bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of conditionally immortalized astrocyte cell lines derived from adult human spinal cord
Authors: Whittemore Scott R. , Neary Joseph T. , Kleitman Naomi , Sanon Henry R. , Benigno Adelaida , Donahue Roger P. , Norenberg Michael D.
Published: December 2004

As an approach to develop both oligodendrocytic and astrocytic cell lines from adult human spinal cord, a cellular preparation of highly enriched oligodendrocytes and their precursors was infected with a replication‐deficient retrovirus containing DNA sequences encoding the temperature‐sensitive mutant of SV40 large T antigen. Six immortal cell lines were obtained. At both permissive (33°C) and non‐permissive (38.5°C) temperatures, all cell lines were positive for vimentin, two demonstrated glial fibrillary acidic protein (GFAP) immunoreactivity, and none expressed oligodendrocyte or micro‐glial markers. The 2 GFAP‐positive cell lines [human spinal cord (HSC)2 and HSC6] were further characterized. Karyotype analysis revealed that both HSC2 and HSC6 cells showed gain of chromosomal material and structural chromosomal abnormalities. However, at non‐permissive temperature both cell lines were indistinguishable from primary human astrocytes by a number of criteria. These properties included glutamine synthetase activity, Na+‐dependent glutamate uptake, K+ flux, purine‐evoked Ca2+ mobilization and entry, and the ability to support neurite outgrowth from embryonic rat retinal explants. The HSC2 and HSC6 cell lines may prove to be valuable models for studying the physiological properties of adult human astrocytes. © 1994 Wiley‐Liss, Inc.
Regulation of an oligodendrocyte progenitor cell line by the interleukin‐6 family of cytokines
Authors: Kahn M. A. , de Vellis J.
Published: December 2004

We report pleiotropic actions of the interleukin‐6 family of cytokines on a rat cerebral cortical oligodendrocyte cell line, Central Glia‐4 (CG‐4). This is a bipotential oligodendrocyte type‐2 astrocyte (O‐2A) progenitor cell line that can be manipulated in vitro to become either a type‐2 astrocyte or to follow a linear sequence of events into becoming a mature oligodendrocyte. Using Northern and Western analyses in conjunction with immunocytochemistry we have demonstrated that ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and interleukin‐6 (IL‐6) cause a transient increase in glial fibrillary acidic protein (GFAP) in oligodendrocyte type‐2 astrocyte (O‐2A) progenitor cells. At maximal cytokine concentrations, the largest increase in GFAP protein levels were observed for CNTF and LIF; albeit, IL‐6 did increase GFAP but the order of magnitude was 6‐7 times less. Moreover, in trophic factor deprived medium, CNTF and LIF protected immature (O4+/MBP−) and mature (MBP+) oligodendrocytes from the apoptotic mode of cell death, while IL‐6 had no effect in enhancing oligodendrocyte cell survival. Analysis of the cytokine‐induced early response genes (ERGs) revealed a strong degree of overlap for CNTF and LIF. The effect of IL‐6 was different in the degree to which the ERGs were up‐regulated and in their temporal patterns of expression. These findings suggest that ERGs may be important, at least in part, for determining the extent of functional overlap observed within this cytokine family. Our findings clearly demonstrate differential regulation of oligodendrocyte survival and differentiation by the IL‐6 family of cytokines.
Denervated skeletal muscle stimulates migration of Schwann cells from the distal stump of transected peripheral nerve: An in vivo study
Authors: Dubový Petr , Svíženská Ivana
Published: December 2004

We have tested the stimulation of Schwann cell migration from the distal stump of a 1 week transected sciatic nerve of adult rats by denervated skeletal muscle. Migrating Schwann cells were distinguished by the presence of non‐specific cholinesterase (nChE) activity and glial fibrillary acidic protein (GFAP) at a distance of about 6 mm among denervated muscle fibres 4 weeks after insertion of the distal stump. In addition, the distal stump was introduced into the open end of a silicone chamber packed with artificial fibrin sponge (Gelaspon®) soaked in homogenate from intact or denervated muscles. A larger amount of migrated Schwann cells was observed in the chambers filled with homogenate from denervated muscles. An alteration in the amounts of Schwann cells migrating into the silicone chambers observed after histochemical staining (nChE or GFAP) was supported by biochemical measurements of the nChE activity. The biochemical assessment of the nChE activity revealed the increased amounts of migrated Schwann cells in proportion to the protein contents of homogenates from the denervated muscles. In addition, heating of homogenate from the denervated muscles resulted in a diminution of Schwann cell migration. Bromodeoxyuridine incorporation did not show an increased proliferation of Schwann cells inside the chambers following application of homogenate from the denervated muscles in comparison with the homogenate from the innervated muscles. Our results suggest a stimulation of Schwann cell migration from the distal stump of the transected sciatic nerve by soluble factor(s) produced by denervated skeletal muscles.
Ryanodine‐induced intracellular calcium mobilisation in cultured astrocytes
Authors: Langley David , Pearce Brian
Published: December 2004

Changes in intracellular Ca2+ concentration were monitored in cultured cortical astrocytes challenged with ryanodine and ATP. Ryanodine elicited a modest, in comparison to ATP, increase in cytosolic Ca2+ concentration in approximately 60% of the cell fields examined. This effect was evident and, in fact, was augmented when incubations were performed in Ca2+ concentration were dose dependent, desensitised rapidly, and could not be mimicked by caffeine. Exposure to ryanodine was without effect on eicosanoid release from these cells nor did it influence Ca2+ mobilisation and eicosanoid release in response to ATP. In contrast, caffeine attenuated part of the ATP‐evoked increase in intracellular Ca2+ concentration in the majority of cells tested and abolished its effect on eicosanoid release.
Regulation of metallothionein‐I+II levels in specific brain areas and liver in the rat: Role of catecholamines
Authors: Gasull Teresa , Giralt Mercedes , Garcia Agustina , Hidalgo Juan
Published: December 2004

