bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunomagnetic removal of neurons from developing chick optic tectum results in glial phenotypic instability
Authors: Galileo Deni S. , Linser Paul J.
Published: May 2005

The ability of embryonic day 12 and 13 optic tectum cells to replace depleted A2B5(+) cells and neurons was tested by immunomagnetic cell separation. Nearly all purified surface A2B5(−) cells were identified as glia by immunoreactivity for either glutamine synthetase of galactocerebroside. Most (∼80%) of the purified A2B5(−) cells became A2B5(+) after 1 day in culture, although no increase in the percentage of A2B5(+) cells (from 45%) was observed in control cultures of unpurified cells. Long‐term monolayer cultures from purified cells contained A2B5(+) cells with mostly flattened glial‐like or round process‐free morphology, whereas those from unpurified cells contained many A2B5(+) neurons. The non‐neuronal A2B5(+) cells frequently reacted with antibodies against glial fibrillary acidic protein and another marker expressed by embryonic brain glia, 5A11. Additionally, some flattened glia‐like cells exhibited elaborate networks of anti‐neurofilament‐M‐reactive filaments. We believe these unusual pheno‐types, which appeared only in cultures of purified A2B5(−) cells, arose in response to the immunomagnetic removal of neurons. In conjunction with previous findings, we conclude that the abnormal phenotypes in purified cell cultures represent glia that were unsuccessful in attempting to replenish the depleted neuronal population. This may reflect restricted developmental potentials that arise during brain ontogeny.
Voltage‐gated currents expressed by rat microglia in culture
Authors: Korotzer Andrew R. , Cotman Carl W.
Published: May 2005

Using the whole‐cell patch‐clamp technique, at least three types of voltage‐gated currents expressed by cultured rat microglia were identified: an inward rectifier K+ current, a delayed rectifier K+ current (IK), and a Na+ current activated by depolarization. The inward rectifier conductance was activated by hyperpolarization to potentials more negative than −80 mV, depended on the external K+ concentration, and declined over time during whole cell recording, as the cell was internally dialyzed. The delayed rectifier current was activated by depolarization to potentials more positive than −40 mV and the rates of activation and deactivation showed a voltage‐dependence similar to such currents seen in other preparations. An inward current possibly carried by Na+ was seen in a small percentage of cells. Recordings had been made from two morphological cell types, namely process‐bearing (“ramified”) and non‐process‐bearing (“ameboid”). Each of these currents was present in microglia of both morphological types. However, microglial morphology, which is thought to represent different states of activation, was significantly related to the types of combinations of currents expressed in a given cell. © 1992 Wiley‐Liss, Inc.
Glial response to dorsal root lesion in the irradiated spinal cord
Authors: Sims T. J. , Gilmore S. A.
Published: May 2005

Exposure of the rat lumbar spinal cord to X‐rays during the early postnatal period results in a marked reduction in the glial populations within the irradiated region. The present study was undertaken to determine what effects this reduction of glia, particularly astrocytes, has on the pattern and characteristics of the scar formation that follows root injury in the normal spinal cord. Morphological assessments 60 days following injury of the right L4 dorsal root revealed a distinct difference in the extent of the astrocyte response between the irradiated and the nonirradiated rats. In the nonirradiated animals, a thick astrocytic scar composed of multiple layers of astrocyte processes formed over the dorsal horn and adjacent portions of the dorsal surface of the cord. This astrocytic response was not confined to the surface of the spinal cord but extended also into the root, i.e., into regions normally considered as PNS. In irradiated rats, the astrocytes did not form a thick scar nor did they extend into the injured root. Instead, they formed a glia limitans and were at most only one or two layers thick over the region of cord comparable to that occupied by the thick astrocytic scar in the nonirradiated rats. Mechanisms involved in the glial response of the irradiated spinal cord to dorsal root injury are discussed, particularly with regard to the possible positive effect that this reduction in scar formation may have on regrowth of injured dorsal root axons into the spinal cord environment. © 1992 Wiley‐Liss, Inc.
Expression of adrenergic receptors in individual astrocytes and motor neurons isolated from the adult rat brain
Authors: Shao Yanping , Sutin Jerome
Published: May 2005

Attempts to show the distribution of adrenergic receptors (ARs) in autoradiographs of a brainstem motor nucleus following elimination of motor neurons yielded the unexpected result of an increase in β‐AR density. This increase was related to the gliosis accompanying the motor neuron degeneration. To determine the cells on which the AR subtypes were located, we dissociated cells from various regions of the adult rat brain and subsequently identified astrocytes by glial fibrillary acidic protein (GFAP) immunofluorescence. Slides containing the astrocytes were prepared for autoradiography using the nonselective β ligand 125I‐iodocyanopindolol (125ICYP) or the α1 ligand 125IBE 2254 (125I‐HEAT). The addition of the selective β1 blocker betaxolol or the β2 blocker ICI 118.551 to the incubation medium to displace 125ICYP binding was used to determine the binding of β‐AR subtypes.

The great majority (> 88%) of isolated astrocytes sampled from the trigeminal motor nucleus, cerebral cortex, striatum, and cerebellum showed β‐AR binding. Astrocytes from the first three regions had similar average densities of β‐ARs, whereas the density in cerebellar astrocytes was 2‐ to 3‐fold greater. The β2‐AR subtypes was proportionally greater than the β1 subtype in each region. Reactive astrocytes isolated from the trigeminal motor nucleus after degeneration of motor neurons showed a β‐AR density nearly 2‐fold greater than resting astrocytes from the same region, with the β1 subtype showing the greater proportional increase. There was no β‐AR binding on trigeminal motor neurons.

