bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional characterization of neurokinin‐1 receptors on human U373MG astrocytoma cells
Authors: Eistetter Harald R. , Mills Ann , Brewster Rachel , Alouani Sami , Rambosson Christine , Kawashima Eric
Published: May 2005

The neurokinin‐1 (NK‐1, substance P) receptor belongs to the class of seven transmembrane domain (7‐TM) receptors that interact with cellular effector systems via guanine nucleotide binding regulatory proteins (G‐proteins). In this study, coupling mechanisms of functional NK‐1 receptoirs endogenously expressed in a human astrocytoma cell line (U373MG) were analyzed. Stimulation with substance P (SP) resulted in 1) a rapid increase in inositol 1,4,5‐trisphosphate (IP3) synthesis; 2) a rise in cytosolic free calcium concentration ([Ca2+]); 3) induction of immediate early gene transcription as monitored by c‐fos and c‐jun expression; and 4) a significant increase in de novo DNA synthesis. Thus, the functional responses induced by stimulation of NK‐1 receptors on U373MG strongly correlate with those observed after treatment of primary astrocytes with SP and make U373MG cells a useful in vitro model system for the analysis of NK‐1 receptor function on astrocytes in vivo. © 1992 Wiley‐Liss, Inc.
Oligodendrocytes and myelin formation along the optic tract of the developing hamster: An immunohistochemical study using the rip antibody
Authors: Jhaveri Sonal , Erzurumlu Reha S. , Friedman Beth , Schneider Gerald E.
Published: May 2005

The monoclonal antibody Rip recognizes an antigen specific to oligoderdrocytes and their processes (Friedman et al: Gila 2:380, 1989). We have used this antibody to document the appearance of oligodendrocytes and the sequence of ensheathment of axons along the optic tract (OT) and within its major target areas in neonatal (P3–P21) and adult hamsters.

Myelination of axons in the visual pathway follows an overall proximo‐distal gradient. On P3, immunopositive, pre‐ensheathing oligodendrocytes are detected in the OT ventral to the lateral geniculate body (LGB) whereas myelin segments are present around OT axons by P5. The first pre‐ensheathing oligodendrocytes are detected medially in the LGB on P7 and myelinated axons in the overlying OT by P11. In the superior colliculus, pre‐ensheathing oligodendrocytes are prsent in the optic fiber layer (SO) on P7, but not in the superficial gray layer (SGS) until P11. Myelination of axons within SO proceeds along a marked rostro‐caudal gradient. On P14, axons in rostral SO are heavily myelinated; thereafter, ensheathment continues caudally within the SO and the SGS.

The progressive invasion of oligodendrocytes along the proximo‐distal axis of the optic pathway, and the corresponding myelination of OT axons, are discussed in the context of a possible inhibitory role of oligodendrocytes in regulating the regenerative propensity of retinotectal axons. © 1992 Wiley‐Liss, Inc.
Serotonin uptake by astrocytes in situ
Authors: Anderson E. J. , McFarland D. , Kimelberg H. K.
Published: May 2005

Co‐localization of glial fibrillary acidic protein (GFAP) and radioactivity was examined after intraventricular injection of [3H]5‐HT in adult rat brains. Radioactivity localized over GFAP‐positive astrocytes was seen, especially when image‐enhancing techniques were applied to the data. Also slices prepared from astrogliotic hippocampi of rats pretreated with kainic acid showed a twofold increased uptake of [3H]5‐HT compared to control slices. This indicates that the uptake of [3H]5‐HT seen in primary astrocyte cultures also occurs for astrocytes in situ. Also, as with astrocyte cultures, only some of the GFAP(+) astrocytes in situ showed localization of radioactivity, supporting the concept of intraregional heterogeneity of astrocyte functions. © 1992 Wiley‐Liss, Inc.
Glial calcium
Authors: Finkbeiner Steven M.
Published: May 2005

This review summarizes current knowledge relating intracellular calcium and glial function. During steady state, glia maintain a low cytosolic calcium level by pumping calcium into intracellular stores and by extruding calcium across the plasma membrane. Glial Ca2+ increases in response to a variety of physiological stimuli. Some stimuli open membrane calcium channels, others release calcium from intracellular stores, and some do both. The temporal and spatial complexity of glial cytosolic calcium changes suggest that these responses may form the basis of an intracellular or intercellular signaling system. Cytosolic calcium rises effect changes in glial structure and function through protein kinases, phospholipases, and direct interaction with lipid and protein constituents. Ultimately, calcium signaling influences glial gene expression, development, metabolism, and regulation of the extracellular milieu. Disturbances in glial calcium homeostasis may have a role in certain pathological conditions. The discovery of complex calcium‐based glial signaling systems, capable of sensing and influencing neural activity, suggest a more integrated neuro‐glial model of information processing in the central nervous system. © 1993 Wiley‐Liss, Inc.
Thyroid hormone up‐regulates thyroid hormone receptor β gene expression in rat cerebral hemisphere astrocyte cultures
Authors: Lebel Jean‐Marc , L'Hérault Sylvie , Dussault Jean H. , Puymirat Jack
Published: May 2005

Oligonucleotide probes complementary to specific regions of three thyroid receptor cDNAs were used to study the effects of thyroid hormone on the expression of the mRNAs encoding two α (α1 and α2) and one β‐thyroid (β1) receptors isoforms in rat cerebral hemisphere astrocyte cultures. Both genes are expressed by type 1 astrocytes. The levels of the α1‐, α2‐, and β1‐mRNAs did not significantly change between day 8 and day 22, in cultures grown in the absence of thyroid hormone. L‐triiodothyronine (L‐T3) treatment of the cultures increased the levels of β1‐mRNAs by fivefold without changing either the levels of the α1‐ and α2‐mRNAs or L‐T3 binding capacity. The effect of L‐T3 on β1‐mRNAs was observed after 4 h of treatment and was independent of protein synthesis, suggesting that this effect is likely to be a direct one. Treatment of the cultures by cytosine arabinosine, a drug that kills dividing cells, specifically decreased level of the α1‐ and α2‐mRNAs by 60% and 38%, respectively. Finally, by immunocytochemistry, we showed that the β1 receptor‐immunoreactivity was either located in the perinuclear region and the cytoplasm or in the nuclei of astrocytes.

