bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of dexamethasone on the differentiation of membrane structure in cultured astrocytes
Authors: Landis Dennis M. D. , Weinstein Lori A. , Skordeles Christine J.
Published: January 2005

Astrocytic processes investing vascular structures or forming the surface of mammalian brain have large numbers of orthogonally packed aggregates of intramembrane particles, termed “assemblies”. Similar particle aggregates are expressed by astrocytes derived from neonatal rat forebrain in secondary culture, but they are much more uniformly distributed across the membranes of the cultured cells. Dexamethasone, a potent glucocorticoid, affects the differentiation of astrocyte membrane structure in two patterns, depending on the rate of proliferation in the culture. When confluent secondary cultures of astrocytes are exposed to 5 μM dexamethasone, the densities of assemblies increase, and in some cells approach the values present in the glial limitans in vivo. However, when rapidly proliferating astrocytes are exposed to dexamethasone during the first week of secondary culture, most of the astrocytes fail to express any assemblies. The rate of astrocyte proliferation is slowed, and a lower cell density is reached during the first 2 weeks of secondary culture in dexamethasone. The suppression of assemblies is transient: as the cultures approach confluence, the proportion of cells expressing assemblies increases to nearly control levels, and the density of assemblies increases to greater than control values in some astrocytes. Certain of the effects of dexamethasone on cultured astrocytes may have relevance for understanding the mechanism(s) of its action in treating cerebral edema.
Astroglial alterations in rat hippocampus during chronic lead exposure
Authors: Selvín‐Testa Asia , Lopez‐Costa J. J. , Nessi De Avin̈n A. C. , Pecci Saavedra J.
Published: January 2005

The present study was performed in order to follow the response of astroglial cells in the rat hippocampus to chronic low‐level lead exposure. The experiments combined immunohistochemistry using anti‐glial fibrillary acidic protein (GFAP) antibody and conventional transmission electron microscopy (EM). Chronic administration with drinking water [1 g% w/v (subclinical dose) of lead acetate dissolved in distilled water] was started through the mother's milk when pups were 7 days old. Following weaning, experimental offspring were treated for 3 months with the same concentration of adulterated water. The group of intoxicated animals and their controls were sacrificed by perfusion‐fixation at 30, 60, and 90 days of exposure. After 60 days of lead treatment, staining of GFAP‐positive cells demonstrated an astroglial transformation from the quiescent to the reactive state, characterized by an increase in GFAP. In control rats no changes in GFAP immunostaining were observed. The intensity of the astroglial response was enhanced after 90 days of lead intoxication, showing an increment of GFAP immunoreactivity. Quantification of these changes was made by computerized image analysis, confirming that the sectional areas of the astroglia in lead‐exposed animals were larger than those in controls. These results are consistent with the ultrastructural alterations. Simultaneously with the increment in gliofilaments, intranuclear inclusions were seen in some astrocytes. The mechanisms by which lead affects astrocytes are unknown. Probably the astroglial changes induced by lead intoxication produce microenvironmental modifications that may disturb the neuronal function.
Heterogeneous astroglial response in the rat spinal cord to long‐term portacaval shunt: An immunohistochemical study
Authors: Bodega G. , Suárez I. , Arilla E. , Rubio M. , Fernández B.
Published: January 2005

Glial fibrillary acidic protein (GFAP) immunoreactivity has been used to study the astroglial response in the rat spinal cord to long‐term portacaval shunt (PCS). The astroglial response in PCS rats is heterogeneous. In general, astrocytes show a loss of GFAP immunoreactivity, as well as shrinking and pyknosis in their nuclei; however, while GFAP reactivity was unchanged in the periependymal region, it was strongly increased in the dorsolateral region of the spinal cord (lateral spinal nucleus, dorsal root entry zone, and the most dorsal region of the dorsal horn). Three possibilities are postulated to explain how astrocytes, in the periependymal and dorsolateral regions, can support the effects of PCS: (a) astrocytes related to glutamatergic pathways ought to possess a more efficient ammonia uptake and detoxification system, (b) long‐term PCS can activate nociceptive pathways (substancePergic fibers), and (c) astrocytes located in periependymal and dorsolateral regions can be exposed to lower concentrations of ammonia because of its diffusion into the cerebro‐spinal fluid close to these regions.
Virulent and attenuated canine distemper virus infects multiple dog brain cell types in vitro
Authors: Pearce‐Kelling Susan , Mitchell William J. , Summers Brian A. , Appel Max J. G.
Published: January 2005

Canine Distemper Virus (CDV) produces an encephalitis in dogs that varies with viral strain. We have studied the cell tropisms of two virulent strains (CDV‐SH and CDV A75–17) and an attenuated strain, Rockborn (CDV‐RO), in cultured canine brain cells. Infected cell types were identified by double immunofluorescent labeling of specific cell markers and viral antigens. All viral strains studied produced infection in astrocytes, fibroblasts, and macrophages. Neurons were not infected by CDV A75–17 but were rapidly infected by CDV‐SH and CDV‐RO. Multipolar oligodendrocytes were very rarely infected by any of the virus strains. In contrast, a morphologically distinct subset of bipolar oligodendrocytes were commonly infected by CDV‐SH and CDV‐RO. The kinetics of infection in the astrocytes, oligodendrocytes, neurons, and macrophages varied between strains. Both CDV‐SH and CDV‐RO rapidly infected bipolar oligodendrocytes, astrocytes, neurons, and macrophages by 14 days post infection while infection by CDV A75–17 was delayed until after 28–35 days post infection. The differences in the growth kinetics and cell tropisms for some brain cells, exhibited by the three viral strains examined in this in vitro study, may relate to the different CNS symptoms that these strains produce in vivo.
Modulation of intracellular Ca++ in cultured astrocytes by influx through voltage‐activated Ca++ channels
Authors: Macvicar Brain A. , Hochman Daryl , Delay Michael J. , Weiss Samuel
Published: January 2005