Astrocytes also showed a significant level of α1‐AR binding. No differences in α1‐AR binding were found in normal astrocytes isolated from the different regions, nor was there an increase in reactive astrocytes. In contrast, trigeminal motor neurons had an α1‐AR density nearly 10 times greater than astrocytes.

In terms of the NE modulation of synaptic responses in motor neurons, the distribution of ARs would permit NE to act indirectly through α1 and β receptors on astrocytes and directly through α1 receptors on motor neurons. © 1992 Wiley‐Liss, Inc.
Calcium dependence of serotonin‐evoked conductance in C6 glioma cells
Authors: Manor David , Moran Nava , Segal Menahem
Published: May 2005

Whole‐cell membrane currents and imaging of intracellular calcium concentrations ([Ca2+]i) were used to investigate the role of calcium in a response to serotonin of C6 glioma cells. Activation of a high‐affinity serotoinin receptor induced a transient rise in calcium concentration in these cells and activated a predominantly potassium conductance, with a small chloride component. Perfusion of the cytoplasm with an internal solution containing high calcium concentration induced similar but prolonged increase of membrane conductance. The responsiveness of C6 cells to serotonin was negatively correlated with the concentration of the unbound calcium chelator BAPTA when BAPTA‐buffered calcium‐containing intracellular solutions were used. Responses to serotonin persisted in the absence of external calcium, decreased gradually, and then recovered partially after replenishment of extracellular calcium. These findings substantiate the direct role of intracellular calcium in mediating the serotonin response, and indicate that serotonin‐induced release of calcium from intracellular stores is sufficient for the activation of conductance in the C6 glioma cell line. © 1992 Wiley‐Liss, Inc.
Postnatal development of dye‐coupling among astrocytes in rat visual cortex
Authors: Binmöller F.‐J. , Müller C. M.
Published: May 2005

Intercellular coupling among astrocytes was studied in rat visual cortex slices from animals aged 1 week to 4 months. Cell coupling via gap junctions was determined by the dye spread of the low molecular weight dye Lucifer Yellow CH injected into electrophysiologically identified cells to adjacent cells. Coupling among glial cells was first detected at postnatal day 11 and was thereafter consistently observed until adulthood. Dye spread was observed up to 300 μm radially from the injected cell covering multiple cortical layers. Following dye injection into a single cell up to several hundred Lucifer Yellow‐positive cells could be observed. Quantitative analysis revealed a similar extent of dye spread at different developmental stages including a quite constant number of dye‐coupled astrocytes from the end of the second postnatal week to adulthood. Double labelling of Lucifer Yellow‐filled cells with an antiserum against the glial fibrillary acidic protein confirmed the astrocytic nature of the injected and coupled cells. Comparison of the density of dye‐coupled cells in a given area and the total number of astrocytes as revealed by immunocytochemical staining suggests that dye‐coupling includes the entire local astrocytic population. It is concluded that coupling among astrocytes via gap junctions in rat visual cortex occurs shortly after birth and reflects one of the first steps in astroglial maturation. © 1992 Wiley‐Liss, Inc.
Immuno‐electron microscopical localization of vimentin and glial fibrillary acidic protein in mouse astrocytes and their precursor cells in culture
Authors: Abd‐El‐Basset Ebtesam M. , Ahmed Ijaz , Kalnins Vitauts I. , Fedoroff Sergey
Published: May 2005

Immuno‐electron microscopy was used to localize the distribution of vimentin and glial fibrillary acidic protein (GFAP) in mouse astrocytes and their precursor cells in primary cultures. In astroblasts and astrocytes, vimentin and GFAP form intermediate filaments (IF), which are heteropolymers, as previously observed in gliomas. Astrocytes and their precursor cells may have IF composed of GFAP‐vimentin heteropolymer or vimentin alone, but IF composed of GFAP only were not seen. It seems that the formation of IF that are GFAP‐vimentin heteropolymers is a feature of normal astroglia development and that the ratio of GFAP to vimentin in these IF reflects the degree of differentiation and functional state of the cell. © 1992 Wiley‐Liss, Inc.
Differential migration of astrocytes grafted into the developing rat brain
Authors: Hatton James D. , Nguyen Mindie H. , Sang U Hoi
Published: May 2005