Taken together with previous data obtained in neuronal cultures where no effect of L‐T3 was observed on the levels of the β1‐mRNAs, our findings indicate that the β1 gene is differentially regulated in neurons and astrocytes. Furthermore, our results are also in favor of specific functions for both types of thyroid receptors during astrocyte differentiation. © 1993 Wiley‐Liss, Inc.
Effects of tumor necrosis factor on inward potassium current and cell morphology in cultured human oligodendrocytes
Authors: McLarnon James G. , Michikawa Makoto , Kim Seung U.
Published: May 2005

The effects of recombinant human tumor necrosis factor‐α (rhTNF‐α) on inward rectifier potassium [K(IR)] currents and on cell morphology have been studied in cultured human oligodendrocytes. Cell‐attached patches were used to isolate and record unitary currents through an inward rectifier K+ channel with a conductance of 23 pS. In control oligodendrocytes the mean open times showed an exponential dependence on patch potential with an e‐fold decrease over a patch hyperpolarization of 28 mV. Treatment of oligodendrocytes with rhTNF (at 250 ng/ml for 24–48 h) had significant actions to diminish the mean open times of K(IR) relative to control values. At cell resting potential the mean open times were reduced by 60% after rhTNF application; the amplitudes of unitary currents or the extrapolated zero‐current potentials were not significantly changed by the cytokine. The rhTNF treatments were not cytotoxic to cultured human oligodendrocytes; however, in some experiments rhTNF caused evident retraction of cell processes. © 1993 Wiley‐Liss, Inc.
A Ca2+ ‐ and pH‐Dependent K+ Channel of rat C6 glioma cells and its possible role in acidosis‐induced cell swelling
Authors: Strupp Michael , Staub Frank , Grafe Peter
Published: May 2005

The aim of the present study was to explore whether a change in membrane K+ conductance contributes to acidosis‐induced swelling of cultured rat C6 glioma cells. Electrophysiological studies were perfomed using whole‐cell and single‐channel recordings in combination with cell volume measurements in cell suspension by flow cytometry. Whole‐cell recordings revealed a voltage‐dependent K+ conductance. The predominant K+ channel in single‐channel recordings with symmetrical high K+ concentrations was inwardly rectifying and had conductances of 35 and 15 pS, respectively. A raised internal free Ca2+ concentration and membrane depolarization increased the open probability of this channel. Internal acidosis (pH 6.4−5.4), on the other hand, reduced open probability and single‐channel conductance. Both whole‐cell and single‐channel K+ currents were blocked by quinidine (0.1−1 mM), which was therefor used to analyze the functional consequences of an inhibition of this conductance for volume. Thereby, quinidine (1 mM) produced a small (5%) and transient cell swelling of C6 glioma cells. In contrast, acidosis (pH 5.6) caused a much larger (about 20%) and maintained swelling. Since quinidine produced only a minor swelling of C6 cells, it is unlikely that inhibition of the K+ conductance caused acidosis‐induced cell swelling. Other mechanisms, such as activation of ion transporters, must therefore be responsible. © 1993 Wiley‐Liss, Inc.
Kainic acid induced alterations in antibody recognition of connexin43 and loss of astrocytic gap junctions in rat brain
Authors: Hossain M. Z. , Sawchuk M. A. , Murphy L. J. , Hertzberg E. L. , Nagy J. I.
Published: May 2005

Intracerebral administration of kainic acid (KA) in rats was previously shown to abolish immunohistochemical labelling for the astrocytic gap junction protein connexin43 (Cx43) at sites depleted of neurons (Vukelic et al: Neurosci Lett 130:120–124, 1991). This response of Cx43 has now been further investigated with a number of different sequence‐specific anti‐Cx43 antibodies. At lesion sites in the thalamus, striatum, and hippocampus examined immunohistochemically with an antibody against amino acids (aa's) 346–363 in the Cx43 sequence, the antibody used in the earlier study, Cx43‐immunoreactivity was increased 5 h after KA injections, absent by 24 h and for up to 2 weeks post‐injection, and began to return to less than normal levels by 2 to 3 weeks post‐injection. Analyses of KA lesion sites with antibodies against other sequences of Cx43 (amino acids 283–298, 253–270, 241–260, 113–123, and 49–61) revealed not only the presence but in some cases an increased density of Cx43 immunoreactivity after a survival time of 1 week. Immunolabelling patterns at these sites consisted of relatively large, coarse profiles rather the fine punctate labelling typically seen in sections of normal brain. In homogenates of KA‐injected striatum analyzed by Western blots, Cx43 was detected at near normal or slightly increased levels at various survival times examined. The 43 kDa phosphorylated form of Cx43 and its faster migrating 41 kDa dephosphorylated form which is generated post‐mortem by a brain phosphatase were both present after standard methods of tissue preparation for Western blot analysis, while only the 43 kDa form was present in normal and KA‐injected striatum after inactivation of brain metabolism by focused cranial microwave irradiation. Ultrastructural investigations of lesions sites within the thalamus revealed a virtual absence of astrocytic gap junctions. These results demonstrate that Cx43 levels initially increase after intracerebral KA treatment, that its molecular organization in resident astrocytes is altered such that epitopes that are normally accessible to antibody are hidden while those that may be hidden or relatively inaccessible are exposed, and that this molecular alteration in Cx43 is associated with loss of astrocytic gap junctions. © 1994 Wiley‐Liss, Inc.
Microtubule‐associated protein 1B (MAP1B) is present in glial cells phosphorylated different than in neurones
Authors: Ulloa Luis , Ibarrola Nieves , Avila Jesúas , Díez‐Guerra F. Javier
Published: May 2005