Fura‐2 and indo‐1 fluorescence measurements were used to examine intracellular Ca++ concentration ([Ca++]i) and its modulation by voltage‐activated influx in murine cortical astrocytes in primary cell culture. Extracellular K+ was increased from 5 to 50 mM to depolarize cells to determine if Ca++ influx through voltage activated Ca++ channels could alter [Ca++]i. In confluent 4 to 6 weeks in vitro astrocyte cultures 50 mM K+ increased [Ca++]i 3‐4‐fold (from 150 nM up to 550 nM); this increase was blocked by nifedipine and enhanced by BayK 8644 indicating that influx was through L‐type channels. However, in 1 to 2 weeks in vitro astrocyte cultures, high K+ reduced [Ca++]i. L‐type channels were apparently present in these cells because high K+ in combination with BayK 8644 increased [Ca++]i. Following pretreatment of 1 to 2 weeks in vitro astrocytes with dibutyryl cAMP (dbcAMP) high K+ increased [Ca++]i in the absence of BayK 8644 indicating enhanced activity of Ca++ channels in agreement with previous voltage‐clamp studies. Ca++ influx through voltage‐activated channels in cultured cortical astrocytes can substantially increase [Ca++]i and these channels can be dynamically modulated by dihydropyridines. Immature astrocytes may express ‘silent’ or inactive Ca++ channels or have a much lower number of channels.
In vitro myelination of regenerating adult rat retinal ganglion cell axons by schwann cells
Authors: Bähr M. , Hopkins J. M. , Bunge R. P.
Published: January 2005

Schwann cell cultures provide a highly favorable substrate for retinal ganglion cell (RGC) survival and axon growth in vitro (Bähr and Bunge, Exp Neurol 106:27, 1989; Hopkins and Bunge, Glia 4:46, 1991). In this report we have extended former studies to obtain axon regeneration, long‐term survival, and myelination of adult rat RGC axons in co‐cultures of retinal explants with purified Schwann cells. By using modified co‐culture conditions, we observed myelination of regenerating adult RGC axons by Schwann cells after 3–4 weeks in vitro. Myelination was associated with a one‐to‐one Schwann cell‐axon relationship, characteristic of the formation of peripheral myelin. Under culture conditions that supported myelination, long‐term survival (more than 12 weeks) of a small population of RGCs was observed. These findings highlight the remarkable ability of Schwann cells to support long‐term survival of adult rat RGCs in the absence of either central nervous system (CNS) target tissue or other peripheral nervous system (PNS) components. This tissue culture system may serve as a model for the systematic study of the molecular mechanisms which are involved in axon regeneration and myelination of adult CNS neurons.
Orientation of the myelin proteolipid protein C‐terminus in oligodendroglial membranes
Authors: Konola Jukka T. , Yamamura Takashi , Tyler Betty , Lees Marjorie B.
Published: January 2005

The topology of the integral membrane proteolipid protein (PLP) has important structural and functional implications for central nervous system myelin. To determine the orientation of the carboxyl‐terminal portion of PLP, cultured mouse oligodendrocytes were probed with polyclonal antibodies raised against a synthetic terminal peptide corresponding to PLP residues 264–276 and with ten separate monoclonal antibodies that react with this region. Cells were examined by double‐label indirect immunofluorescence for the presence of the PLP C‐terminus and either oligodendrocyte‐specific surface or intracellular antigens. To detect surface antigens, both living and paraformaldehyde‐fixed cells were incubated with primary antibodies and then stained with fluorochrome‐conjugated second antibodies. Antigens located within the cytoplasmic space were identified after fixation and permeabilization of cells. Live‐labeled oligodendrocytes were stained brightly for myelin‐oligodendrocyte glycoprotein, galactocerebroside, and other surface markers but did not stain for the PLP C‐terminus or the intracellular proteins myelin basic protein and beta‐tubulin. Fixation alone was sufficient for partial permeabilization of oligodendrocytes to antibodies and resulted in limited staining of the PLP C‐terminus and intracellular proteins. The permeabilized oligodendrocytes stained intensely for the PLP C‐terminus, myelin basic protein, and betatubulin. Finally, trypsinization of living oligodendrocytes eliminated surface myelin‐oligodendrocyte glycoprotein staining but did not change the immunostaining properties of the PLP C‐terminus. These results provide evidence that the carboxyl‐terminus of PLP is located at the cytoplasmic face of oligodendroglial membranes.
The abundance of Aplysia gliagrana depends on Ca2+ and/or Na+ concentrations in sea water
Authors: Keicher Erick , Maggio Katia , Hernandez‐Nicaise Mari‐Luz , Nicaise Ghislain
Published: January 2005