Fetal and neonatal astrocytes migrate in specific patterns when transplanted into the adult rat host brain. However, it is unclear whether these astrocytes demonstrate the same degree of mobility during early brain development. In the present study, neonatal cortical, hippocampal, and hypothalamic astrocytes were collected from the brains of 1‐ to 3‐day‐old rats and placed in tissue culture. After 14 to 21 days, cultures enriched in astrocytes were harvested and labelled with either the fluorescent dye Fast Blue or fluorescein‐labelled latex beads. They were then transplanted into the right frontal cerebrum of neonatal rats at 2,5,8, and 11 days postpartum. Seven days after transplantation, animals were sacrificed and their brains were fixed by immersion in aldehydes, sectioned on a cryostat, and examined with fluorescence microscopy. Transplanted astrocytes migrated along the corpus callosum, internal capsule, glial limitans, ventricular linings, and hippocampal structure. Labelled cells were also found in the contralateral hemisphere in day 2 brains. Migration in a radial fashion from the injection site toward the periphery was a particularly obvious pattern, and was most pronounced in these younger hosts. In days 5 and 8 rat brains, astrocyte migration became more restricted to the hemisphere of implantation. In 11‐day‐old host brains hemispheric restriction and other region‐specific influences became manifest and specifically modulated migration. Radial migration was absent in the 11‐day‐old host group except for cells of cortical origin. The observed results demonstrate that neonatal cortical, hippocampal, and hypothalamic astrocytes transplanted into the neonatal cerebrum migrate in patterns that are more extensive than in the adult brain. This suggests that cellular migration in the neonatal brain is governed by factors that are less restrictive than those regulating migration in the adult brain. In particulur, our observations imply that radial glia may provide migratory substrates for transplanted astrocytes, and region‐specific regulation of migration may begin around 11 days after birth. © 1993 Wiley‐Liss, Inc.
Excitatory amino acid‐induced phosphoinositide hydrolysis in Müller glia
Authors: López‐Colomé Ana María , Ortega Arturo , Romo‐De‐Vivar Mónica
Published: May 2005

The presence of excitatory amino acid (EAA) receptors coupled to phosphoinositide metabolism in primary cultures of Müller (glial) cells from the chick retina was established. The order of potency of analogues for stimulating [3H]inositol phosphate (IP) accumulation was quisqualate (QA) > L‐glutamate (L‐Glu) = kainate (KA) > N‐methyl‐D‐aspartate (NMDA) > L‐aspartate (L‐Asp) with EC50 in the range of 1‐100 μM. 1‐Aminocyclopentane‐1,3‐dicarboxylate (trans‐ACPD), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isox‐azolepropionate (AMPA), 2‐amino‐3‐phosphonopropionate (AP3), and 2‐amino‐4‐phosphonobutyrate (AP4) showed no effect either on basal concentration or on stimulated accumulation of [3H]IPs. The effect of EAA was potently inhibited by the ionotropic NMDA receptor antagonists 2‐amino‐5‐phosphonopentanoate (AP5), 3‐[(RS)‐2‐carboxy‐piperazin‐4‐yl]‐propyl‐1‐phosphonate (CPP), and (+)‐5‐methyl‐10,11‐dihydro‐5H‐diben‐zo[a,d]cyclohepten‐5‐10‐imine (MK‐801); L‐Glu antagonists at non‐NMDA receptors, the quinoxalines NBQX and DNQX, inhibited weakly the response to L‐Glu, KA, and NMDA, and more potently that to QA. The translocation of protein kinase C was also stimulated by EAA with the same pharmacological profile, and was partially inhibited by kynurenate (KYN). L‐Glu and KA induced 45Ca2+ influx, which was decreased by KYN and CNQX. EAA‐induced [3H]IPs accumulation was decreased by verapamil but not by nifedipine, and slightly diminished by dantrolene. Results demonstrate that EAA‐induced phosphoi‐nositide hydrolysis in Müller cells shows pharmacological differences with that in astrocytes and neuronal cells and could be triggered by a different mechanism. © 1993 Wiley‐Liss, Inc.
Oligodendrocyte development and differentiation in the rumpshaker mutation
Authors: Fanarraga M. L. , Sommer I. U. , Griffiths I. R. , Montague P. , Groome N. P. , Nave K.‐A. , Schneider A. , Brophy P. J. , Kennedy P. G. E.
Published: May 2005

The jimpy rumpshaker (jprsh) mutation is an amino acid substitution in exon 4 (Ile186→Thr) of the proteolipid protein (PLP) gene on the X chromosome. Affected mice show moderate hypomyelination of the central nervous system (CNS) with increased numbers of oligodendrocytes in the white matter of the spinal cord, a feature distinguishing them from other PLP mutations such as jp, in which premature cell death occurs with reduced numbers of oligodendrocytes. Myelin sheaths of jprsh immunostain for myelin basic protein (MBP) and DM‐20, but very few contain PLP. This study examines the differentiation of oligodendrocytes cultured from the spinal cords of young mutant and wild type mice using various surface and cytoplasmic antigenic markers to define the stage of development. The majority of oligodendrocytes from mutant mice progress normally to express MBP; approximately 30%, relative to wild type, contain DM‐20 at the in vivo age of 16 days, but very few immunostain for PLP or the O10 and O11 markers. The morphology of mutant cells in respect to membrane sheets and processes appears similar to normal. The jprsh oligodendrocyte is, therefore, characterized by a failure to express the markers indicative of the most mature cell; however, it is probably able to achieve a normal period of survival. These data, taken in conjunction with previous results, suggest that the PLP gene has at least two functions; one, probably involving PLP, is concerned with a structural role in normal myelin compaction; the other, perhaps related to DM‐20 (or another lower molecular weight proteolipid), is essential for cell survival. The mutation in jprsh at residue 186 suggests that this region, which is common to PLP and DM‐20, is not critical for this latter function. © 1993 Wiley‐Liss, Inc.
Endogenous opioid systems and the growth of oligodendrocyte progenitors: Paradoxical increases in oligodendrogenesis as an indirect mechanism of opioid action
Authors: Hauser Kurt F. , Gurwell Julie A. , Bhat Narayan R.
Published: May 2005