A panel of four anti‐MAP1B antibodies have been used to study the presence and post‐translational modification of MAP1B in primary cultures of glial cells. Two antibodies (150 and 125) recognize phosphorylated epitopes whereas the other two (531 and 842) recognize non‐phosphorylated phosphorylatable epitopes on the MAP1B molecule. Immunofluorescence and Western blot analysis with antibodies 531 and 842 revealed the presence of small amounts of MAP1B‐like immunoreactivity in type 1 astrocytes and a greater content in more differentiated glial cells found in long‐term cultures. By immunofluorescence, these latter cells gave positive immunostaining with antibody 125, which recognizes a phosphorylated epitope phosphorylated by casein kinase II. Antibody 150, which reacts to a phosphorylated epitope on the MAP1B molecule, did not show any detectable immunoreactivity in glial cells cultures, either by immunofluorescence or Western blot. All four antibodies recognized hippocampal neurones in culture, with especially intense immunostaining in cell bodies and axons, and reacted strongly with protein present in hippocampal neurones extracts showing an electrophoretic mobility similar to that of brain MAP1B. In mixed optic nerve glial cell cultures, anti‐galactocerebroside (Ga1C) positive cells gave also positive staining with antibodies 531 and 125. We propose that MAP1B is present in cultures of glial cells in moderate amounts and with a phosphorylation state different than in neurones. Thus, less differentiated glial cells, such as type 1 astrocytes, have a small amount of MAP1B, mainly in a non‐phosphorylated form, which is spread diffusely in the cytoplasm and probably does not interact with microtubules. More differentiated glial cells, such as oligodendrocytes, show a greater content in MAP1B which, at least in part, is phosphorylated by a casein kinase II‐like activity. © 1994 Wiley‐Liss, Inc.
Characteristics of Na+ current in Schwann cells cultured from frog sciatic nerve
Authors: Miyazaki T. , Tasaka J. , Sakai S. , Hashiguchi T. , Padjen A. L. , Tosaka T.
Published: May 2005

Characteristics of voltage‐dependent currents in cultured frog Schwann cells were investigated by the whole‐cell clamp technique. An inward current was detectable at a membrane potential level more positive than‐50 mV and reached a maximum value at about‐10 mV, while no rectifying channel was present. The inward current was carried by Na+ ions, because the extrapolated reversal potential of the current agreed with the calculated ENa, and the current was sensitive to tetrodotoxin. The membrane potential for half‐maximal inactivation was‐82 mV. The inactivation curve indicated that more than 90% of the Na+ channels were inactivated at the resting membrane potential, suggesting that the cultured frog Schwann cells could not generate an action potential under physiological conditions. The time constant for the inactivation at a maximum current was 5.3 ms (‐10 mV, 13°C).

The electrophysiological characteristics of the Na+ current in the cultured frog Schwann cells were compared with those in other tissues. This Na+ current was quantitatively different from that observed in the amphibian node of Ranvier but was similar to that in the mammalian Schwann or glial cells, especially in the more hyperpolarized half‐maximal inactivation potential and in the slower inactivation time course. © 1994 Wiley‐Liss, Inc.
The immortalized astroglial cell line RC7 is a new model system for the study of nerve growth factor (NGF) regulation: Stimulation by interleukin‐1β and transforming growth factor‐β1 is additive and affected differently by dibutyryl cyclic AMP
Authors: Hahn Martina , Lorez Hanspeter , Fischer Güunther
Published: May 2005

Nerve growth factor (NGF) synthesis was studied with an astroglial cell line derived from rat cerebellar astrocytes by transfection with a simian virus 40 T containing retroviral vector. As in primary astrocytes, NGF synthesis/secretion could be stimulated dose‐dependently with interleukin‐1βsZ (IL‐1βsZ) and transforming growth factor‐1βsZ (TGF‐βsZ1). We therefore have used this cell line as a model system to analyze putative intracellular signalling pathways underlying the effects of these factors. Protein kinase C inhibitors (calphostin and Ro 31–8830) as well as a lipoxygenase inhibitor (nordihydroguaiaretic acid) did not affect stimulation of NGF synthesis/secretion by IL‐1βsZ or TGF‐βsZ1. However, dibutyryl cyclic AMP partly inhibited the stimulation by TGF‐βsZ1 but did not affect that evoked by IL‐1βsZ. This finding, together with the fact that IL‐1βsZ and TGF‐βsZ1 stimulate NGF production/secretion in an additive manner, indicates that different intracellular signalling pathways are involved in the mediation of IL‐1βsZ and TGF‐βsZ1 induced NGF production/secretion. © 1994 Wiley‐Liss, Inc.
Media that support the growth and differentiation of oligodendrocytes do not induce olfactory ensheathing cells to express a myelinating phenotype
Authors: Doucette R. , Devon R.
Published: May 2005