The glial cells surrounding the identified giant nerve cell bodies R2 or LP1 of Aplysia punctata were studied by quantitative electron microscopy. They contain specific, electron‐dense but non‐osmiophilic membrane‐bound granules, approximately 0.3 μm in diameter, called gliagrana. Similar glial granules are more often found in marine than in freshwater molluscs, possibly because they represent a calcium store used to compensate excess Na+ in the extracellular milieu of marine species and to regulate perineuronal calcium concentration. In agreement with this hypothesis, the abundance of gliagrana (=number of glial granules per μm2) is found to be higher in animals adapted to low Ca2+ artificial sea water than in animals kept in high Ca2+ (or low Na+) conditions. This finding is not observed after 1 week but after 2 weeks of adaptation.
Zinc toxicity and induction of the 72 kD heat shock protein in primary astrocyte culture
Authors: Swanson Raymond A. , Sharp Frank R.
Published: January 2005

Zinc is a potent inducer of the 72 kD heat shock protein (HSP72). In brain, pathological conditions such as ischemia and seizures increase extracellular zinc. The present study examines the effect of zinc on HSP72 expression in rat primary cortical astrocyte culture. Astrocytes were grown to confluence and exposed to zinc chloride in CO2‐equilibrated Earle's buffered salt solution. Expression of HSP72 was examined using immunocytochemistry. HSP72 was induced with zinc concentrations of 5 to 100 μM after 4 h exposures, or 200 to 300 μM after 15 min exposures. At the lower concentrations expression occurred in small clusters of contiguous cells. At concentrations high enough to cause cell death, HSP72‐positive astrocytes formed a continuous margin around patches of dead cells. These patterns of HSP72 expression are similar to the patterns seen after cerebral ischemia in vivo. Exposure to zinc at 100 μM for 4 h or 400 μM for 15 min caused greater than 90% cell death. Increases in extracellular zinc may contribute to HSP72 induction and astrocyte death under ischemia and other pathological conditions in brain. © 1992 Wiley‐Liss, Inc.
Comparative biochemical, morphological, and immunocytochemical studies between C‐6 glial cells of early and late passages and advanced passages of glial cells derived from aged mouse cerebral hemispheres
Authors: Lee Kendall , Kentroti Susan , Billie Holly , Bruce Clifford , Vernadakis Antonia
Published: January 2005

We have used C6 glial cells (2B clone), early and late passage, as well as advanced passages (8–17) of glial cells derived from aged (18‐month‐old) mouse cerebral hemispheres (MACH), as model systems for studying glial properties. In this study passages 20–24 were considered “early” and passages 73–90 were considered “late.” Activities of glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP) were used as biochemical markers for astrocytes and oligodendrocytes, respectively. Glial phenotypes were identified immunocytochemically using double staining for glial fibrillary acidic protein (GFAP) and A2B5 antigen (type 1 and type 2 astrocytes) or galactocerebroside (GalC) and A2B5 antigen (oligodendrocytes); cells positive for A2B5 and negative for both GFAP and GalC were considered to be precursor cells. Cultures were grown either in DMEM supplemented with 10% fetal bovine serum or in serum‐free chemically defined medium (CDM) supplemented with insulin and transferrin. We report that early‐passage C6 glial cells continue to be bipotential cells and when grown in the absence of serum express high GS and CNP activities correlating with the high number of GFAP‐ and GalC‐positive cells, respectively. Late‐passage cells continued to be committed to the type 2 astrocytic phenotype regardless of media composition (± serum). MACH cultures consist of protoplasmic type 1 astrocytes, differentiated type 2 astrocytes, and oligodendrocytes as well as glial progenitor cells. When these cultures were grown in CDM + transferrin, both GS and CNP activities increased, suggesting that transferrin has provided the signal for progenitor cells present in these cultures derived from aged brain to differentiate into type 2 astrocytes and oligodendrocytes. © 1992 Wiley‐Liss, Inc.
Differential proliferative response of human and mouse astrocytes to gamma‐interferon
Authors: Yong Voon Wee , Tejada‐Berges Trevor , Goodyer Cynthia G. , Antel Jack P. , Yong Flons P.
Published: January 2005

We have previously shown that gamma‐interferon promoted the proliferation of adult human astrocytes isolated from brain biopsy specimens. In contrast, in the present study, astrocytes derived from neonatal mouse brains and treated with recombinant murine gamma‐interferon responded by a decreas (average of 50% at 100 U/ml) in proliferation. The basal rate of proliferation as assessed by bromodeoxyuridine incorporation was markedly increased in neonatal mouse astrocytes when compared to the adult human cells, suggesting that age, and the corresponding metabolic activity of cells, could be important determinants in the mitogenic response of astrocytes to cytokines. However, subsequent examinations of fetal human and adult mouse astrocytes, with comparable basal rate of proliferation to neonatal mouse and adult human cells respectively, showed gamma‐interferon to promote DNA synthesis in fetal human astrocytes while inhibiting that of adult mouse astrocytes. The results suggest species differences in the proliferative response of human and mouse astrocytes to the cytokine gamma‐interferon. © 1992 Wiley‐Liss, Inc.
Clonal segregation of oligodendrocytes and astrocytes during in vitro differentiation of glial progenitor cells
Authors: Lubetzki Catherine , Goujet‐Zalc Cecile , Demerens Corinne , Danos Olivier , Zalc Bernard
Published: January 2005