Endogenous opioids inhibit nervous system development by inhibiting the proliferation of certain neuronal and glial progenitors. To determine whether opioids affect the growth of preoligodendrocytes, the effects of the endogenous opioid [Met5]‐enkephalin were examined in preoligodendrocytes in primary mixed‐glial and preoligodendrocyte‐enriched (>98% pure) cultures. Proliferating preoligodendrocytes in mixedglial or preoligodendrocyte‐enriched cultures were continuously treated for a total of 40 h with either basal growth media (controls), 1 μM [Met5]‐enkephalin, 1 μM [Met5]‐enkephalin plus the opioid antagonist naloxone (3 μM), or naloxone alone (3 μM), and incubated in [3H]‐thymidine (0.2 μCi/ml/4–6 h) after 34–36 h of opioid exposure. Opioid dependent changes in DNA synthesis were assessed autoradiographically in O4‐immunoreactive oligodendrocyte progenitors. Naloxone alone significantly decreased the rate of DNA synthesis and number of O4‐immunoreactive preoligodendrocytes in mixed‐glial cultures. However, naloxone and/or [Met5]‐enkephalin did not affect DNA synthesis or the number of O4‐immunoreactive preoligodendrocytes in cultures enriched in preoligodendrocytes. The results suggest that astrocytes, or perhaps another cell type, play a permissive role in opioid‐dependent alterations in preoligodendrocyte proliferation. Endogenous opioids affect the genesis of neural cells by both direct and indirect mechanisms. © 1993 Wiley‐Liss, Inc.
Synaptic transmission in the hippocampus: Critical role for glial cells
Authors: Keyser David O. , Pellmar Terry C.
Published: May 2005

The importance of glial cells in controlling the neuronal microenvironment has been increasingly recognized. We now demonstrate that glial cells play an integral role in hippocampal synaptic transmission by using the glial‐specific metabolic blocker fluoroacetate (FAC) to selectively inhibit glial cell function. FAC inhibits evoked intracellular postsynaptic potentials (PSPs; IC50 = 39 μM) as well as population PSPs (IC50 = 65 μM) in field CA1 of the guinea pig hippocampal slice. Spontaneous synaptic transmission is concurrently decreased. These effects are time and dose dependent. ATP concentrations in glial but not neuronal elements are also significantly reduced with FAC treatment. Simultaneous application of the metabolic substrate isocitrate with FAC prevents both the reduction in glial ATP concentrations and the decrease in evoked PSPs. Given that isocitrate is selectively taken up by glia, these data further support a glial specific metabolic action of FAC. Additionally, FAC has no postsynaptic effects as peak responses to iontophoretically applied glutamate are unchanged. However, the decay of both iontophoretic and evoked PSPs are prolonged following FAC treatment suggesting inhibition of glutamate uptake may contribute to the FAC‐induced depression of synaptic potentials. These results show, for the first time, that glial cells are critical for maintenance of synaptic transmission and suggest a role for glial cells in the modulation of synaptic efficacy. © 1994 Wiley‐Liss, Inc.
GD3+ cells in the adult rat optic nerve are ramified microglia rather than 0–2Aadult progenitor cells
Authors: Wolswijk Guus
Published: May 2005

The adult central nervous system (CNS) contains a population of adult oligodendrocyte‐type‐2 astrocyte (O‐2A) progenitor cells (O‐2Aadult progenitor cells). These cells may provide a source of the new oligodendrocytes that are needed to repair demyelinated lesions. In order to examine the role of O‐2Aadult progenitor cells in the regeneration of the oligodendrocyte population following demyelinating damage, it is essential to be able to identify such cells unambiguously in sections of adult CNS tissue. The present study examined whether antibodies to the ganglioside GD3 specifically label O‐2Aadult progenitor cells in cultures and sections of adult optic nerve, since previous studies on the developing CNS had suggested that O‐2Aperinatal progenitor cells were GD3+ in vitro and in vivo. Evidence is presented indicating that, although O‐2Aadult progenitor cells in vitro were labelled with the R24 mAb (an anti‐GD3 mAb), all GD3+ cells in sections of adult optic nerve bound the OX‐42 mAb and the B4 isolectin derived from Griffonia Simplicifolia, and thus were not O‐2Aadult progenitor cells, but ramified microglia. The data suggest that O‐2Aadult progenitor cells become GD3+ when placed in culture and that ramified microglia lose GD3‐expression in vitro. © 1994 Wiley‐Liss, Inc.
A physiological measure of carbonic anhydrase in müller cells
Authors: Newman Eric A.
Published: May 2005

Carbonic anhydrase activity was characterized in freshly dissociated Müller cells of the salamander retina. Intracellular pH was monitored using ratio imaging of the indicator dye BCECF as extracellular Pco2 was varied. The extracellular solution was switched rapidly (141 ms rise time) from a HEPES buffered to a CO2‐HCO−3 buffered solution (both pH 7.4). Introduction of CO2‐HCO−3 produced a rapid cell acidification. Cell pH dropped from a steady‐state pH of 7.02 in HEPES solution to pH 6.81 in CO2‐HCO−3. Methazolamide, a carbonic anhydrase inhibitor, dramatically reduced the initial rate of acidification, demonstrating that the acidification was produced by the carbonic anhydrase‐catalyzed hydration of CO2. The initial rate of acidification, 52.6 pH units per min (0.88 pH units per s), was reduced ∼ 150‐fold to 0.36 pH units per min by 10−3 M methazolamide. Half‐maximal inhibition occurred at a methazolamide concentration of 5.6. 10−7 M. The carbonic anhydrase inhibitor acetazolamide (10−3 M) also greatly reduced the rate of cell acidification. The latency to the onset of carbonic anhydrase inhibition was 660 ms for methazolamide and 7.5 s for acetazolamide. The carbonic anhydrase inhibitor benzolamide (10−4 M, 4 min exposure), which is poorly membrane permeant, had little effect on the rate of cell acidification, indicating that the site of carbonic anhydrase action was intracellular. The activity of Müller cell carbonic anhydrase may help to buffer extracellular CO2 variations in the retina. © 1994 Wiley‐Liss, Inc.
Stimulation of in vitro myelin synthesis by microglia
Authors: Hamilton Steven P. , Rome Leonard H.
Published: May 2005