The glial cells that ensheath olfactory axons are referred to as ensheathing cells. In vivo, these non‐myelinating glial cells express a mixture of astrocyte‐specific and Schwann cell‐specific phenotypic features with the former cellular phenotype predominating, but in vitro can assemble a myelin sheath when co‐cultured with dorsal root ganglion neurons. Thus, certain in vitro conditions induce ensheathing cells to express a phenotype more like that of a myelinating Schwann cell. The present study addresses whether ensheathing cells will express a myelinating phenotype in neuron‐free cultures when fed for 1 to 5 weeks with media shown to promote the growth and differentiation of oligodendrocyte progenitor cells. The ensheathing cell cultures were initiated using the nerve fiber layers (NFL) of rat olfactory bulb primordia. Oligodendrocyte cultures were established from newborn rat neopallium and from the tissue that remained after removing the NFL from the developing olfactory bulb (i.e., the OB‐NFL). The cultures were double‐labelled with rabbit polyclonal antibodies to S100 or glial fibrillary acidic protein in combination. With the mouse monoclonal antibodies 04, BRD1 [anti‐galactocerebroside (anti‐GAL‐C)], and anti‐myelin basic protein (MBP). In some experiments the ensheathing cells were labelled with PKH26 prior to being co‐cultured with oligodendrocytes of the OB‐NFL. None of the media induced ensheathing cells to express either GAL‐C or MBP. However, when 0.5 mM dibutyrylcyclic‐AMP (dBcAMP) was added to the medium, ensheathing cells became GAL‐C + ve, but remained MBP‐ve. The molecular mechanisms that regulate the expression of a myelinating phenotype by ensheathing cells appear to be different from those that operate in oligodendrocytes. © 1994 Wiley‐Liss, Inc.
Myelin‐associated glycoprotein is not detectable in perikaryal myelin of spiral ganglion neurons of adult mice
Authors: Martini Rudolf
Published: May 2005

The expression of the adhesion molecules N‐CAM (neural cell adhesion molecule), L1, myelin‐associated glycoprotein (MAG), and P0 has been investigated by postembedding‐immunoelectron microscopy in spiral ganglia of adult mice in which both axons and neuronal cell bodies are myelinated. L1 was absent from both axonal and perikaryal myelin of spiral ganglion neurons but was expressed on unmyelinated nerve fibers. P0 was expressed in compacted parts of both types of myelin but was absent from non‐compacted myelin. MAG was not detectable in perikaryal myelin but was strongly expressed in axonal myelin both periaxonally and in non‐compacted regions. Conversely, N‐CAM was detectable at the interface between myelin and neuronal perikarya and in non‐compacted regions of perikaryal myelin, whereas it was hardly detectable in axonal myelin. This inverse expression of MAG and N‐CAM in perikaryal and axonal myelin points to the possibility that N‐CAM may functionally replace MAG in perikaryal myelin of spiral ganglion neurons. © 1994 Wiley‐Liss, Inc.
Confocal imaging of glial cells in the intact rat optic nerve
Authors: Butt A. M. , Colquhoun K. , Berry M.
Published: May 2005

Astrocytes and oligodendrocytes in the isolated intact mature rat optic nerve have been computer imaged in three dimensions by laser scanning confocal microscopy of single cells, dye‐filled with lysinated rhodamine dextran (LRD). Our results illustrate the first application of these techniques to an intact CNS white matter tract and provide comparative data for previous studies on neonatal rat optic nerve (Butt and Ransom: Glia 2:470–475,1989; Butt and Ransom: J Comp Neurol 338:141–158, 1993). The combined use of intracellular injection of LRD and confocal imaging significantly improves the resolution of glial cell structure, particularly that of mature astrocytes, for a number of reasons. (1) Single mature dye‐filled glia can be imaged, because LRD does not pass through gap junctions. (2) The entire process field of astrocytes can be visualized in a single two‐dimensional image. (3) Cell images can be rotated through 360° in all planes to provide a new perspective of glial cell structure in the intact tissue. (4) Reconstruction of optical sections, within a narrow focal plane, provides a high definition and resolution of the finer details of glial morphology. Using these techniques, three astrocyte subclasses were distinguished on morphological criteria. It is the conclusion of this study that the majority of these forms represent a single population of fibrous astrocytes which are well‐suited to perform the multiple functions attributed to astrocytes in the CNS. The morphology of mature myelin‐forming oligodendrocytes was also described. The results confirm that the structural characteristics of both astrocyte subtypes and oligodendrocytes described in younger rat optic nerves by Butt and Ransom (Glia 2:470–475,1989; J Comp Neurol 338:141–158, 1993) accurately reflect those of the mature glial population. © 1994 Wiley‐Liss, Inc.
Similarities and differences between fish oligodendrocytes and schwann cells in vitro
Authors: Bastmeyer Martin , Jeserich Gunnar , Stuermer Claudia A. O.
Published: May 2005

In light of the striking differences between oligodendrocytes of the optic nerve/tract of adult goldfish and their mammalian counterparts, a further characterization of goldfish oligodendrocytes was performed. A comparison with Schwann cells was included because fish optic nerve/tract‐derived oligodendrocytes bear remarkable similarities to this type of glial cell. Fish optic nerve/tract‐derived oligodendrocytes that had differentiated into 04 and 6D2‐positive cells and thus expressed early myelin marker molecules were found to incorporate BrdU and to divide in vitro over a period of weeks. For the induction of more advanced markers of myelinogenesis such as the CNS‐specific myelin protein 36K, oligodendrocytes required extensive contact with axons. Other agents, such as fetal calf or carp serum, substrate components, or forscolin failed, however, to induce 36K expression. 04/6D2‐positive oligodendrocytes could be distinguished from fish 6D2‐positive Schwann cells derived from cranial nerves by their antigenic pheno‐type: Schwann cells but not oligodendrocytes exhibited the low affinity NGF receptor. While both cell types carry the cell adhesion molecules NCAM, E 587 antigen, and the L2/HNK‐1 epitope, only Schwann cells possess a further adhesion molecule, Neurolin. © 1994 Wiley‐Liss, Inc.
Astrocytes exhibit regional specificity in gap‐junction coupling
Authors: Lee Sung H. , Kim Warren T. , Cornell‐Bell Ann H. , Sontheimer Harald
Published: May 2005