The role of the catecholamines noradrenaline, adrenaline and dopamine on metallothionein (MT) levels of specific areas of the rat brain has been studied. MT‐I or MT‐I+II levels were measured by radioimmunoassay using specific antibodies that cross‐react only slightly with human MT‐III (growth inhibitory factor, GIF). The inhibition of tyrosine hydroxylase with α‐methyl‐p‐tyrosine (MPT), which depletes brain dopamine, noradrenaline, and adrenaline, increased MT levels in all brain areas studied (frontal cortex, cortex, medulla oblongata plus pons, midbrain, striatum, hippocampus, hypothalamus, and cerebellum) when considering the results of two separate experiments. The α‐ and β‐receptor blockers, phentolamine, and propranolol, alone or together, did not increase brain MT levels in any area of the brain, suggesting that the effect of MPT in vivo is related to inhibition of the synthesis of dopamine rather than of noradrenaline and adrenaline. Dopamine, noradrenaline, and serotonin increased MT‐I levels in primary cultures of neurons, whereas decreased them in astrocyte‐enriched primary cultures. Since MT‐I levels are about ten times higher in astrocytes than in neurons, the increased brain MT levels induced by MPT may reflect the supperssion of the normal inhibitory effect of dopamine on astrocyte MT levels. The increase in MT concentrations induced in most parts of the brain by immobilization stress was not prevented by MPT, phentolamine, or propranolol, suggesting that it was not mediated by the central monoamines.
Type II sodium channels in spinal cord astrocytes in situ: Immunocytochemical observations
Authors: Black Joel A. , Westenbroek Ruth , Ransom Bruce R. , Catterall William A. , Waxman Stephen G.
Published: December 2004

The expression of sodium channel α‐subunit isoforms in astrocytes in adult rat spinal cord and optic nerve was examined utilizing immunocytochemical methods with antibodies generated against conserved and subtype‐specific sequences of the sodium channel. In adult rat spinal cord, astrocytes within the dorsal and ventral funiculi were immunolabelled with antibody SP20, which recognizes a conserved sequence within sodium channel types I, II, and III. In addition, astrocytes within these spinal cord white matter tracts were immunostained with antibody SP11‐II, which recognizes sodium channel type II. Antibodies SP11‐I and SP32‐III, which are directed against subtype‐specific sequences in sodium channel types I and III, respectively, did not label astrocytes in the dorsal and ventral funiculi of the spinal cord. In optic nerves, astrocytes were immunostained with antibody SP20. However, no detectable labelling of cells within the optic nerve was observed with antibodies SP11‐I, SP11‐II, and SP32‐III. These observations demonstrate that sodium channel II is expressed by astrocytes in spinal cord white matter. Moreover, these data suggest that regional factors regulate the level of sodium channel isoform expression in astrocytes.
Blood monocytes and spleen macrophages differentiate into microglia‐like cells on monolayers of astrocytes: Morphology
Authors: Sievers Jobst , Parwaresch Reza , Wottge Hans‐Ulrich
Published: December 2004

Several morphological and functional properties of microglial cells, the resident immunoeffector cells of the central nervous system (CNS), differ from those of monocytes/macrophages in other tissues. Microglia are assumed to derive from the myelomonocytic lineage, possibly as a distinct subpopulation that diverges from a common cell line early in ontogeny, invades the CNS, proliferates, and differentiates into ameboid and then ramified microglia. We tested the hypothesis that some morphological and functional properties of microglia are induced in myelomonocytic cells by nervous tissue, specifically astrocytes. In the present in vitro studies we compared the differentiation of microglia, blood monocytes, and spleen macrophages on acellular substrates and on monolayers of astrocytes and fibroblasts.

On acellular substrates, microglial cells at first acquire an ameboid morphology; later they show a few short, unbranched processes. On monolayers of pure astrocytes, microglial cells at first also differentiate inot ameboid cells, but after 5 to 7 days they start to develop processes with large lamellopodial tips. These lengthen and branch continuously during the next 2 weeks in vitro, demarcating a round to oval territory around the small ellipsoid cell body. By contrast, on monolayers of fibroblasts the microglial cells develop an ameboid morphology, but do not grow the typical long branched processes of the ramified form. Blood monocytes and spleen macrophages behave indistinguishably from microglia both on acellular and cellular substrates, i.e., on astroglia they develop the ramified form, while on fibroblasts they retain the ameboid shape. When microglia, macrophages, or monocytes are cultured on coverslips on top of astrocytic monolayers, i.e., physically separated from the astroglia, but exposed to the medium conditioned by astrocytes, a significant proportion of them also develop the ramified shape. These findings indicate that the ramified shape of microglia is induced by astrocytes. Since this morphology can also be induced in blood monocytes and macrophages, we take this to be further evidence for the proposition that microglial cells are derived from the myelomonocytic lineage, and, moreover, that properties of resident macrophages are largely determined by tissue components of their host organ.
Ca2+ influx into leech glial cells and neurones caused by pharmacologically distinct glutamate receptors
Authors: Hoschstrate Peter , Schlue Wolf‐Rüdiger
Published: December 2004

The effect of glutamatergic agonists on the intracellular free Ca2+ concentration ([Ca2+]i) of neuropile glial cells and Retzius neurones in intact segmental ganglia of the medicinal leech Hirudo medicinalis was investigated by using iontophoretically injected fura‐2. In physiological Ringer solution the [Ca2+]i levels of both cell types were almost the ssame (glial cells: 58 ± 30 nM, n = 51; Retzius neurones: 61 ± 27 nM, n = 64).