Central nervous system myelin is elaborated by oligodendrocytes, which have been studied extensively in cell culture. Dissociated brain cultures allow in vitro analysis of events in myelinogenesis, including cell‐cell interactions. Microglia, the primary phagocytic cell of the central nervous system, appear in developing fiber tracts prior to the onset of myelination in vivo. To gain insight into potential oligodendrocytemicroglial interactions during development, these cells were co‐cultured and various parameters of myelin synthesis were measured. In co‐culture, microglia stimulated the synthesis of sulfatide, a myelin‐specific galactolipid, in oligodendrocytes, as well as the expression of the myelin‐specific proteins myelin basic protein and proteolipid protein. Activity of the oligodendrocyte cytoplasm‐specific enzyme 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase was not elevated, suggesting that the effects of microglia were not due to stimulation of oligodendrocyte proliferation. This was confirmed by the inability of microglia to induce significant DNA synthesis. Conditioned medium from cultured microglia provided a similar stimulatory activity, suggesting that the increase in myelin synthesis does not require contact between oligodendrocytes and microglia. These findings suggest a stimulatory role for microglia during myelinogenesis. © 1994 Wiley‐Liss, Inc.
Bacterial endotoxin induces [Ca2+]i transients and changes the organization of actin in microglia
Authors: Bader Marie‐France , Taupenot Laurent , Ulrich Gabrielle , Aunis Dominique , Ciesielski‐Treska Jaroslava
Published: May 2005

We have employed amoeboid microglia purified from primary cultures of neonatal rat brain to examine the effect of bacterial lipopolysaccharide (LPS), a potent activator of immune cells, on intracellular calcium concentration ([Ca2+]i) in brain macrophages. In single brain macrophages loaded with indo 1, pulse administration of LPS elicited a rapid and transient increase in [Ca2+]i From a total of 70 cells examined, all responded to LPS with a similar [Ca2+]i transient, indicating a good homogeneity of the cell population with regard to the LPS response. It was concluded that the rise of cytosolic [Ca2+]i originated from intracellular stores because the response to LPS occured similarly in the presence or in the absence of extracellular Ca2+. A second administration of LPS to the same cells resulted in a second but reduced [Ca2+]i transient. In contrast to the first response to LPS, this second response was totally dependent on the presence of Ca2+ in the extracellular medium. The first response to LPS was strongly inhibited by ruthenium red and could be suppressed in a reversible manner by pre incubating the cells with caffeine in the absence of Ca2+ in the extracellular medium. These results indicate that caffeinesensitive intracellular Ca2+ stores may be the major source of Ca2+ in the response of brain macrophages to LPS. The possible release of Ca2+ from phosphatidylinositol(3,4,5)‐trisphosphate (IP3)‐sensitive stores in brain macrophages was also evaluated by stimulating cells with the IP3‐mobilizing agonist histamine. Brain macrophages were heterogeneous with regard to the histamine response since histamine induced a [Ca2+]i rise in only 30% of cells examined.

The increase in [Ca2+]i triggered by LPS may in turn activate several intracellular events involved in the transition from one microglial functional state to another. We examined the effect of LPS on the state of organization of actin, which is an essential component of chemoattractant signal transduction. Immunofluorescence staining with anti‐actin antibody which recognizes actin in both filamentous and nonfilamentous configurations, indicated that LPS produced drastic changes in the actin organization. LPS‐treated cells appeared more intensely fluorescent than untreated cells due to the concentration of diffuse fluorescence near the center of the cell. In addition, prominent fluorescent dots were present in the subplasmalemmal region in cells stimulated with LPS. This specific LPS‐induced reorganization of actin was also observed in cells preincubated in the external medium without Ca2+. Thus, it is likely that the LPS‐induced mobilization of Ca2+ from intracellular stores may be responsible for the changes observed in the actin organization. © 1994 Wiley‐Liss, Inc.
Astrocyte‐schwann cell interactions in culture
Authors: Ghirnikar Roopa S. , Eng Lawrence F.
Published: May 2005