Astrocytes are coupled to each other via gap‐junctions both in vivo and in vitro. Gap‐junction coupling is essential to a number of astrocyte functions including the spatial buffering of extracellular K+ and the propagation of Ca2+ waves. Using fluorescence recovery after photo‐bleach, we quantitatively assayed and compared the coupling of astrocytes cultured from six different central nervous system (CNS) regions in the rat: spinal cord, cortex, hypothalamus, hippocampus, optic nerve, and cerebellum. The degree of fluorescence recovery (% recovery) and time constant of recovery (∼) served as quantitative indicators of coupling strength. Gap‐junction coupling differed markedly between CNS regions. Coupling was weakest in astrocytes derived from spinal cord (43% recovery, τ∼400 s) and strongest in astrocytes from optic nerve (91% recovery, τ∼226 s) and cerebellum (95% recovery, τ∼100 s). As indicated by the degree of recovery, coupling strength among CNS regions could be ranked as follows: spinal cord < cortex < hypothalamus < hippocampus = optic nerve = cerebellum. Gap‐junction coupling also differed between CNS regions with respect to its sensitivity to inhibition by the uncoupling agent octanol. Kd values for 50% inhibition by octanol ranged from 188 μM in spinal cord astrocytes to 654 μM in hippocampal astrocytes. Sensitivity of gap‐junctions to octanol could be ranked as follows: spinal cord = cortex = hypothalamus> cerebellum> optic nerve> hippocampus. The observed differences in coupling indicate differences in the number of gap‐junction connections in astrocytes cultured from the six CNS regions. These differences may reflect the adaptation of astrocytes to varying functional requirements in different CNS regions. © 1994 Wiley‐Liss, Inc.
Fura‐2 signals evoked by kainate in leech glial cells in the presence of different divalent cations
Authors: Munsch Thomas , Nett Wolfgang , Deitmer Joachim W.
Published: May 2005

The glutamate‐agonist kainate evokes Ca2+ transients in both neurones and glial cells. Owing to the membrane depolarization elicited by kainate, a Ca2+ influx could occur through voltage‐gated Ca2+ channels or through the kainate‐gated cation channels directly. We have measured ratio signals of the calcium indicator dye fura‐2, injected into giant glial cells of the leech Hirudo medicinalis, as response to kainate (5‐20μ) in the presence of different divalent cations. The responses to kainate increased during the first 2‐4 kainate applications, both in unclamped and in voltage‐clamped cells. The fura‐2 fluorescence ratio (F 350/F380) still increased when Ca2+ was replaced by Ba2+ but was suppressed in Ca2+ ‐free saline and in the presence of Ni2+ (2 mM). Co2+ and Mn2+ (2 mM) also reduced the kainate‐induced fura‐2 fluorescence signals, due to entry of these divalent cations into the cells and subsequent quenching the fluorescence of the intracellular dye. It is concluded that Ni2+ blocks the kainate‐induced membrane depolarization and Ca2+transient but apparently does not enter the cells, while Ba2+, Co2+, and Mn2+ appear to permeate the membrane, presumably through the kainategated channels. © 1994 Wiley‐Liss, Inc.
Experimental gliopathy in the adult rat CNS: Effect on the blood‐spinal cord barrier
Authors: Krum Janette M.
Published: May 2005

The expression of certain blood‐brain barrier (BBB) properties in CNS endothelial cells appear to be dependent on astroglial interactions in vitro. However, evidence for direct astroglial support of BBB function in vivo is controversial. To determine if perivascular astroglial damage or loss would compromise BBB function in situ, localized astroglial degeneration was produced in adult rat spinal cords by systemic injections of the anti‐metabolite 6‐aminonicotinamide (6‐AN). Between 1 and 5 days after 6‐AN administration microvessels in the lumbar spinal cord (blood‐spinal cord barrier) were examined for the expression of several BBB markers and for leakage of endogenous and exogenous proteins by means of immunocytochemical and histochemical procedures. Glial cells throughout the gray matter were swollen after 24 h, and by 5 days post‐injection perivascular astroglia in laminae VI‐VIII appeared completely degenerated. Microvessels were undamaged and continued to express BBB markers such as GLUT‐I, ‐γ‐glutamyltranspeptidase, andendothelial barrier antigen in this region in a manner comparable to control animals. These results suggest that differentiated, BBB‐competent microvascular endothelia in situ may not depend on continuous astroglial support to maintain these particular BBB characteristics. However, the BBB to protein appeared to be compromised; the gray matter was immunoreactive for serum albumin and some areas were permeable to intravascularly injected horseradish peroxidase (HRP). No increase in microvascular transport vesicles was apparent, and no open, tracercontaining interendothelial junctions were detected using standard ultrastructural methods. Some venous structures were surrounded by hemorrhages and HRP reaction product. Thus, astrocytic injury may alter venous, and possibly microvascular, permeability to macromolecules. © 1994 Wiley‐Liss, Inc.
Trimethyltin‐induced expression of GABA and vimentin immunoreactivities in astrocytes of the rat brain
Authors: Andersson Helena , Luthman Johan , Olson Lars
Published: May 2005