In both cell types glutamate, kainate, and quisqualate induced an increase in [Ca2+]i which was inhibited by 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX). This increase was caused by a Ca2+ influx from the extracellular space because the response was greatly diminished upon removal of extracellular Ca2+.

The glutamate receptors of neuropile glial cells and Retzius neurones differed with respect to the relative effectiveness of the agonists used, as well as with regard to the inhibitory strenght of DNQX. In Retzius neurones the agonist‐induced [Ca2+]i increase was abolished after replacing extracellular Na+ by organic cations or by mM amounts of Ni2+, whereas in glial cells the [Ca2+]i increase was largely preserved under both conditions. It is concluded that in Retzius neurones the Ca2+ influx is predominantly mediated by voltage‐dependent Ca2+ channels, whereas in neuropile glial cells the major influx occurs via the ion channels that are associated with the glutamate receptors.
Normal and pathological expression of GFAP promoter elements in transgenic mice
Authors: Galou M. , Pournin S. , Ensergueix D. , Ridet J.‐L. , Tchélingérian J.‐L. , Lossouran L. , Privat A. , Babinet C. , Dupouey P.
Published: December 2004

The expression of the glial fibrillary acidic protein (GFAP), a component of astroglial intermediate filaments, is regulated under developmental and pathological conditions. In order to characterize DNA sequences involved in such regulations, we produced transgenic mice bearing 2 kb of the 5′ flanking region of the murine GFAP gene linked to the Escherichia coli β‐galactosidase (β‐gal) reporter gene. Seven transgenic lines were obtained. We observed that the regulatory elements present in the transgene GFAP‐nls‐LacZ direct an expression in the neural and non‐neural tissue and target in vivo an unexpected subpopulation of astrocyte. In the developing brain, β‐gal activity and GFAP appeared simultaneously and in the same region, on embryonic day 18 (E18), suggesting that the 2 kb of the promoter contains the regulatory sequences responsible for the perinatal vimentin/GFAP switch. In addition, we demonstrated that the 2 kb sequence of the GFAP promoter used in the transgene possess elements which are activated after a surgical injury, thus permitting to study some aspects of reactive gliosis in these transgenic mice. These transgenic lines provide a useful tool by enabling further studies of astroglial and, probably, neuronal physilogies.
GM‐CSF promotes proliferation of human fetal and adult microglia in primary cultures
Authors: Lee Sunhee C. , Liu Wei , Brosnan Celia F. , Dickson Dennis W.
Published: December 2004

Proliferation of microglia/macrophages is a common finding in many central nervous system diseases. To identify mitogenic signals for human microglia, we examined primary cultures of human fetal and adult microglia after stimulation with cytokines, colony stimulating factors (CSFs), or LPS, using proliferating cell nuclear antigen (PCNA) expression as an index of cell proliferation. The results showed that both M‐CSF and GM‐CSF induced microglial proliferation in fetal and adult human cultures, but that GM‐CSF provided a much stronger stimulus. At 96 h post‐stimulation, the mean PCNA labeling index was 2.4 for M‐CSF and 13.3 for GM‐CSF in fetal microglia; in adult microglia, the PCNA labeling index was 4.7 for M‐CSF and 9.0 for GM‐CSF. The effect of GM‐CSF on fetal microglia was dose dependent and synergistic with M‐CSF. LPS abolished the basal level of PCNA labeling in adult microglia, but in fetal microglia, caused a slight increase in PCNA labeling (1.9) at 96 h and consistently enhanced microglial cell survival and differentiation into highly branched cells. The production of GM‐CSF in purified human fetal astrocyte and microglial cultures was examined after stimulation with LPS, TNF‐α, or IL‐1β. Unlike M‐CSF, neither cell type produced GM‐CSF in unstimulated cultures; however, when stimulated with IL‐1β, astrocytes expressed GM‐CSF mRNA and protein, which accumulated in the culture through 72 h. In microglia, LPS was the only effective inducing agent. An immunocytochemical study performed to identify in vivo sources of GM‐CSF revealed selective labeling of reactive astrocytes in active lesions of multiple sclerosis and senile plaques of Alzheimer's disease. Our data demonstrate that both fetal and adult human microglia are capable of proliferation in response to CSFs, GM‐CSF being the more effective stimulus.
Cultured rat astrocytes possess Na+‐Ca2+ exchanger
Authors: Takuma Kazuhiro , Matsuda Toshio , Hashimoto Hitoshi , Asano Shoichi , Baba Akemichi
Published: December 2004