After injury, either as a result of trauma or degenerating/demyelinating diseases, axons of the central nervous system (CNS) normally fail to regenerate. Transplantation of glial cells, particularly Schwann cells, into areas of injury or demyelination has been considered a promising approach to promote recovery. However, the extent of Schwann cell interaction with CNS axons is greatly influenced by the presence of astrocytes which redefine the CNS‐PNS (peripheral nervous system) boundary in a lesioned CNS, thereby preventing invasion of Schwann cells. The molecular basis for this restrictive effect of astrocytes on Schwann cells is not known. In the present study, we have cocultured astrocytes and Schwann cells to develop an in vitro model to characterize this interaction. Astrocytes in contact with Schwann cells appeared hypertrophied and showed increased staining for glial fibrillary acidic protein (GFAP). In cocultures maintained for 2‐3 weeks, segregation of the two cell types was observed, Schwann cells appeared in groups, and each group was surrounded and separated from one another by astrocytic processes. Since the behavior of these two cell types observed in culture is very similar to their interaction seen in vivo, this coculture model may be useful in further studying the relationship between astrocytes and Schwann cells. © 1994 Wiley‐Liss, Inc.
Axons regulate the expression of shaker‐like potassium channel genes in Schwann cells in peripheral nerve
Authors: Chiu S. Y. , Scherer S. S. , Blonski M. , Kang S. S. , Messing A.
Published: May 2005

We examined potassium channel gene expression of two members of the Shaker subfamily, MK1 and MK2, in sciatic nerves from rats and mice. In Northern blot analysis, MK1 and MK2 probes detected single transcripts of ∼8 kb and ∼9.5 kb, respectively, in sciatic nerve and brain from both species. Polymerase chain reaction amplification of a cDNA library of cultured rat Schwann cells using MK1‐ and MK2‐ specific primers produced DNA fragments that were highly homologous to MK1 and MK2. To determine whether these channel genes were axonally regulated, we performed Northern blot analysis of developing, permanently transected, and crushed rat sciatic nerves. The mRNA levels for both MK1 and MK2 increased from P1 to P15 and then declined modestly. Permanent nerve transection in adult animals resulted in a dramatic and permanent reduction in the mRNA levels for both MK1 and MK2, whereas normal levels of MK1 and MK2 were restored when regeneration was allowed to occur following crush injury. In all cases, MK1 and MK2 mRNA levels paralleled that of the myelin gene P0/sb. Elevating the cAMP in cultured Schwann cells by forskolin, which mimics axonal contact but not myelination, did not induce detectable leveles of MK1 and MK2 mRNA by Northern blot analysis. Further, the level of MK1 mRNA in the vagus nerve, which contains relatively fewer myelinating Schwann cells and relatively more non‐myelinating Schwann cells than the sciatic nerve, is reduced relative to the sciatic nerve. In conclusion, we have identified two Shaker‐like potassium channel genes in sciatic nerves whose expressions are regulated by axons. We suggest that MK1 and MK2 mRNA are expressed in high levels only in myelinating Schwann cells and that these Shaker‐like potassium channel genes have specialized roles in these cells.
Postmitotic oligodendrocytes generated during postnatal cerebral development are derived from proliferation of immature oligodendrocytes
Authors: Skoff Robert P. , Ghandour M. Said , Knapp Pamela E.
Published: May 2005

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

In early myelinating regions of the cerebrum, glycolipid positive cells account for the majority of the 3H‐thymidine labeled cells. This data, when combined with the quantification of proliferating astrocytes (ASs) from previous immunocytochemical and electron microscopic studies, indicate that oligodendroblasts and astroblasts constitute the vast majority of the proliferating glia in the brain and in optic nerve at times when ASs and OLs are being generated. In normal postnatal cerebral development, the immature ASs and OLs which proliferate are the direct, immediate precursors for most postmitotic ASs and OLs.
Osmoregulatory changes in Myo‐inositol content and Na+/Myo‐inositol contransport in rat cortical astrocytes
Authors: Strange Kevin , Emma Francesco , Paredes Ana , Morrison Rebecca
Published: May 2005

Exposure of cortical astrocytes to 325, 350, or 390 mosM culture media for 48 h caused a 1.4‐, 2.1‐, and 3.5‐fold increase, respectively, in cellular content of the compatible osmolyte myo‐inositol. Elevated myo‐inositol levels accounted for ∼56‐100% of the solute needed by the cells for complete volume regulation under hypertonic conditions. Myo‐inositol accumulation was associated with 4‐5‐fold (peak rate) and 1.8‐2‐fold (steady‐state rate) increases in the rate of Na+ ‐dependent myo‐inositol uptake when cells were acclimated to 390 mosM culture medium for 12 h or 24‐96 h, respectively. When medium osmolality was elevated by 25 mosM, peak and steady‐state increases in myo‐inositol uptake of 1.7‐fold and 1.3‐fold, respectively, were observed. Exposure to 390 mosM medium for 12‐48 h induced a 3‐8‐fold increase in cotransporter mRNA levels suggesting that the increase in myo‐inositol uptake is brought about by increased cotransporter gene expression. Abrupt return of hypertonic cells to an isotonic medium induced a rapid increase in myo‐inositol efflux and a return of cotransporter mRNA to control values in <2 h. In contrast, the cotransporter remained fully activated at hypertonic levels for 16 h. Between 16‐24 h after the transfer, the rate of myo‐inositol uptake returned to control values. The remarkable sensitivity of the cotransporter to hypertonic stress indicates that upregulation of myo‐inositol transport in glial cells is likely to occur in a variety of disease states that cause an elevation of plasma osmolality. Slow downregulation of the cotransporter may be responsible in part for the slow loss of myo‐inositol and cerebral edema that occurs with too rapid correction of chronic plasma hypertonicity.
Transitory disappearance of microglia during the regeneration of the lizard medial cortex
Authors: Lopez‐Garcia C. , Nacher J. , Castellano B. , de la Iglesia J. A. Luis , Molowny A.
Published: May 2005

In normal lizards, microglial cells populate the medial cortex (a zone homologous to the hippocampal fascia dentata), with a preferential distribution along the border between the granular cell layer and the plexiform layers.