Adult Sprague‐Dawley rats were given a single dose of trimethyltin chloride (TMT). Three days following treatment, a neuronal alteration was observed in the CA3c pyramidal cell layer of hippocampus which was not accompanied by any apparent astrocyte reaction. At 1 as well as 2 weeks after treatment, a gliosis in hippocampus, piriform, and entorhinal cortices was detected by glial fibrillary acidic protein (GFAP) immunohistochemistry. Concomitant with the enhanced astrocytic GFAP, astrocytes were swollen and expressed immunoreactivity to vimentin and γ‐aminobutyric acid (GABA). The astrocytic GABA immunoreactivity may reflect a trimethyltin‐induced alteration in astrocyte phenotype, or alterations in compartmentalization and/or metabolism of GABA. © 1994 Wiley‐Liss, Inc.
Downregulation of in vitro neurotoxicity of brain macrophages by prostaglandin E2 and a β‐adrenergic agonist
Authors: Théry Clotilde , Dobbertin Alexandre , Mallat Michel
Published: May 2005

Brain macrophages (BM), a subpopulation of microglia, have the ability to kill neurons by producing reactive oxygen intermediates. Cocultures of neurons and macrophages derived from the cerebral cortex of rat embryos were used to look for regulation ofBM neurotoxicity. Isoproterenol (10−7 M), a β‐adrenergic agonist, induced a significant inhibition of BM neurotoxicity and this effect was abolished in the presence of propranolol, a β‐adrenergic antagonist. BM neurotoxicity was also reduced in the presence of prostaglandin E2 (10−8, 10−6 M), a metabolite derived from arachidonic acid. These results suggest endogenous mechanisms of neuroprotection operating either during development or following lesions. © 1994 Wiley‐Liss, Inc.
Cortical type 2 astrocytes are not dye coupled nor do they express the major gap junction genes found in the central nervous system
Authors: Belliveau Daniel J. , Naus Christian C. G.
Published: May 2005

The O‐2A progenitor cell first described from the rat optic nerve is a bipotential precursor of oligodendrocytes and type 2 astrocytes. Each cell expresses specific markers that distinguish them as unique cell types. O‐2A progenitors cultured in high serum preferentially differntiate into type 2 astrocytes and when exposed to defined medium or low serum develop along the oligodendrocyte lineage. We analyzed the gap junction gene expression of type 2 astrocytes to determine if they are coupled to form a syncytium, like their type 1 astrocyte counterparts. Dye coupling experiments demonstrated that cortical type 2 astrocytes are not coupled, while type 1 astrocytes in the same culture dish are highly coupled. Immunocytochemistry revealed the presence of Cx43 in type 1 astrocytes but we could not detect Cx26, 32, or 43 protein in type 2 astrocytes. In situ hybridization did not detect mRNA for any of the three connexin genes in type 2 astrocytes. These data demonstrate that type 2 astrocytes do not express the major gap junction genes found in the central nervous system. The precise function of type 2 astrocytes is not known but the lack of gap junction genes expression suggests that their functions are different from the spatial buffering capacity of type 1 astrocytes.
A comparative Mac‐1 immunocytochemical and lectin histochemical study of microglial cells in the normal and athymic mice
Authors: Htain Wynn‐Wynn , Leong Seng‐Kee , Ling Eng‐Ang
Published: May 2005

The number of microglial cells in the supraventricular part of the corpus callosum stained with Mac‐1 antibody (against CR3 antigens) and the intensity of staining were studied in both the homozygous athymic nude mouse (nu/nu) and normal BALB/c mouse (+/+). For quantitative analysis, the mean microglial cell counts (expressed in terms of packing density) from 40 μm thick immunostained sections were obtained and tested by analysis of variance. The Mac‐1 positive cells in neonatal nude mice were slightly less intensely stained than those of their normal littermates. Such was not noticeable in the 13‐week‐ and 1‐year‐old animals. The mean number of immunopositive microglial cells per 0.0324 mm2 was significantly less in the 5‐day‐old (P < 0.001) and 13‐week‐old (P < 0.05) nude mice when compared to normal mice of corresponding ages. The difference was insignificant in the 1‐year‐old nude and normal mice. The distribution of Mac‐1 labelled lnicroglia in different areas of the brain of the postnatal nude and normal mouse was also examined. In the brain areas examined, e.g., the olfactory bulb, cerebral and cerebellar cortex, the number of microglia in the nude mouse was considerably reduced. The study of lectin labelled sections also showed a much smaller number of labelled microglial cells in the athymic mouse. This was especially obvious in the 5‐day‐old nude mouse when compared to the normal BALB/c mouse (P < 0.01). The lesser counts of microglia in athymic mice may be attributed to the lack of T lymphocytes and the thymus gland. The latter is known to produce thymic hormones especially thymosin, essential for the maturation of monocytes considered to be the precursor cells of microglia.
Human retinal microglia: Expression of immune markers and relationship to the Glia limitans
Authors: Provis Jan M. , Penfold Philip L. , Edwards Antony J. , van Driel Diana
Published: May 2005