Na+‐Ca2+ exchange activity in its reverse mode was demonstrated in cultured rat astrocytes. Combination of ouabain (1 mM) and monensin (20 ±M) caused a marked increase in 45Ca2+ uptake in astrocytes. 45Ca2+ uptake was also stimulated by lowering the external Na+ concentration. Ouabain plus monensin‐stimulated 45Ca2+ uptake was blocked by 3,4‐dichlorobenzamil (IC50, 16 ±M), an inhibitor of Na+‐Ca2+ exchanger, but not by nifedipine (0.1 ±M). The stimulated‐45Ca2+ uptake was observed even in K+‐free medium, and external K+ at 5‐10 mM caused a 2.2‐fold increase in the uptake. Microspectrofluorimetry using the Ca2+‐sensitive dye fura‐2 showed that ouabain plus monensin increased intracellular Ca2+ concentration in single astrocytes. The Ca2+ signal was dependent on external Ca2+ (EC50, 1.4 mM), and blocked by 20 ±M 3,4‐dichlorobenzamil, but not by Ca2+ channel blockers (Cd2+, 20 ±M; Ni2+, 100 ±M). Antiserum of cardiac Na+‐Ca2+ exchanger recognized 160 and 120‐135 kDa proteins on SDS‐polyacrylamide gel electrophoresis of astrocyte homogenate. Northern blot analysis revealed the presence of mRNA for the exchanger protein in astrocytes. These findings indicate that Na+‐Ca2+ exchanger which is modulated by K+ is present in cultured rat astrocytes.
Subcellular heterogeneity of voltage‐gated Ca2+ channels in cells of the oligodendrocyte lineage
Authors: Kirischuk S. , Scherer J. , Möller T. , Verkhratsky A. , Kettenmann H.
Published: December 2004

We studied the distribution of voltage‐gated Ca2+ channels in cells of the oligodendrocyte lineage from retinal and cortical cultures. Influx Of ca2+ via voltagegated channels was activated by membrane depolarization with elevated extracellular K+ concentration ([K+]e) and local, subcellular increases in cytosolic free Ca2+ concentration ([Ca2+]in) could be monitored with a fluometric system connected to a laser scanning confocal microscope. In glial precursor cells from both retina and cortex, small depolarizations (with 10 or 20 mM K+) activated Ca2+ transients in processes indicating the presence of low‐voltage‐activated Ca2+ channels. Larger depolarizations (with 50 mM K+) additionally activated high‐voltage‐activated Ca2+ channels in the soma. An uneven distribution of Ca2+ channels was also observed in the mature oligodendrocytes; Ca2+ trasients in processes were considerably larger. Recovery of Ca2+ levels after the voltage‐induced influx was achieved by the activity of the plasmalemmal Ca2+ pump, while mitochondria played a minor role to restore2+ levels after an influx through voltageoperated channels. During the development of white matter tracts, cells of the oligodendrocyte lineage contact axons to form myelin. Neuronal activity is accompanied by increases in [K+]e; this may lead to Ca2+ changes in the processes and the Ca2+ increase might be a signal for the glial precursor cell to start myelin formation. © 1995 Wiley‐Liss, Inc.
Transformation of type 1 astrocytes with N‐ethyl‐N‐nitrosourea: Establishment of an in vitro system and the role of the p53 gene
Authors: Hiraga Shoju , Arita Norio , Ohnishi Takanori , Izumoto Shuichi , Taki Takuyu , Yamamoto Hiroshi , Higuchi Masahide , Hayakawa Toru
Published: December 2004

N‐ethyl‐N‐nitrosourea (ENU)‐induced gliomas, animal models of human gliomas, are most frequently oligodendrocytic, while human gliomas tend to be astrocytic. To facilitate a detailed study of human glial carcinogenesis, we developed an in vitro system using type 1 astrocyte transformation with ENU. Type 1 astrocytes from fetal Wistar rat brain were treated by a single dose of ENU. Transformed colonies appeared 50 days after exposure to single doses of ENU greater than 150 μg/mL. Cloned cells from these colonies retained the immunohistochemical characteristics of type 1 astrocytes. They showed rapid growth and high saturation densities, colony formation in low (2%) serum medium and gave rise to tumors when injected into nude mice. When p53 expression was studied at each passage, a single cell positive for mutant p53 protein emerged 40 days after ENU treatment. In the next 1–3 passages, the mutant p53 positive cell formed piled‐up colonies and exhibited dominant growth. Northern blot analysis showed markedly increased accumulations of p53 mRNA in transformed cells. This in vitro transformation system of type 1 astrocytes provides a valuable tool for further investigations of astrocyte carcinogenesis. © 1995 Wiley‐Liss, Inc.
Glial cell transplants that are subsequently rejected can be used to influence regeneration of glial cell environments in the CNS
Authors: Blakemore W. F. , Crang A. J. , Franklin R. J. M. , Tang K. , Ryder S.
Published: December 2004

Transplantation of glial cells into demyelinating lesions in CNS offers an experimental approach which allows investigation of the complex interactions that occur between CNS glia, Schwann cells, and axons during remyelination and repair. Earlier studies have shown that (1) transplanted astrocytes are able to prevent Schwann cells from participating in CNS remyelination, but that they are only able to do so with the cooperation of cells of the oligodendrocyte lineage, and (2) transplanted mouse oligodendrocytes can remyelinate rat axons provided their rejection is controlled by immunosuppression. On the basis of these observations, we have been able to prevent the Schwann cell remyelination that normally follows ethidium bromide demyelination in the rat spinal cord by co‐transplanting isogeneic astrocytes with a potentially rejectable population of mouse oligodendrocyte lineage cells. Since male mouse cells were used it was possible to demonstrate their presence in immunosuppressed recipients using a mouse Y‐chromosome probe by in situ hydridisation. When myelinating mouse cells were rejected by removal of immunosuppression, the demyelinated axons were remyelinated by host oligodendrocytes rather than Schwann cells, whose entry was prevented by the persistence of the transplanted isogeneic astrocytes. The oligodendrocyte remyelination was extensive and rapid, indicating that the inflammation associated with cell rejection did not impede repair. If this host oligodendrocyte remyelination was prevented by local X‐irradiation, the lesion consisted of demyelinated axons surrounded by processes from the transplanted astrocytes. By this approach, it was possible to create an environment which resembled the chronic plaques of multiple sclerosis. Thus, these experiments demonstrate that in appropriate circumstances the temporary presence of a population of glial cells can alter the outcome of damage to the CNS. © 1995 Wiley‐Liss, Inc.
GFAP‐positive hippocampal astrocytes in situ respond to glutamatergic neuroligands with increases in [Ca2+]i
Authors: Porter James T. , McCarthy Ken D.
Published: December 2004