Intraperitoneal injection of the neurotoxin 3‐acetylpyridine (3AP) induces a selective lesion in the medial cortex with a rapid degeneration of the granular layer and its zinc‐enriched axonal projection. Within 6‐8 weeks, the granular layer is, however, repopulated by a new set of neurons generated in the subjacent ependyma and the cell debris is removed. The aim of this study was to determine to what extent microglia were involved in the scavenging processes during the regeneration process. To this end we studied the brains of regenerating lizards at different times after 3AP lesion, visualising microglial cells by the nucleoside diphosphatase (NDPase) histochemical reaction.

Surprisingly, we found that stained microglial cells disappeared 6–8 hours after 3AP injection and remained absent until 10–15 days after injection. One month postlesion an increased population of microglial cells was found scattered throughout all plexiform layers of the cortex. Thorough examination of semithin and ultrathin sections confirmed the absence of microglia in the medial cortex of recent lesioned animals but the presence of an exuberant population after 1 month postlesion. In the tissue, phagocytotic scavenging was carried out by radial ependymocytes, not by microglia.
Immunocytochemical demonstration of glycogen phosphorylase in Müller (glial) cells of the mammalian retina
Authors: Pfeiffer Brigitte , Grosche Jens , Reichenbach Andreas , Hamprecht Bernd
Published: May 2005

Glycogen phosphorylase (GP) was immunocytochemically detected in Müller cells of the rabbit and rat retina using a monoclonal antibody raised against bovine brain GP. Immunofluorescence and immunoenzymatic procedures were applied on isolated, Müller cells and sections of paraformaldehyde‐fixed, paraffin‐embedded retinas. All methods used revealed positive immunostaining. GP immunoreactivity was most intense in the Müller cell endfeet and the pericarya, corresponding to the nerve fibre layer and the inner nuclear layer in the retina. The presence of GP in Müller cells stresses the important role of these glial cells in the energy metabolism of the mammalian retina.
Astrocyte and microglial motility in vitro is functionally dependent on the hyaluronan receptor RHAMM
Authors: Turley E. A. , Hossain M. Z. , Sorokan T. , Jordan L. M. , Nagy J. I.
Published: May 2005

RHAMM (Receptor for Hyaluronic Acid Mediated Motility) has been identified as a receptor for the extracellular matrix component hyaluronan (HA) and was recently shown to be essential for the locomotion of normal and transformed peripheral cells. Until now the potential role of RHAMM in the motility of neural‐derived cells has not been investigated. Here, we report that cultured primary astrocytes, astrocyte cell lines, and microglia express this receptor and exhibit RHAMM‐dependent motility. Immunocytochemical localization of RHAMM showed that it was often present as aggregates at the periphery of cells in contact with one another or concentrated on protruding processes of isolated cells. Glial cells contained 50 and 72 kDa forms of RHAMM, and both of these forms were found to have HA binding capacity. Time lapse imaging of cell locomotion revealed a significant inhibition of motility and process elongation by neutralizing anti‐RHAMM antibodies and by peptides corresponding to the HA binding domains of RHAMM. These results demonstrate that RHAMM serves a role in glial cell locomotion in vitro and provide the basis for investigations of the motile behavior of glial cells in vivo after CNS injury.
Evidence for an NGF‐induced autocrine neurotrophic potential of glial cells in nervous system development
Authors: Zimmermann Astrid , Sutter Arne , Stephani Ulrich
Published: May 2005

NGF receptor bearing peripheral glial cells, purified from chicken embryo dorsal root ganglia, were found to secrete neurite growth promoting activity (NGPA) but no NGF (detection limit 1 pM). If, however, minute concentrations of NGF (2 pM) were added to the glial cultures, an autocrine response was observed. Levels of NGF‐like activity in the medium rose up to 50‐fold. Induction of this autocrine response occured within a narrow range of NGF concentrations. NGPA production was not affected by addition of NGF. Coculture with neurons influenced the neurotrophic factor production. The data suggest that glial cells can “sense” traces of NGF and regulate its availability during neural development.
Expression of myelin transcription factor I (MyTI), a “Zinc‐Finger” DNA‐binding protein, in developing oligodendrocytes
Authors: Armstrong Regina C. , Kim Jin G. , Hudson Lynn D.
Published: May 2005