The immunoreactivity, morphology and relationship to the glia limitans of microglia were investigated in flatmounts and sections of normal human retina, using immunogold histochemistry, electron microscopy (EM), and antibodies directed against CD45, major histocompatability complex class I (MHC‐I), MHC‐II, and human macrophage antigens. Immunoreactivity was evident for all antibodies tested, including MHC‐I, which labeled both microglia and retinal vascular endothelium. Most consistent labeling was obtained using antibodies to CD45, MHC‐II, and anti‐human macrophage (S22) antigen. Immunoreactive cells were seen in the perivascular space (perivascular cells), where they were closely adherent to the vessel profile, and in the retinal parenchyma (microglia). Some parenchymal microglia were also vessel associated and by EM were seen to be closely related to the glia limitans (paravascular microglia). Paravascular microglia were shown by optical densitometry, to express higher levels of MHC antigens than neighboring, non‐vessel associated, parenchymal microglia. In addition, paravascular microglia were macrophage (S22) antigen positive, while other parenchymal microglia did not express macrophage antigens. Quantitative data indicate that similar populations of microglia are immunoreactive to CD45, MHC‐I, and MHC‐II, while relatively few microglia (approximately 10%) are immunoreactive for human macrophage (S22) antigens, supporting previous suggestions that microglia are a heterogeneous population. © 1995 Wiley‐Liss, Inc.
Resident microglia and hematogenous macrophages as phagocytes in adoptively transferred experimental autoimmune transferred experimental autoimmune encephalomyelitis: An investigation using rat radiation bone marrow chimeras
Authors: Rinner W. A. , Bauer J. , Schmidts M. , Lassmann H. , Hickey W. F.
Published: May 2005

Hematogenous macrophages are known to be involved in the induction of tissue damage in the central nervous system (CNS) as well as of clinical symptoms in experimental autoimmune encephalomyelitis (EAE). Although resident microglia can become phagocytic under certain circumstances, little is known about the role of these cells in brain inflammation in vivo. We thus studied EAE in the model of radiation bone marrow chimeras that allows us to distinguish donor‐derived hematogenous cells from resident effector cells. Inflammation in the CNS was qualitatively and quantitatively similar in chimeras compared to fully histocompatible Lewis rats. Although activated resident microglial cells were outnumbered four‐to sevenfold in EAE lesions by hematogenous macrophages, the number of resident microglia with ingested myelin was equal to that of macrophages containing myelin debris. Phagocytic resident microglia, expressing the macrophage activation marker ED1, showed ramified as well as amoeboid morphology. From our studies the following conclusions can be drawn. First, a considerable proportion of resident microglia upregulated ED1. Second, resident microglia provide a small but substantial source of brain macrophages in EAE as compared to the large influx of macrophages. Third, our results suggest that macroglia, due to their strategic position within the CNS, are more effective in removal of myelin debris compared to hematogenous macrophages. © 1995 Wiley‐Liss, Inc.
Interleukin 1‐β‐ and tumor necrosis factor‐α‐mediated regulation of ICAM‐1 gene expression in astrocytes requires protein kinase C activity
Authors: Ballestas Mary E. , Benveniste Etty N.
Published: May 2005

Several adhesion molecules including intracellular adhesion molecule‐1 (ICAM‐1) are expressed by astrocytes, the predominant glial cell of the central nervous system (CNS). Such molecules are important in the trafficking of leukocytes to sites of inflammation, and in lymphocyte activation. ICAM‐1 is constitutively expressed by neonatal rat astrocytes, and expression is enhanced by the proinflammatory cytokines interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), and interferon‐γ (IFN‐γ), with IL‐1β and TNF‐α being the strongest inducers. In this study, we have examined the nature of the second messengers involved in ICAM‐1 gene expression induced by the cytokines IL‐1β and TNF‐α. Our results indicate that stimuli related to protein kinase C (PKC) such as the phorbol ester phorbol 12‐myristate 13‐acetate (PMA) and calcium ionophore A23187 increase ICAM‐1 mRNA expression, whereas cyclic nucleotide analogs and PKA agonists have no effect. Pharmacologic inhibitors of PKC such as H7, H8, and calphostin C inhibit ICAM‐1 gene expression inducible by IL‐1β and TNF‐α. Prolonged treatment of astrocytes with PMA results in a time‐dependent downregulating of the PKC isoforms α, δ, and ϵ, and a concomitant diminution of ICAM‐1 mRNA expression induced by IL‐1β, TNF‐α, and PMA itself at specific time points post‐PMA treatment. These data, collectively, demonstrate a role for various PKC isoforms in IL‐1β and TNF‐α enhancement of ICAM‐1 gene expression in rat astrocytes. © 1995 Wiley‐Liss, Inc.
Astrocytic gap junction removal, connexin43 redistribution, and epitope masking at excitatory amino acid lesion sites in rat brain
Authors: Ochalski P. A. Y. , Sawchuk M. A. , Hertzberg E. L. , Nagy J. I.
Published: May 2005

We previously reported that kainic acid (KA) lesion sites in rat brain exhibit an absence of astrocytic gap junctions at 1 week post‐lesion. Loss of immunocytochemical reactivity with a sequence‐specific antibody against the astrocytic gap junctional protein connexin43 (Cx43) suggested epitope masking since persistence of Cx43 was observed on Western blots. Here we determined the fate of Cx43 at various times after thalamic KA and straital NMDA lesions. In normal tissue and at 6 hr post‐KA lesion, Cx43 immunoreactivity predominated at typical astrocytic gap junctions. Immunolabelled junctions were still seen at 3 days, with epitope masking already present, and were virtually absent by 6 days post‐lesion. Gap junction remodeling was indicated by the appearance of intercellular immunostained annular profiles and uncharacteristically extensive gap junctions between symmetrically immunolabelled membranes and between labelled astrocytic and unlabelled oligodendrocytic membranes. Labelled multivesicular clusters emerged at 2 days, were numerous at 3 days and constituted the sole Cx43 sequestration site by post‐lesion day 6. Ultrastructural disruption and gap junction disassembly progressed more slowly in NDMA‐injected tissue where immunoreactivity persisted, albeit at markedly decreasing levels until the final survival time examined (16 days). Intense Cx43 immunolabelling was seen in filopodia of putative reactive astrocytes at the lesion periphery at 6–8 days and was associated at 16 days with an increased number of gap junctions primarily between fine astrocytic processes. These results demonstrate that massive neuronal loss alone or in conjunction with direct actions of excitotoxins on astrocytes precipitates an astrocytic reaction accompanied initially by removal of their gap junctions followed by redistribution of Cx43, and suggest that the astrocytic syncytium may undergo reorganization in a manner leading to isolation of the lesion site. © 1995 Wiley‐Liss, Inc.
Molecular cloning of IL‐2Rα, IL‐2Rβ, and IL‐2Rγ cDNAs from a human oligodendroglioma cell line: Presence of IL‐2R mRNAs in the human central nervous system
Authors: Otero Glen C. , Merrill Jean E.
Published: May 2005