To study the clonal lineage of the glial progenitor population, isolated from newborn rat brain (Lubetzki et al. J Neurochem 56:671, 1991), we combined somatic transgenesis using a retroviral vector encoding a modified bacterial β‐galactosidase with nuclear localization, and triple immunofluorescence labeling with A2B5, anti‐galactosylceramide, and anti‐glial acidic fibrillary protein antibodies. This allowed clonal analysis of the postnatal glial lineage with precise phenotypic identification of each cell within the lacZ‐positive clones. When infected cells were cultivated under constant conditions, in the presence of either 1% or 10% fetal calf serum (FCS)‐containing medium, all the 250 lacZ‐positive clusters examined were homogeneous, i.e., either oligodendroglial or astroglial. Mixed astrocyte—oligodendroglial clones were observed when cells cultivated in the presence of 1% FCS were switched to a 10% FCS‐containing medium, confirming the bipotentiality of glial progenitor cells (Temple and Raff Nature 313:223, 1985). However, even under the switch culture conditions, segregation into homogeneous clones of either oligodendrocytes or astrocytes still predominated, and the percentage of mixed clones dropped from 25 to 8 or to 3, when the switch took place at 8, 16, or 22 days in vitro, respectively. Two additional observations lead us to suggest that microenvironmental factors are responsible for the clonal segregation of glial progenitor cells: 1) the uneven distribution of oligodendrocyte and astrocyte clusters, the latter being seen mostly on the edge of the coverslips; and 2) the presence, in the vicinity of an homogeneous lacZ‐positive clone, of some lacZ‐negative cells expressing the same phenotype. © 1992 Wiley‐Liss, Inc.
The origin and nature of ramified and amoeboid microglia: A historical review and current concepts
Authors: Ling Eng‐Ang , Wong Wai‐Chow
Published: January 2005

The origin of ramified microglia has been a longstanding controversial issue, with 4 major schools of thought, which state that they are derived (1) from invasion of mesodermal pial elements, (2) from neuroectodermal matrix cells together with the macroglia, (3) from pericytes, and (4) from invasion of monocytes in early development. This paper is in support of the last‐mentioned hypothesis. It is known that ramified microglial cells do not divide under normal circumstances, and since our studies in the corpus callosum have shown that these cells do not appear until the fifth postnatal day, it is reasoned that they must be derived from some preexisting mitotically active cells. The putative precursor is the preponderant amoeboid microglia in the same region. Our experimental studies with the carbon labelling technique have demonstrated for the first time that blood monocytes invade into the early postnatal brain to become amoeboid microglia, which then differentiate into ramified microglia. Just like other tissue macrophages, the monocyte‐derived amoeboid microglia exhibit features indicative of phagocytic activities. These include the content of hydrolytic enzymes, uptake of carbon, and a characteristic surface morphology, as seen by scanning electron microscopy. The transformation of amoeboid microglia into ramified microglia, which occurs between the second and third postnatal week, is considered to be a regressive phenomenon, as shown by the diminution of their content of hydrolytic enzymes and the downregulation of membrane antigen. Apart from their primary role as active phagocytes, their involvement in Alzheimer's disease (AD) is evidenced recently by the fact that the cells are specifically marked by antibodies present in the cerebrospinal fluid of AD patients. In conclusion, ramified microglial cells are derived from monocytes, but through an intermediate amoeboid microglia stage as active macrophages in the perinatal period.
Bone marrow derived elements and resident microglia in brain inflammation
Authors: Lassmann Hans , Schmied Mascha , Vass Karl , Hickey William F.
Published: January 2005

Infection of the central nervous (CNS) system by the human immunodeficiency virus (HIV) depends on the migration of infected hematogenous cells into the brain. We thus used quantitative light and electron microscopic immunocytochemistry to study the homing and turnover of bone marrow derived cells in the CNS in radiation bone marrow chimeras under normal conditions and in experimental autoimmune encephalomyelitis (EAE) as an experimental model of brain inflammation. Our studies suggest the following conclusions. First, the central nervous system is continuously patrolled by a small number of T‐lymphocytes and monocytes. Meningeal and perivascular monocytes are slowly replaced by hematogenous cells under normal conditions, and this turnover is accelerated in the course of inflammation. In contrast, resident microglia represent a very stable cell pool, which in adult animals is only exceptionally replaced by hematogenous cells, even after recovery from severe brain inflammation. Second, although in bone‐marrow‐chimeric animals resident microglia, astrocytes, and ependymal cells are not able to present antigen to Lewis T‐lymphocytes, the inflammatory reaction in EAE is qualitatively and quantitatively similar in these animals compared to fully histocompatible Lewis rats. Finally, resident microglia express the macrophage activation antigen ED1. Thus, microglia cells appear to function as effector cells in EAE lesions.
Characterization of microglia and macrophages in the central nervous system of rats: Definition of the differential expression of molecules using standard and novel monoclonal antibodies in normal CNS and in four models of parenchymal reaction
Authors: Flaris Nicolaos A. , Densmore Tamara L. , Molleston Michael C. , Hickey William F.
Published: January 2005