It is becoming increasingly clear that astrocytes play very dynamic and interactive roles that are important for the normal functioning of the central nervous system. In culture, astrocytes express many receptors coupled to increases in intracellular calcium ([Ca2+]i). In vivo, it is likely that these receptors are important for the modulation of astrocytic functions such as the uptake of neurotransmitters and ions. Currently, however, very little is known about the expression or stimulation of such astrocytic receptors in vivo. To address this issue, confocal microscopy and calcium sensitive fluorescent dyes were used to examine the dynamic changes in astrocytic [Ca2+]i, within acutely isolated hippocampal slices. Astrocytes were subsequently identified by immunocytochemistry for glial fibrillary acidic protein. In this paper, we present data indicating that hippocampal astrocytes in situ respond to glutamate, kainate, α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA), 1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid (t‐ACPD), N‐methyl‐D‐aspartate (NMDA), and depolarization with increases in [Ca2+]i. The increases in [Ca2+]i occurred in both the astrocytic cell bodies and the processes. Temporally the changes in [Ca2+]i were very dynamic, and various patterns ranging from sustained elevations to oscillations of [Ca2+]i were observed. Individual astrocytes responded to neuroligands selective for both ionotropic and metabotropic glutamate receptors with increases in [Ca2+]i. These findings indicate that astrocytes in vivo contain glutamatergic receptors coupled to increases in [C2+]i and are able to respond to neuronally released neurotransmitters. (c) 1995 Wiley‐Liss, Inc.
Binding and action of glucagon in cultured mouse astrocytes
Authors: Cockram C. S. , Kum W. , Ho S. K. S. , Zhu S. O. , Young J. D.
Published: December 2004

This study investigates glucagon binding in primary cultures of differentiated mouse astrocytes and the effect of glucagon on intracellular cAMP accumulation.

Binding of 125I‐glucagon (0.53 nM) to mouse astrocyte suspensions reached equilibrium after 10 min at 22°. Equilibrium binding corresponded to 46 · 15 pmol/mg protein (n = 3) representing approximately 10,000 occupied sites per cell at the tracer concentration used. Dissociation occurred with a half‐time of 2.5 min at 22°C and was not accelerated in the presence of unlabelled glucagon (1 M). Scatchard analysis suggested the presence of more than one class of binding site. The Ka for the higher affinity sites was 5.7–7.4 × 106 M−1. The Ka for the lower affinity sites was 3.6–5.3 104 M−1. The results suggest the presence of approximately 43,000 high affinity sites per cell. Binding was inhibited by unlabelled glucagon with an IC50 of 50 nM but unaffected by insulin and somatostatin. However, no 125I‐glucagon binding could be detected when intact monolayer cells attached to culture dishes were used.

Glucagon stimulated cyclic‐AMP accumulation in both cell suspensions and intact monolayer cells in a dose‐dependent fashion. However high concentrations were required when compared to the receptor‐binding studies.

Marked degradation of 125I‐glucagon by astrocytes during binding experiments was observed and this was inhibited by unlabelled glucagon but also by insulin and desoctapeptide insulin. © 1995 Wiley‐Liss, Inc.
Glial cell types, lineages, and response to injury in rat and fish: Implications for regeneration
Authors: Sivron Tomer , Schwartz Michal
Published: December 2004

Axons of the mammalian central nervous system do not regenerate spontaneously after axonal injury, unlike the central nervous system axons of fish and amphibians and the peripheral nervous system of mammals, which possess a good regenerative ability (Grafstein: The Retina: A Model for Cell Biology Studies, Part II, 1986; Kiernan: Biol Rev 54:155–197, 1979; Murray: J Comp Neurol 168:175–196, 1976; Ramony Cajal: Degeneration and Regeneration of the Nervous System, 1928; Reier and Webster: J Neurocytol 3:591–618, 1974; Sperry: Physiol Zool 23:351–361, 1948). It was previously believed that intrinsic differences between the central nervous system neurons of mammals and fish account for their differences in regenerative ability. The past decade, however, has seen an accumulation of evidence, indicating that mammalian central nervous system neurons are able to regenerate injured axons, at least to some extent. This was first demonstrated by Aguayo and colleagues (David and Aguayo: Science 214:931–933, 1981; Kierstead et al: Science 246:255–257, 1989), who showed that injured mammalian central nervous system axons can grow for a considerable distance into an autograft of a peripheral nerve. It was also demonstrated that injured rabbit optic axons can regenerate into their own environment (i.e., into the distal part of the injured optic nerve), if the injured nerve is treated so as to make it conducive for growth (Lavie et al: J Comp Neurol 298:293–314, 1990; Eitan et al: Science 264:1764–1768, 1994). It is generally believed today that the glial cells surrounding the axons play a crucial role in determining the regenerative capacity of any central nervous system. In this review, we discuss various aspects of glial cells in the context of regeneration, with special emphasis on similarities and differences between the glial cells of rat and fish optic nerves, representing a nonregenerative and a regenerative system, respectively. © 1995 Wiley‐Liss, Inc.
Differential Na+ channel β1 subunit mRNA expression in stellate and flat astrocytes cultured from rat cortex and cerebellum: A combined in situ hybridization and immunocytochemistry study
Authors: Oh Youngsuk , Waxman Stephen G.
Published: December 2004