In the present study, we analyzed human adult brain, fetal spinal cord, and an interleukin‐2 (IL‐2)‐responsive human oligodendroglioma subclone, TC620.6A2, for the presence of mRNAs for the α, β, and γ chains of the interleukin‐2 receptor (IL‐2Rα, IL‐2Rβ, and IL‐2Rγ). IL‐2Rβ mRNA, but notIL‐2Rα or IL‐2Rγ was detectable by Northern blot analysis in adult human brain tissues. Northern blot analysis of TC620.6A2 and human fetal tissues revealed mRNAs of 1.5 kb and 1.3 kb that hybridized to the IL‐2Rα cDNA at low to medium stringency. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) experiments were done on the TC620.6A2 cell line utilizing primers to IL‐2Rα, IL‐2Rβ, and IL‐2Rγ. Southern blot analysis of the TC620.6A2 RT‐PCR reactions detected products identical in size to the peripheral blood lymphocyte (PBL) positive controls at high stringency. Several of the TC620.6A2 IL‐2Rα, IL‐2Rβ, and IL‐2Rγ cDNAs were cloned and sequenced. The sequences were found to be identical to the known IL‐2R sequences. To our knowledge, these experiments are the first to demonstrate the presence of authentic IL‐2R mRNAs in a human oligodendrocyte‐like cell line. Demonstration of mRNA for IL‐2Rβ in human adult brain, IL‐2Rα in fetal brain, and IL‐2Rα, IL‐2Rβ, and IL‐2Rγ in a malignant neural cell line suggests the possibility of a role for IL‐2/IL‐2R interactions in development and disease. © 1995 Wiley‐Liss, Inc.
Three different thyroid hormone receptor isoforms are detected in a pure culture of ovine oligodendrocytes
Authors: Fierro‐Renoy Juan Francisco , Szuchet Sara , Falcone Maria , Macchia Enrico , Degroot Leslie
Published: May 2005

Thyroid hormones are important for the normal development of the central nervous system. In humans, the period around the end of the intrauterine life and the first few months of neonatal life is critically dependent on the presence of normal amounts of thyroid hormone. There are significant events occurring during this time; mydelination is one. Myelin is synthesized by oligodendrocytes. A panel of site‐ specific polyclonal antibodies against α‐1 thyroid hormone receptor (TR), α‐2 variant TR, and β‐1 TR isoforms has been employed to investigate the presence of TR isoforms in a pure culture of ovine oligodendrocytes by the avidin‐biotin peroxidase immunocytochemical method. Strong nuclear staining was obtained with all the anti‐TR antibodies; no reaction products were detected in the cytoplasm or cellular processes. By contrast, an anti myelin basic protein antibody gave strong cytoplasmic and process staining; no nuclear staining was seen. These latter results served to 1) confirm that the cells under study are oligodendrocytes; and 2) prove that the nuclear staining with anti‐TR antibodies is specific. Preimmune sera were totally negative. Scatchard analysis of [125I] T3 binding by isolated oligodendrocyte nuclei demonstrated the existence of high‐affinity low‐capacity T3 binding sites with a Ka of ≈ 6 × 10−9 M and a maximal binding capacity of ≈ 20 fmol/100 μg of DNA. Our results demonstrate that differentiated oligodendrocytes express α‐1 and α‐2 variant and β‐1 isoforms of TR at the protein level and support the notion of a direct impact of thyroid hormones on oligodendrocytes in their regulation of myelin synthesis. © 1995 Wiley‐Liss, Inc.
Regulation of neurofibromin expression in rat sciatic nerve and cultured Schwann cells
Authors: Wrabetz Lawrence , Feltri Maria Laura , Kim Haesun , Daston Maryellen , Kamholz John , Scherer Steven S. , Ratner Nancy
Published: May 2005

Loss of function mutations at the NF1 locus may act intrinsically in Schwann cells to cause the formation of benign Schwann cell tumors (neurofibromas) in patients with type 1 neurofibromatosis. To identify contexts in Schwann cells in which such mutations may play an important role, we measured the levels of NF1 mRNA and neurofibromin in rat sciatic nerve during development, after axotomy, and in cultured rat Schwann cells. NF1 mRNA was present in developing sciatic nerve throughout the period of active Schwann cell proliferation and myelination. After nerve transection, no alteration in NF1 message level was detected, but neurofibromin levels increased, as assessed by immunohistochemistry and Western blotting, suggesting that, in vivo, neurofibromin expression in Schwann cells is post‐transcriptionally induced during Wallerian degeneration. Cultured rat Schwann cells constitutively expressed NF1 mRNA and neurofibromin. Schwann cell proliferation induced by exposure to serum and forskolin was not associated with changes in NF1 mRNA or neurofibromin expression, whereas Schwann cell proliferation induced by extracts of embryonic brain membranes was associated with increased NF1 message and neurofibromin expression. Thus, Schwann cells, both in vivo and in vitro, express NF1 mRNA constitutively; the expression of NF1 mRNA and neurofibromin is modulated by only some mitogenic stimuli in Schwann cells. © 1995 Wiley‐Liss, Inc.