The production of myelin by oligodendrocytes requires the coordinated, massive synthesis of myelin components, a program that is dependent on transcriptional controls. Myelin transcription factor I (MyTI) was named for its ability to recognize the proteolipid protein (PLP) gene, the most abundantly transcribed central nervous system myelin gene (Kim and Hudson: Mol. Cell Biol. 12:5632, 1992). MyTI is a zinc‐dependent, DNA‐binding protein of the Cys2‐His‐Cys class. The pattern of MyTI expression, documented in the present study, suggests that MyTI may be instrumental in early stages of oligodendrocytic development and myelin production. MyTI mRNA transcripts are more highly expressed in oligodendrocyte progenitors than in differentiated oligodendrocytes. In vitro and in vivo analyses show that MyTI immunoreactivity is stronger in oligodendrocytic progenitors than in mature oligodendrocytes which have already accumulated PLP. In oligodendrocyte progenitors, MyTI immunoreactivity appears as speckles within the nucleus, suggestive of an association of MyTI with a function that is spatially segregated into discrete nuclear domains. MyTI continues to be expressed in cells transcribing PLP. However, as oligodendrocytes accumulate PLP, MyTI immunoreactivity becomes restricted to the cytoplasm and progressively diminishes. Since MyTI has two widely separated sets of DNA‐binding domains and initial MyTI expression markedly precedes PLP expression, we hypothesize the following model: MyTI may play a role in assembling transcriptionally active complexes of PLP, perhaps by bending the DNA of the promoter region to induce an appropriate conformation to enable subsequent binding the DNA of the promoter region to induce an appropriate conformation to enable subsequent binding of additional regulatory proteins. © 1995 Wiley‐Liss, Inc.
Astrocytes modulate retinal vasculogenesis: Effects on endothelial cell differentiation
Authors: Jiang Baoen , Alibezhadian M. , Caldwell Ruth B.
Published: May 2005

In the developing retina, the microvessels form by differentiation of endothelial precursor cells in a process referred to as vasculogenesis. Experiments using in vivo and in vitro model systems were designed to determine the specific influence of astrocytes on this process. Immunolocalization analyses of retinal vasculogenesis in vivo showed that astrocytes spread within the nerve fiber layer of the neonatal rat retina just ahead of the forming vessels. Then, endothelial precursor cells align themselves in register with the astrocytes. In contact with astrocytes, precursor cells differentiate as vascular endothelium, as indicated by lumen formation and patency to red blood cells. Experiments in vitro using cell culture and conditioned medium approaches showed that cell‐cell contact between rat brain astrocytes and bovine retinal endothelial cells results in release of soluble factors, inhibiting endothelial cell growth and inducing morphological differentiation in capillary‐like structures. Thus, it is suggested that astrocytes lay down the pattern for vasculogenesis and induce the elongation and alignment of endothelial precursor cells into a prevascular meshwork. In contact with astrocytes, precursor cells differentiate as vascular endothelium. Furthermore, this cell‐cell contact with astrocytes apparently inhibits endothelial cell growth and stimulates their elongation, alignment, and morphogenic differentiation by means of the release or activation of soluble, growth factor‐like substances. © 1995 Wiley‐Liss, Inc.
Characterization of a voltage‐dependent potassium channel in quid Schwann cells reconstituted in planar lipid bilayers
Authors: Noceti F. , Ramírez A. N. , Possani L. D. , Prestipino G.
Published: May 2005

An affinity column prepared with noxiustoxin (NTx), a K+ channel blocker from the venom of the Mexican scorpion Centruroides noxius, was used to purify a functional channel from a detergent extract of Schwann cell membrane of the giant axon of the squid Loligo vulgaris. The purified protein was reconstituted as a functional unit in a planar lipid bilayer and tested with a sequence of potentials to obtain information about single‐channel amplitude and kinetics. The reconstituted channel showed delayed rectifier behavior with a slope conductance of 10 pS under 5:1 asymmetric KCl concentrations and a clear tendency to open under negative potentials. The zero‐current potential was +36 mV, which fitted well with the Nernst equation for the CIS/TRANS K+‐concentration ratio of 5:1. The channel also showed a strong sensitivity to tetraethylammonium and its activity was inhibited by NTx, as expected from the purification procedure. The behavior of this protein in the presence of 0.5 mM ATP (cis side) was also tested, significantly increasing current fluctuations across the membrane. In order to compare the modulation of the Schwann cell K+ channel with that of the axonal K+ channel, a purified protein from the squid axon membrane was also tested in the presence of ATP. This 10–11 pS, delayed rectifier channel from the squid giant axon (Prestipino et al., FEBS Lett. 250:570–574, 1989) was also tested in the presence of ATP and showed a similar rise in activity. © 1995 Wiley‐Liss, Inc.
Cytokine‐induced expression of type II nitric oxide synthase in astrocytes is downregulated by ATP and glutamate
Authors: Murphy Sean , Lin Hsin Lee , Park Song Kyu
Published: May 2005

Combinations of cytokines and/or phorbol ester induce expression of Type II nitric oxide synthase (NOS) mRNA in astrocyte cultures via protein kinase mediated pathways (Simmons and Murphy: GLIA 11:227, 1994; Feinstein et al.: J Neurochem 62:811,1994). Agonists that activate receptors linked to protein kinases did not reproduce this effect of cytokines in astrocytes. On the contrary, ATP and glutamate treatment of astrocytes prior to a combination of interleukin‐1ß and interferon‐γ markedly reduced (30–50%) subsequent NOS mRNA expression. The effect was not seen if treatment coincided with or followed cytokine activation, suggesting that ATP and glutamate were not destabilizing NOS mRNA. The effects of ATP and glutamate were additive and could be mimicked by selective receptor agonists, but were insensitive to a specific inhibitor of protein kinase C. The inhibition of cytokine‐induced NOS mRNA expression caused by these agents was not the result of interference with the activation/translocation of nuclear factor‐ϰB by interleukin‐1ß. These results suggest that exposure of astrocytes to ATP and glutamate, both of which increase markedly in a variety of neuropathologies, could modulate the subsequent responsiveness of these cells to NOS‐inducing stimuli. As such, this may be an important regulatory mechanism in the expression of Type II NOS in vivo. © 1995 Wiley‐Liss, Inc.