Astrocytes have been shown to express voltage‐sensitive Na+ channels, but the molecular structure of these channels is not yet known. Recent studies have demonstrated the expression of rat brain voltage‐sensitive Na+ channel mRNAs in astrocytes. In this study, we used a combined non‐radioactive in situ hybridization immunocytochemistry method to investigate the expression of voltage‐sensitive Na+ channel ß1 subunit (Naß1) mRNA in definitively identified, GFAP‐positive astrocytes cultured from two different regions of the rat brain, cerebrum and cerebellum. In general, two morphologically distinct types of GFAP‐positive astrocytes were observed in culture: flat, fibroblast‐like and stellate, process‐bearing. We observed a differential expression ofNaß1 mRNA in GFAP‐positive astrocytes: (1) stellate astrocytes expressed Naß1 mRNA, although the level of Naß1 mRNA expression was variable, and (2) flat astrocytes generally did not express Naß1 mRNA. Moreover, Bergmann‐like cells from cerebellum did not express Naß mRNA, while the granule cells associated with Bergmann‐like cell expressed Naß mRNA. These observations indicate that Naß mRNA is differentially expressed in rat astrocytes with various morphologies in vitro. © 1995 Wiley‐Liss, Inc.
Indicator expression directed by regulatory sequences of the glial fibrillary acidic protein (GFAP) gene: In vivo comparison of distinct GFAP‐lacZ transgenes
Authors: Johnson William B. , Ruppe Mary D. , Rockenstein Edward M. , Price Jennifer , Sarthy Vijay P. , Verderber Lisa C. , Mucke Lennart
Published: December 2004

An increase in the expression of the glial fibrillary acidic protein (GFAP) gene by astrocytes appears to constitute a crucial component of the brain's response to injury because it is seen in many different species and features prominently in diverse neurological diseases. Previously, we have used a modified GFAP gene (C‐339) to target the expression of β‐galactosidase (β‐gal) to astrocytes in transgenic mice (Mucke et al.; New Biol 3:465–474 1991). To determine to what extent the in vivo

Throughout this text, “in vitro” refers to cells grown in culture in culture and “in vivo” to living animals.

expression of GFAP‐driven fusion genes is influenced by intragenic GFAP sequences, the E. coli lacZ reporter gene was either placed downstream of approximately 2 kb ofmurine GFAP 5′ flanking region (C‐259) or ligated into exon 1 of the entire murine GFAP gene (C‐445). Transgenic mice expressing C‐259 versus C‐445 showed similar levels and distributions of β‐gal activity in their brains. Exclusion of intragenic GFAP sequences from the GFAP‐lacZ fusion gene did not diminish injury‐induced upmodulation ofastroglial β‐gal expression or increase β‐gal expression in non‐astrocytic brain cells. These results demonstrate that 2 kb ofmurine GFAP 5′ flanking region is sufficient to restrict transgene expression primarily to astrocytes and to mediate injury‐responsiveness in vivo. This sequence therefore constitutes a critical target for mediators of reactive astrocytosis. While acute penetrating brain injuries induced focal increases in β‐gal expression around the lesion sites in C‐259, C‐445, and C‐339 transgenic mice, infection of C‐339 transgenic mice with scrapie led to a widespread upmodulation of astroglial β‐gal expression. Hence, GFAP‐lacZ transgenic mice can be used to monitor differential patterns of astroglial activation in vivo. These and related models should facilitate the assessment of strategies aimed at the in vivo manipulation of GFAP expression and astroglial activation. © 1995 Wiley‐Liss, Inc.
The receptor for tumor necrosis factor on murine astrocytes: Characterization, intracellular degradation, and regulation by cytokines and theiler's murine encephalomyelitis virus
Authors: Aránguez Isabel , Torres Concepción , Rubio Nazario
Published: December 2004

Previous reports have shown that tumor necrosis factor (TNF) exerts a role on the physiology of astrocytes under inflammatory situations. The signalling for biological effects of this and other cytokines are usually exerted through cell surface receptors. In this study, we have demonstrated the presence of a surface TNF α receptor type I in murine astrocytes of both SJL/J and BALB/c origin, using 125I‐labelled recombi‐nant mouse TNFα. A linear Scatchard plot indicates the presence of only one type of receptor with a MW of 58 kDa (Type I TNF receptor) that binds the ligand with a Kd of 1 10−9 M. There are 3,000 copies of this receptor on untreated astrocytes. The results also indicate that receptor‐bound TNF is rapidly internalized at 37°C and degraded intracellularly to a principal molecular species which elutes from HPLC reverse‐phase columns at 38% acetonitrile rather than at 60% as native TNFα does. The binding is up‐regulated by increasing the number of receptors (but not its affinity) by treatments with Theiler's murine encephalomyelitis virus (TMEV), Con A and inflammatory cyto‐kines such as IL‐1, IL‐6 and INF‐β. It is not influnced by vaccinia virus, IL‐2, or LPS. This receptor may contribute to the initiation of perpetuation of the immune response which mediates the demyelinating inflammation induced by Theiler's virus. © 1995 Wiley‐Liss, Inc.
Disruption of the blood‐brain interface in neonatal rat neocortex induces a transient expression of metallothionein in reactive astrocytes
Authors: Penkowa Milena , Moos Torben
Published: December 2004

