Some of the in vitro observations could be reproduced in vivo. Junctions between ependymal cells disappeared and intercellular spaces appeared between these cells and the cerebral parenchyma at the basolateral side of the ependymal layer. At the apical side, morphological modifications of junctions and cilia were less evident. As these experimental conditions resemble those existing during demyelination the morphological changes described may account for perturbations of the physiological functions of the ependymal cell.
Production and disposition of 1‐methyl‐4‐phenylpyridinium in primary cultures of mouse astrocytes
Authors: Di Monte Donato A. , Wu Ellen Y. , Irwin Ian , Delanney Louis E. , Langston J. William
Published: January 2005

Dopaminergic neurons are a primary target for 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) neurotoxicity. However, the conversion of MPTP to its neurotoxic 1‐methyl‐4‐phenylpyridinium metabolite (MPP+) is likely to occur in astrocytes via the monoamine oxidase (MAO)‐dependent formation of the 1‐methyl‐4‐phenyl‐2,3‐dihydropyridinium intermediate (MPDP+). The main purpose of this study was to characterize the molecular mechanism(s) by which MPP+, once generated by astrocytes, may reach the extracellular space to become available for the active accumulation into dopaminergic neurons. Primary cultures of mouse astrocytes were used as an in vitro model system. After the addition of MPTP, levels of MPP+ were found to increase at constant rates both intracellularly at time points when no sign of cytotoxicity was evident. In contrast, MPDP+ levels remained quite stable during 4 days of incubation in the presence of MPTP. Finally, when astrocytes were allowed to accumulate MPP+ by pretreatment with either MPTP or MPP+ and then were incubated in fresh medium not containing MPTP or MPP+, intracellular levels of MPP+ rapidly declined and corresponding amounts of this compound were found in the incubation medium. Results of this study are compatible with the following conclusions: (1) the MPP+ accumulated in the extracellular compartment during incubations with MPTP is not released from astrocytes as a consequence of its own cytotoxic effects; (2) MPP+ can be formed extracellularly presumably via autoxidation of MPDP+ after this latter compound has been generated within astrocytes and has crossed astrocyte membranes; and (3) despite its charged chemical structure, MPP+ can cross the plasma membrane toward the extracellular space after being formed within astrocytes.
Isomorphic activation of astrocytes in the somatosensory thalamus
Authors: Wells Joseph , Vietje Brad P. , Wells David G. , Paradee Janelle
Published: January 2005

Structural recovery in the rat somatosensory thalamus after the loss of one of its major inputs provided a model for studying the changes in astrocytes associated with reactive synaptogenesis. The temporal separation of the initiation of Wallerian degeneration and reactive synaptogenesis permitted astrocytic changes to be correlated either with the removal of degeneration, early in the recovery sequence, or with synaptogenesis, later in recovery. Over a period of post‐lesion times ranging from 3 days to 13.5 months, GFAP‐positive astrocytic fibers were quantified and the population density of S‐100‐positive astrocytic cell bodies was determined in the ventral posterolateral nucleus (VPL). The relative area of astrocytic cell bodies was measured at an early peak of the increased GFAP immunoreactivity (4–5 days post‐lesion). The normal side of VPL (c‐VPL) was compared to the deafferented side of VPL (d‐VPL) and the ratio d‐VPL/c‐VPL was determined. Astrocytes in d‐VPL underwent a minimal isomorphic activation with little or no hypertrophy or proliferation but with a large increase in GFAP immunoreactivity. Prior to the initiation of synaptogenesis, there was a decrease both in GFAP immunoreactivity and in the population density of VPL astrocytes. The decreases in the recovery curves suggested that a suppression of the influence of astrocytes may have been important for sprouting and/or synaptogenesis. In other systems, where synaptogenesis was initiated early in the recovery sequence, the suppression of astrocytes that was related to synaptogenesis may have been masked by astrocytic changes related to the removal of degeneration.
Co‐cultures of microglia and astrocytes from kainic acid‐lesioned adult rat hippocampus: Effects of glutamate
Authors: Bowman Charles L. , Swann John W. , Severin Charles M. , Romanowski Marcus R.
Published: January 2005

The long‐standing question concerning the direct actions of glutamate on the membrane potential of astroglial cells in the central nervous system was addressed using the in vitro kainic acid‐lesioned hippocampal slice preparation and primary cell co‐cultures of astrocytes and microglia derived from such lesions. The ultrastructure of the lesioned hippocampus was examined to aid in the identification of the cells appearing in culture.

In culture, microglia appeared as flat cells, less than 1 μm in thickness at the edge of the cell, but thicker (about 5 μm) near the nucleus. The cytoplasm was packed with granular inclusions. Microglia appeared in two morphological forms, amoeboid and ramified. The amoeboid form was characterized by a cell body with a single process, and was always observed 1 day after starting the cell culture. Such cells became less frequent after 1 week in culture. The ramified form appeared as a rounded cell, devoid of processes, and were frequently observed in older cultures (> 1 week). Microglia did not round up after exposure to dibutyrylcyclic adenosine monophosphate (cAMP), and did not stain for glial fibrillary acidic protein (GFAP). An ultrastructural examination of the lesion demonstrated that microglia were present and that they contained many cytoplasmic granules similar to lipofuscin‐containing granules. No filaments were observed in the cytoplasm of microglia. By contrast, the cytoplasm of astrocytes in culture had far fewer granules, rounded up to dibutyryl‐cAMP, exhibited multiple processes, and stained for GFAP. In slices, astrocytes had no lipofuscin‐containing granules, but numerous cytoplasmic filaments were present.

The membrane potential of the rounded‐up cells in culture varied between −10 mV and −95 mV (while electrically unresponsive cells in the lesion varied between −47 and −95 mV). In culture, glutamate evoked a depolarization or a hyperpolarization of the membrane potential of 87% of the rounded‐up cells tested. Ionophoretic application of glutamate depolarized the membrane potential of 50% of the cells tested in the lesion.

The results show that co‐cultures of microglia and astrocytes can be obtained routinely from lesions within the adult central nervous system, and that glutamate can affect the membrane potential of astrocytes in cultures derived from such adult tissue. These findings suggest that responses to glutamate, recorded from unresponsive cells (presumptive astrocytes) in the lesioned slice preparation, are likely the result of direct action of this neurotransmitter on these cells. © 1992 Wiley‐Liss, Inc.