This report describes the development of a new panel of monoclonal antibodies produced following immunization of mice with cultured rat microglial cells. Using these new reagents and previously defined antibodies that bind to microglia or macrophages, the responses of parenchymal microglia, perivascular “microglial” cells, and infiltrating macrophage/monocytes were examined in 4 divergent models of central nervous system reaction. These were brain abscess, experimental allergic encephalomyelitis, Wallerian degeneration, and stab wound. No single new anitbody was specific only for microglia; all antibodies positively staining microglial cells also labeled various subsets of macrophage/monocytic cells in normal tissues of the immune system. Moreover, the results indicate that microglia are capable of different levels and a variety of types of response, as defined by the molecules they elaborate. These findings suggest that these CNS resident cells belong to the extended monocyte/macrophage/dendritic cell family and that they do not respond in a stereotypic manner to all forms of CNS insult.
Microglial MHC antigen expression after ischemic and kainic acid lesions of the adult rat hippocampus
Authors: Finsen B. R. , Jørgensen M. B. , Diemer N. H. , Zimmer J.
Published: January 2005

By taking advantage of the specific neuronal and connective organization of the hippocampus and the different susceptibility of hippocampal neurons to transient cerebral ischemia or intraventricular injections of kainic acid (KA), we examined the microglial reactions to different types of neuronal injury. In all areas with neuronal or axonal degeneration, the microglial cells reacted by specific degeneration‐related morphological transformations and expression of class I major histocompatibility complex (MHC) antigen. Subpopulations of microglial cells also expressed class II MHC antigen and leukocyte common antigen (LCA) in relation to (1) degenerating nerve cell bodies in the dentate hilus and the CA1 and CA3 pyramidal cell layers, (2) postischemic degeneration of dendrites in the stratum radiatum of CA1, and (3) combined dendritic and axonal degeneration in the stratum radiatum of the KA‐lesioned CA3. MHC II and LCA expression was not observed in relation to degeneration of the CA3‐derived Schaffer collaterals in CA1 after KA‐induced CA3 lesions. In the case of ischemia the degeneration‐related reactions were preceded by an early, generalized microglial reaction, which also included areas without subsequent signs of neural degeneration. This reaction, which was transient and characterized by subtle morphological changes and induction of class I MHC antigen only, was presumably triggered by a general postischemic pertubation of the cerebral microenvironment, and not by actual neural degeneration. In conclusion, we found that microglial expression of class I MHC antigen was a sensitive marker of both the general pertubation after ischemia and axonal degeneration distant from the areas of actual nerve cell death. Induction of microglial LCA and class II MHC antigen expression, together with protracted expression of class I, was, characteristic of a protracted reaction, only found in areas with degeneration of nerve cell bodies and dendrites.
Heterogeneity of microglial and perivascular cell populations: Insights gained from the facial nucleus paradigm
Authors: Streit Wolfgang J. , Graeber Manuel B.
Published: January 2005

We reflect here on the development of a neuroimmunological concept which has been formulated over the past 5 years through studying microglial cell responses in the facial nerve system. A simple axotomy of the adult rat facial nerve which causes regeneration of facial motor neurons and little, if any, cell death can activate microglial cells just as easily as a full‐blown degeneration of the entire nucleus induced by toxic ricin. In both instances, the prompt microglial reaction is characterized by a series of structural and phenotypic changes which are in many ways similar to an immune response, e.g., there is cell proliferation and upregulation of MHC antigens. However, since white blood cells do not participate in the retrograde response of facial motor neurons, we have adopted a notion which views microglia as a CNS‐wide network of immunocompetent cells whose morphological dissimilarities from leukocytes are a result of their unique adaptation to the CNS architecture. We have continued our in vivo investigations of the phagocytic and immunophenotypic properties of microglial and perivascular cells during the retrograde reaction of facial motor neurons by using intraneural injections of fluorogold (FG) and ricin followed by lectin and immunostaining for microglia. Two new findings can be added to the microglial neuroimmune network: (1) Microglia take up FG only after motor neuron degeneration, whereas perivascular cells may take up FG under nondegenerating conditions. (2) Immunologically important molecules, such as MHC class II, CD4, and leukocyte common antigens, are expressed by different microglial subpopulations. Thus there is functional and phenotypic heterogeneity among immunocompetent cells of the CNS.
Electrophysiological behavior of microglia
Authors: Kettenmann H. , Banati R. , Walz W.
Published: January 2005

The present knowledge of voltage‐ and ligand‐activated ion channels of cultured microglial cells is described and its relevance is discussed. All microglial cells cultured from rat or mouse brain express an inward rectifying K+ channel but no outward currents. This expression is not changed by the length of the cultivation period, nor is it different in freshly isolated cells. It makes the microglial cells distinct from peritoneal macrophages, which possess an outward rectifying K+ channel. In bone marrow, 2 populations of cells could be distinguished electrophysiologically, one with the channel pattern of macrophages and one with that of microglial cells. This finding is interesting in light of the fact that it is presently hypothesized that the differentiation of monocytes into microglia takes place exclusively during embryonic development but not in the adult. The available data thus support the hypothesis that within the bone marrow a population of macrophage precursor cells exists with a possible lineage relationship to brain macrophages. The lack of outward currents in the microglial cells has the functional consequence that even a small inward current leads to a large membrane depolarization, since K+ outward currents are not activated with the depolarization. The microglial cell is thus very sensitive to depolarizing events. We found that ATP induced an inward current and an increase in the conductance, whereas ADP, AMP, and adenosine did not. These relative potencies indicate that microglia possess a P2 purinoceptor linked to an ion channel. The amplitude of the inward current elicited by ATP is about 80 pA and is sufficient to depolarize microglial cells close to 0 m V. Quite obviously, ATP is the agent that is activating ion channels that cause a pronounced depolarization, and as a result of the peculiar ion channel pattern of microglia, has pronounced effects on the membrane potential. Since ATP is released from cells during tissue injury, it has the potential to act as an initial trigger for the transition from one microglial functional state to another.
Reactive glia as rivals in regulating neuronal survival
Authors: Giulian Dana
Published: January 2005