Exposure of the adult rat brain parenchyma to zinc induces an increase in the intracerebral expression of the metal‐binding protein, metallothionein, which is normally confined to astrocytes, ependymal cells, choroid plexus epithelial cells, and brain endothelial cells. Metallothionein is expressed only in diminutive amounts in astrocytes of the neonatal rat brain, which could imply that neonatal rats are devoid of the capacity to detoxify free metals released from a brain wound. In order to examine the influence of a brain injury on the expression of metallothionein in the neonatal brain, PO rats were subjected to a localized freeze lesion of the neocortex of the right temporal cortex. This lesion results in a disrupted blood‐brain interface, leading to extravasation of plasma proteins. From 16 h, reactive astrocytosis, defined as an increase in the number and size of cells expressing GFAP and vimentin, was observed surrounding the neocortical lesion site. Astrocytes and pial cells situated adjacent to the area of injury also became positively stained for metallothionein. At 3–6 days post‐lesion, the highest level of reactive astrocytes expressing metallothionein was observed. Neo‐Timm staining revealed that histochemically reactive zinc had disappeared from the lesion site. Extracellular albumin and metallolhionein‐positive astrocytes were absent approximately 2 weeks after the lesion, whereas reactive astrocytosis was still observed. These results show that a lesion of the neonatal rat brain induces a transient expression of metallothionein in reactive astrocytes, probably as a response to metals released from the site of the brain injury. © 1995 Wiley‐Liss, Inc.
The human blood‐brain barrier glucose transporter (GLUT1) is a glucose transporter of gray matter astrocytes
Authors: Morgello Susan , Uson Ronald R. , Schwartz Elissa J. , Haber Richard S.
Published: December 2004

Human and monkey brain sections were examined by immunohistochemical light and electron microscopy to determine the distribution of GLUT1, a glucose transporter isoform associated with erythrocytes and endothelial cells of the human blood‐brain barrier. Protein immunoblotting of fractionated human brain membranes was performed to determine the distribution of molecular forms of the transporter. GLUT1 staining was abundant in erythrocytes and cerebral endothelium of gray and white matter but was also present diffusely in gray matter neuropil when viewed by light microscopy. Immunoelectron microscopy confirmed the gray matter and vascular localization of GLUT1, with specific GLUT1 staining seen in erythrocytes, gray and white matter endothelial cells, astrocyte foot processes surrounding gray matter blood vessels, and in astrocyte processes adjacent to synaptic contacts. No astrocytic staining was identified in white matter. Astrocyte GLUT1 staining was identified only in mature gray matter regions; undifferentiated regions of preterm (22–23 weeks gestation) cortex had GLUT1 staining only in blood vessels and erythrocytes, as did germinal matrix. Immunoblots of adult human frontal cortex revealed that two forms of GLUT1 (45 and 52 kDa) were present in unfractionated brain homogenates. Immunoblots of vessel‐depleted frontal lobe revealed only the 45 kDa form in gray matter fractions, and depleted in membranes prepared from white matter regions. We conclude that the GLUT1 isoform of glucose transporter is present both in endothelium of the blood‐brain barrier and in astrocytes surrounding gray matter blood vessels and synapses. Furthermore, the form present in astrocytes is likely to have a lower molecular weight than the form found in cerebral endothelium. The GLUT1 transporter may play an important role not only in astrocyte metabolism, but also in astrocyte‐associated pathways supporting neuronal energy metabolism. © 1995 Wiley‐Liss, Inc.
Hypoxia‐induced vascular endothelial growth factor expression in normal rat astrocyte cultures
Authors: Ijichi Akihiro , Sakuma Shirou , Tofilon Philip J.
Published: December 2004

Vascular endothelial growth factor (VEGF) is an endothelial cell‐specific mitogen, which also enhances vascular permeability. Because this angiogenic factor has been suggested to play a role in brain tumor biology, we have begun to investigate the regulation of VEGF expression in cultures of rat type I astrocytes. In this report, we have focused on the influence of hypoxia on VEGF expression. Under standard in vitro conditions (21% O2) VEGF expression in astrocytes is barely detectable by northern analysis. However, after exposure to 0.2% O2 for as little as 3 h VEGF mRNA levels are markedly increased reaching a maximum by approximately 8 h of exposure. Treatment of astrocytes with CoCl2 or desferrioxamine results in a similar induction of VEGF, suggesting that the oxygen sensor regulating VEGF expression in astrocytes is a heme‐containing molecule. Although acute treatment with TPA (6 h) induces VEGF expression, chronic exposure to TPA (24 h) to deplete PKC activity does not reduce the hypoxia‐induced VEGF expression. These data indicate that VEGF induction in astrocytes can proceed through PKC‐dependent and ‐independent pathways. Furthermore, chronic exposure to TPA or treatment with herbimycin A results in the enhancement of the hypoxia‐mediated increase in VEGF mRNA levels. These results suggest that PKC and herbimycin‐sensitive tyrosine kinase may serve as negative regulators of the hypoxia‐activated signal transduction pathway that leads to the induction of VEGF expression. However, treatment of astrocytes with the nonspecific kinase inhibitors H7 and H8 reduced the level of VEGF induction by hypoxia, indicating that some type of kinase activity is required in this signaling pathway. © 1995 Wiley‐Liss, Inc.
Interferon‐γ and lipopolysaccharide reduce cAMP responses in cultured glial cells: Reversal by a type IV phosphodiesterase inhibitor
Authors: Patrizio Mario , Costa Tommaso , Levi Giulio
Published: December 2004