Reactive gliosis is a response noted after nearly every type of CNS injury and involves both activated microglia and astroglia. Although many investigators believe that reactive glia in some way regulate the survival of injured neurons, the influence of glial elements upon damaged neural tissues remains uncertain. To examine relationships between reactive glia and neurons, secretion products from both microglia and astroglia are tested for their effects upon the survival of cultured neurons. Microglia are found to secrete neurotoxic agents, while astroglia are a source of neuronotrophic factors. Similar patterns of soluble factor production are noted for astroglia‐rich or microglia‐rich regions of rat neocortex damaged by ischemia. These observations suggest that microglia and astroglia compete for control of neuronal survival. Importantly, microglial neurotoxins might hinder the recovery of neurologic function at sites of inflammation.
Detection of lysosomal cysteine proteinases in microglia: Flow cytometric measurement and histochemical localization of cathepsin B and L
Authors: Banati R. B. , Rothe G. , Valet G. , Kreutzberg G. W.
Published: January 2005

The activation and differentiation of microglia is a prominent pathophysiological process in numerous inflammatory and demyelinating diseases of the central nervous system, including Alzheimer's disease and the AIDS encephalopathy. The tissue damage during these diseases has partly been attributed to lipid peroxidating reactive oxygen intermediates for which activated microglia are a major source. The destruction of tissue may also involve the release of proteolytic enzymes, such as the lysosomal cysteine proteinases cathepsin B and L, which are present notably in phagocytic cells. The cathepsins B and L are endopeptidases with a substrate specificity including important proteins, like myelin basic protein, extracellular matrix components, or the class II major histocompatibility complex. Because of this pathophysiological relevance the cathepsins B and L were chosen for histochemical demonstration in isolated and cultured rat microglia and measurement by a new flow cytometric method.

Cathepsin B/L activity was measured flow cytometrically in single viable cells by the intracellular cleavage of non‐fluorescent (Z‐Phe‐Arg)2‐rhodamine 110 to the green fluorescent monoamide Z‐Phe‐Arg‐rhodamine 110 and rhodamine 110. In microglia we measured a cathepsin B/L activity that was 2.5 times higher than in thioglycolate‐elicited, i.e., inflammatory peritoneal rat macrophages. In elicited peritoneal macrophages the formation of fluorescent product was 6.2 times higher than in unstimulated resident peritoneal macrophages, demonstrating that the activation and differentiation of mononuclear phagocytes is accompanied by an increased cathepsin B/L enzyme activity.

The subcellular localization of cathepsin B/L activity in plated viable microglia was demonstrated histochemically by the use of Z‐Ala‐Arg‐Arg‐4‐methoxy‐2‐naphthylamide. Its blue fluorescent cleavage product 4‐methoxy‐2‐naphthylamide was found in lysosomes.

Our study shows that activated microglia are an important potential source of cathepsin B/L. This is particularly interesting as enzymatically active cathepsins have recently been found extracellularly at high levels in the senile plaques of Alzheimer's disease, which are known to contain many activated microglia. The release of proteinases by microglia may play a crucial role in the pathomechanism of tissue‐destructing diseases in the brain.
Receptor subtype involved and mechanism of norepinephrine‐induced stimulation of glutamate uptake into primary cultures of rat brain astrocytes
Authors: Fahrig Thomas
Published: January 2005

Glutamate uptake into rat brain astrocytes is potently stimulated by addition of norepinephrine (NE). This effect is mediated by α1‐adrenergic receptors expressed by these cells (Hansson and Rönnbäck: Life Sci 44:27, 1989; Brain Res 548:215, 1991). The present study was undertaken in order to identify the adrenergic receptor subtype involved, and to determine the sequence of events following receptor activation. NE increased glutamate uptake rates in a dose‐ and time‐dependent manner (EC50 = 6 μM). Both, the selective α1‐receptor antagonist prazosine (IC50 = 2.5 μM) and the α1b‐adrenergic receptor subtype specific alkylating agent chlorethylclonidine (CEC, 100 μM) prevented NE (100 μM) evoked stimulation of glutamate uptake. Furthermore, omission of Ca2+ from the extracellular medium had no significant influence on NE‐induced increase in glutamate uptake, indicating that the stimulatory effect is mediated by α1b‐adrenergic receptors. Treatment of cells with pertussis toxin (PTX) for 24 h or with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) for 30‐45 min prior to NE addition abolished the NE‐mediated effect on glutamate uptake. Addition of TPA alone resulted in a rapid increase of glutamate uptake, which declined to control levels when TPA was applied 30 min prior to uptake initiation by glutamate. The increase in glutamate uptake elicited by TPA and NE added at the same time showed no additivity of the stimulatory effect resulting from treatment with each agent alone. Treatment of cell cultures with 1 mM Ba2+, which has been shown to block outwardly directed K+ ‐currents in astrocytes (Barres et al.: Glia 1:10, 1988), influenced glutamate transport principally in the same manner as found for TPA and NE, respectively. Again no additivity of Ba2+ and NE effects on glutamate uptake was observed. Finally, NE; TPA; and Ba2+ ‐stimulated glutamate uptake exhibited nearly identical time‐response curves.
Appearance of GFAP‐positive cells in adult human brain cultures spontaneously decelerated in growth
Authors: Peržel'Ova Anna , Mareš Vladislav
Published: January 2005

Cell cultures were derived from adult human brain biopsies [from cortical gray (cultures 9‐HB‐G and 33‐HB‐G) and white (culture 14‐HB‐W) and stroke‐injured white matter (culture 33‐HB‐IW)]. The morphology and growth rate of cultured cells were examined and correlated with the presence of vimentin and glial fibrillary acidic protein (GFAP). The cultures from various brain matters differed in cell morphology and rate of growth but not in GFAP and vimentin staining. Cells of primary and rapidly proliferating cultures were GFAP‐negative and vimentin‐positive. Spontaneous growth deceleration occurred in culture 14‐HB‐W within passages 5 to 10 and in cultures 9‐HB‐G, 33‐HB‐G, and 33‐HB‐W within passages 17 to 20. This deceleration, as well as the successive complete growth arrest, were accompanied by an appearance of GFAP‐positive cells and an elevated intensity for vimentin staining. We propose that GFAP‐positive astrocytes originate from glial precursor cells that migrate from the explants and differentiate under prolonged subcultivation.
Inducible nitric oxide synthase activity of cloned murine microglial cells
Authors: Corradin Sally Betz , Mauël Jacques , Donini Susanne Denis , Quattrocchi Emilia , Ricciardi‐Castagnoli Paola
Published: January 2005

Nitric oxide (NO) is a short‐lived diffusable molecule now believed to participate in multiple physiologic functions in the CNS including neurotransmission and the maintenance of vascular tone. Previously, we reported that cell lines obtained by retroviral immortalization of tissue macrophages (Mϕ;) could be induced to synthesize nitrite (NO), a stable end product of the NO synthetic pathway. We have further characterized the induction and activity of this pathway in a panel of seven microglial clones derived from primary embryonic mouse brain cultures. Like Mϕ;, these clones were found to release high levels of NO‐2 in response to recombinant interferon‐γ (rIFN‐γ) as a priming signal together with either bacterial lipopolysaccharide (LPS) or exogenous recombinant tumor necrosis factor‐α (rTNF‐α). As previously demonstrated for Mϕ;, phagocytosis of zymosan particles during induction of enzyme activity enhanced subsequent NO production, which is of interest in light of the postulated phagocytic role of microglia within the CNS. Biochemical characterization of enzyme activity in intact microglial clones and in isolated cytosolic fractions indicates that the microglial NO synthase present in these murine cell clones represents the Mϕ;‐like isotype. These findings suggest that microglial cells could represent a major source of NO within the CNS.
The molecular genetics of myelination: An update
Authors: Lemke Greg
Published: January 2005

Recent molecular genetic studies have provided new insights into the structure and function of 2 of the major integral membrane proteins of myelin—the proteolipid protein (PLP) and protein zero (Po)—and have uncovered a third such protein—PMP22/gas3. The rumpshaker mouse has been shown to carry a point mutation in the PLP gene that uncouples a deleterious effect on CNS myelin assembly, which these mice exhibit, from oligodendrocyte degeneration and cell death, which they do not. The developmental importance of the Po protein in PNS myelination has been dramatically demonstrated by the analysis of loss‐of‐function mutations engineered through the expression of antisense RNA and through the insertional inactivation of the Po gene by homologous recombination in embryonic stem cells and the generation of Po‐deficient mice. The cloned promoter of the Po gene has been shown to drive quantitative, Schwann cell‐specific expression of heterologous genes in transgenic mice. The PMP22/gas3 gene, previously cloned from fibroblast cell lines, has been found to encode an axonally regulated Schwann cell protein that is assembled into PNS myelin. Importantly, this gene appears to be the target of mutations that result in the Trembler alleles in mice, and in Charcot‐Marie‐Tooth disease Type 1a, the most common inherited peripheral neuropathy in humans.
Fish optic nerve oligodendrocytes support axonal regeneration of fish and mammalian retinal ganglion cells
Authors: Bastmeyer Martin , Bähr Mathias , Stuermer Claudia A. O.
Published: January 2005

Segments from adult fish and rat retinae were explanted on myelin‐marker expressing oligodendrocytes derived from the regenerating goldfish optic nerve. Fish axons grew in high density and even rat retinal axons regenerated to considerable length on the surface of the fish oligodendrocytes, suggesting that this type of fish glia has axon‐growth promoting surface components that exert their influence across species boundaries. One interesting surface component of the fish oligodendrocytes as demonstrated here is the E 587 antigen, which is related to the L1 family of cell adhesion molecules.