The aim of the present study was to determine whether two classical macrophage activators, bacterial lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) could affect the accumulation of the second messenger cAMP in cultured rat microglia and astrocytes. Purified microglia and astrocyte secondary cultures obtained from the neonatal rat were grown for 3 days in basal medium Eagle (BME) + 10% fetal calf serum (FCS). Exposure of microglia to LPS resulted into a dose‐ and time‐dependent decrease in the accumulation of cAMP induced by receptor‐mediated (isoproterenol or prostaglandin E2) or direct (forskolin) activation of adenylate cyclase. The inhibitory effect of LPS was rapid (a 10 min preincubation was sufficient to approach a maximal effect), occurred at low doses (IC50 = 1.2 ng/ml), and was not abrogated by pertussis toxin. A selective inhibitor of type IV phosphodiesterase (rolipram, 100 nM) prevented the effect of LPS on cAMP accumulation, while inhibitors of other forms of phosphodiesterase were unable to do so. IFN‐γ (100 u/ml) also caused a depression of the evoked cAMP accumulation in microglia after a 10 min preincubation, and its effect was prevented by rolipram, as in the case of LPS. Astrocytes differed from microglia in that LPS (1–100 ng/ml) did not inhibit the accumulation of cAMP induced by either isoproterenol or forskolin; on the other hand, IFN‐γ did have an inhibitory effect (though less pronounced than in microglia) that could be prevented by rolipram. Our observations indicate that two potent activators of microglia acting at different receptors, LPS and IFN‐γ, can diminish the accumulation of cAMP through a common mechanism, the stimulation of a specific form of cAMP phosphodiesterase. The fact that IFN‐γ, but not LPS, was effective in astrocytes suggests that LPS receptors are scarcely, if at all, expressed in these cells, or that they are differently coupled to second messengers. Selective inhibitors of type IV phosphodiesterase might prevent some of the obnoxious actions of LPS or IFN‐γ in the living organism. © 1995 Wiley‐Liss, Inc.
Multiply myelinated axons in the optic nerve of mice deficient for the myelin‐associated glycoprotein
Authors: Bartsch Udo , Montag Dirk , Bartsch Susanne , Schachner Melitta
Published: December 2004

We recently reported that some retinal ganglion cell axons in mice deficient for the myelin‐associated glycoprotein are concentrically surrounded by more than one myelin sheath. In the present study, we demonstrate that myelin sheaths displaced from the axon reveal a normal ultrastructure of compact myelin, with the only exception that multiple myelination of axons frequently correlates with the presence of unfused regions of major dense lines. Supernumerary sheaths terminated on other sheaths or on astrocyte cell surfaces in a pattern closely resembling the morphology of a true paranode. The thickness of compact myelin of multiply myelinated axons was significantly increased when compared with axons of similar caliber surrounded by a single myelin sheath. Our observations demonstrate that maintenance of compact myelin and paranodal regions is not dependent on direct axonal contact and that the presence of more than one concentric myelin sheath around an axon results in dysregulation of the axon‐to‐fiber ratio. © 1995 Wiley‐Liss, Inc.
Isoform‐specific expression of sodium channels in astrocytes in vitro: Immunocytochemical observations
Authors: Black J. A. , Westenbroek R. , Minturn J. E. , Ransom B. R. , Catterall W. A. , Waxman S. G.
Published: December 2004

The expression of sodium channel α‐subunit isoforms in astrocytes cultured from P‐0 rat spinal cord and P‐7 rat optic nerve was examined utilizing immunocytochemical methods with antibodies generated against conserved and isoform‐specific amino acid sequences of the rat brain sodium channel. In spinal cord cultures at 5 days in vitro (DIV), both stellate and flat astrocytes were immunostained with antibody SP20, which recognizes a conserved sequence common to sodium channel types I, II/IIA, and III. Antibody SP11‐I, which is directed against a subtype‐specific sequence in sodium channel I, did not yield detectable staining in spinal cord astrocytes. Antibody SP11‐II, which is directed against a subtype‐specific sequence in sodium channel II, immunostained both stellate and flat spinal cord astrocytes, although with less intensity than SP20. Antibody SP32‐III, which is directed against a subtype‐sequence in sodium channel III, immunostained stellate but not flat spinal cord astrocytes. SP20, SP11‐II, and SP32‐III staining persisted in stellate spinal cord astrocytes through 14–21 DIV, while SP20 and SP11‐II immunostaining in flat spinal cord astrocytes was attenuated with time in culture. In optic nerve cultures at 5 DIV, SP20 staining was present in both stellate and flat astrocytes, but at reduced levels compared to spinal cord astrocytes. With increased time in culture, SP20 staining was maintained in stellate optic nerve astrocytes but was gradually lost in flat optic nerve astrocytes. Stellate optic nerve astrocytes exhibited low levels of staining with SP11‐I, SP11‐II, and SP32‐III. Flat optic nerve astrocytes lacked or displayed very low SP11‐II staining, and SP11‐I and SP32‐III staining was not detectable. These observations demonstrate that cultured astrocytes are immunoreactive to antibodies generated against conserved and isotype‐specific peptide sequences of rat brain sodium channels, and further suggest that there are different patterns of sodium channel expression between flat vs. stellate astrocytes and in astrocytes derived from different regions of the CNS. © 1995 Wiley‐Liss, Inc.