In long term cocultures of oligodendrocytes and retinal axons, the fish glial cells were found to enwrap rat axons. This suggests that the oligodendrocytes of the regenerating goldfish optic nerve/tract may, despite striking differences, represent the equivalent to mammalian optic nerve oligodendrocytes. © 1993 Wiley‐Liss, Inc.
A cell surface antigen expressed by astrocytes and their precursors
Authors: Szigeti Vilma , Miller Robert H.
Published: January 2005

Increasing evidence suggests that astrocytes of the mammalian CNS are a heterogeneous population of cells that express a number of common characteristics. In most cases, astrocytes or their precursors contain a class of intermediate filaments, composed in large part of glial fibrillary acidic protein (GFAP). While the expression of GFAP immunoreactivity is a specific characteristic of astrocytes in the vertebrate CNS, not all astrocytes contain detectable levels of glial filaments, particularly during early development. We have isolated a monoclonal antibody termed 7B11 which binds to the surface of astrocytes and glial precursors, but not to other major types of neural cells. The 7B11 antigen is expressed by astrocytes in the adult CNS in vivo and in cultures of developing CNS tissue, but not on cells of the peripheral nervous system. During early development, 7B11 immunoreactivity appears prior to the expression of GFAP and is retained as punctate staining on the surface of most classes of astrocytes. During CNS maturation, however, 7B11 immunoreactivity is lost from the surface of Bergmann glia in the cerebellum, suggesting that differentiative events lead to functionally and anti‐genically distinct classes of CNS glial cells. In the adult spinal cord, biochemical analysis suggests that the epitope recognized by 7B11 is associated with a group of polypeptides of apparent molecular weights 200‐160, 140, and 92 kD. The cellular distribution of 7B11 expression suggests that astrocytes and their precursors share a distinct cell surface antigenic property and that the expression of 7B11 immunoreactivity may be a useful operational marker for astrocytes in the absence of detectable GFAP expression. © 1993 Wiley‐Liss, Inc.
Differences in glycogen metabolism in astroglia‐rich primary cultures and sorbitol‐selected astroglial cultures derived from mouse brain
Authors: Dringen Ralf , Hamprecht Bernd
Published: January 2005

Recently it has become possible by chemical selection using sorbitol instead of glucose in the culture medium to produce pure astroglial cultures from astroglia‐rich primary cultures from mouse brain. The glycogen‐degrading enzyme glycogen phosphorylase in brain is localized in astrocytes and ependymal cells. In view of this fact it appeared necessary to study the influence of glucose and other hexoses on the glycogen metabolism in these cultures lacking the influence of other cell types in comparison to the astroglia‐rich primary cultures containing several types of cells. The sorbitol‐fed selected cultures and the glucose‐deprived astroglia‐rich primary cultures contain less than 10% of the glycogen encountered in glucose‐fed primary cultures. During incubation with glucose the glycogen content of the selected cultures and the glucose‐deprived primary cultures increases by more than one order of magnitude. Nevertheless, not all cells are found to have accumulated glycogen. The time course of the replenishment of glycogen is similar in both types of culture, although maximal levels reached in the selected cultures are 3 times those in the astroglia‐rich primary cultures. This difference might be explained by the fact that the ratio of the maximal activities of glycogen synthase and glycogen phosphorylase in selected cultures was found to be twice that in the unselected cultures. During glucose deprivation the glycogen content is reduced in both culture systems with half‐maximal contents being reached at 15 min (primary culture) and 45 min (selected culture). Both types of culture can also utilize mannose for the synthesis of glycogen and the production of lactate. In contrast, fructose or galactose are poor substrates, or none at all, for the generation of glycogen. In conclusion, the absence of non‐astroglial cells and the high glycogen content after glucose feeding makes pure astroglial cultures an even better system for investigating the regulation and the function of astrocytic glycogen than astroglia‐rich primary cultures. © 1993 Wiley‐Liss, Inc.
Bergmann glia require continuous association with purkinje cells for normal phenotype expression
Authors: Fisher Marilyn , Trimmer Patricia , Ruthel Gordon
Published: January 2005

Bergmann glia (Bg) respond to the early postnatal Purkinje cell (Pc) death in Lurcher (Lc) mutant mouse cerebellum by down‐regulating expression of the enzyme glycerol‐3‐phosphate dehydrogenase (GPDH). To determine whether glial GPDH expression requires the continued presence of Pcs in adults, we used single intracerebellar injections of kainic acid to kill Pcs in wild‐type mice from 7 weeks to 11 months old. Bg at all ages tested responded to Pc loss by down‐regulating GPDH expression. To learn whether a high level of GPDH could be reinduced following down‐regulation in Lc Bg, we grafted wild‐type fetal Pcs into Lc cerebella. The influence of grafted Pcs on GPDH expression is host‐age and implant‐position dependent. Only Pcs implanted into hosts less than 6 weeks old were later found to be associated with GPDH‐positive Bg. Grafted Pcs that migrated into the anterior folia of young hosts were more likely to be associated with GPDH‐positive Bg than Pcs migrating to other positions. EM analysis showed that Bg ensheathment of grafted Pcs is thinner and more discontinuous, but qualitatively similar to normal. The results suggest that the interaction between host Bg and grafted Pcs can sustain elevated GPDH expression in Bg that have not yet down‐regulated, but is not adequate to reinduce expression in those cells that have. © 1993 Wiley‐Liss, Inc.