bluuebunny/crossref_metadata_2025 huggingface dataset:
13C NMR spectroscopy study of cortical nerve cell cultures exposed to hypoxia
Authors: Müller T. B. , Sonnewald U. , Westergaard N. , Schousboe A. , Petersen S. B. , Unsgård G.
Published: January 2005

Primary cultures of cerebral cortical GABA‐ergic neurons growing on top of a preformed layer of astrocytes (co‐cultures) were incubated with [1‐13C]glucose and exposed to a low oxygen atmosphere (2% O2) for 17 hr. 13C, 1H, and 13P nuclear magnetic resonance (NMR) spectroscopy was performed on perchloric acid (PCA) extracts of cells and of media collected from these cultures. In the control groups incorporation of 13C label into glutamine, citrate, and lactate could be demonstrated in both cell extracts and culture media. Labeled GABA and glutamate were only observed in cell extracts. During hypoxia high energy phosphates decreased but lactate production and glucose consumption increased. There was a decreased amount of citrate and glutamine in cell extracts and media of the hypoxic co‐cultures. There was a change in distribution of the 13C label within the GABA molecule, with an increase of labeling in the C‐2 position. This change in 13C distribution was not found in glutamine present in the media where it is a precursor for GABA in neurons. Instead a decrease in the corresponding C‐4 position was observed. These results suggest that energy depletion during hypoxia leads to reduced export from the astrocytic tricarboxylic acid (TCA) cycle as demonstrated by a decreased amount of citrate and changed distribution of 13C in glutamine. The change in the distribution of label in GABA from cell extracts as compared to glutamine in the medium may indicate that neurons are synthesizing GABA using precursors supplied from their own TCA cycle and not from precursors supplied by astrocytes.
Lesion‐specific pattern of immunocytochemical distribution of growth‐associated protein B‐50 (GAP‐43) in the cerebellum of weaver and PCD‐mutant mice: Lack of B‐50 involvement in neuroplasticity of Purkinje cell terminals?
Authors: Bäurle J. , Oestreicher A. B. , Gispen W. H. , Grüsser‐Cornehls U.
Published: January 2005

The growth‐associated protein B‐50 (GAP‐43) is thought to play a major role in the development and regeneration of neurons. The participation of B‐50 in neuronal plasticity is well documented, especially for monoaminergic systems. However, such an important role for B‐50 in GABAergic systems has not been substantiated to date. This study was performed to obtain detailed information about the identity of B‐50 immunopositive axons and terminals in the cerebellum and to test the involvement of this protein during plastic changes as observed in the projections of GABAergic Purkinje cells to the lateral vestibular nucleus (LVN). For this purpose mutant mice with specific cerebellar cell loss were used. Weaver mutants (B6CBA wv/wv), PCD‐mutants (B6C3Fe pcd/pcd), and their corresponding wild‐type mice were investigated with immunocytochemical and immunoblot procedures at the age of 8‐‐23 days and 5‐‐6 months using polyclonal and monoclonal antibodies to B‐50. Substantial differences in B‐50 distribution wee detected between normals and mutants and between young and adult animals. These results demonstrate that the labeling of B‐50 is mainly related to the outgrowth of parallel fibers and to a minor degree on the ingrowth of non‐GABAergic cerebellar afferents. There was no immunocytochemical indication that B‐50 is related to Purkinje cells or accompanies the plasticity of the GABAergic innervation of the LVN. © 1994 Wiley‐Liss, Inc.
Phagocytic activity of macrophages and microglial cells during the course of acute and chronic relapsing experimental autoimmune encephalomyelitis
Authors: Bauer J. , Sminia T. , Wouterlood F. G. , Dijkstra C. D.
Published: January 2005

The EDI monoclonal antibody recognizes an antigen in lysosomal membranes of phagocytes. The expression of this antigen in cells increases during phagocytic activity. Here we describe the expression of ED1‐immunoreactivity during the various stages of both acute (monophasic) and chronic relapsing experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. During the first attack of acute and chronic relapsing EAE, ED1‐immunoreactivity was present in macrophages and in cells which displayed morphologic features of activated microglial cells (i.e., cells with thick short processes). At the ultrastructural level these cells were seen to contain phagocytosed myelin structures in lysosomes. ED1‐immunoreactivity in these cells was present in the cytoplasm near lysosomes. During the remission phase of acute EAE and the relapse phase of chronic relapsing EAE, ED1‐positive cells with dendritic morphology not only were present in or nearby lesions, but were also found at sites distant from lesions throughout large parts of the brain. These cells had a morphology comparable to microglial cells in normal brain. A major difference between animals which were in remission and animals which on day 25 were suffering from a relapse, was that the latter showed the presence of lesions with darkly stained round ED1‐positive macrophages and activated microglial cells. These results indicate that during a relapse, newly recruited bloodborne macrophages infiltrate the brain and, together with activated lymphocytes and microglial cells, recommence a new demyelination process. © 1994 Wiley‐Liss, Inc.
Migration of genetically labeled glioma cells after implantation into murine brain
Authors: Yamada M. , Shimizu K. , Miyao Y. , Hayakawa T. , Nakajima K. , Nakahira K. , Nakagawa H. , Mikoshiba K. , Ikenaka K.
Published: January 2005

Murine RSV‐M glioma cells were genetically labeled with a retroviral BAG vector carrying the Escherichia coli β‐galactosidase gene. The X‐gal‐positive stable cell line RSV‐M/BAG was obtained by the FDG‐FACS method. To examine the behavior of glioma cells in the brain, we homografted RSV‐M/BAG cells into the brain of C3H/HeN mice as cell suspensions. Individual grafted glioma cells were easily detected by histochemical staining for B‐galactosidase (β‐gal). Three days after grafting, the β‐gal‐positive cells were mainly found in the subependymal zone of the lateral ventricle. In addition, some solitary labeled cells were found at locations distant from the injection sites. On the seventh day after implantation, tumor masses were observed and graft‐derived glioma cells were migrating bilaterally along the fibers in the corpus callosum. Other labeled cells extended into the brain parenchya via the perivascular (Virchow‐Robin) spaces. Rapid and extensive migration of individual glioma cells was thus clearly demonstrated by intracerebral transplantation of RSV‐M/BAG cells. © 1994 Wiley‐Liss, Inc.
Intracranial infusion of monoamine‐activated α2‐Macroglobulin decreases dopamine concentrations within the rat caudate putamen
Authors: Hu Y.‐Q. , Dluzen D. E. , Koo P. H.
Published: January 2005

Monoamine‐activated α2‐macroglobulin (α2M) has been shown to inhibit choline acetyltransferase in basal forebrain neurons as well as neurotrophin‐dependent neuronal functions. The objective of this study was to determine whether monoamine‐activated α2M can affect the caudate putamen (CP) dopaminergic system in vivo. Male rats received intracranial infusions of methylamine‐activated α2M (0.6 nmole) and contralateral infusions of its vehicle, phosphate‐buffered saline (PBS). Five days following infusion, the animals were killed, the CP dissected into three rostral‐caudal segments, and assayed for dopamine (DA) using a high‐performance liquid chromatography system. Within the two rostral CP segments (the approximate site of cannula placement), statistically significant (26%) reductions of DA concentrations were obtained on the α2M‐infused side of the CP with 90–100% of the animals showing decreases. At a more distal (caudal) site of the CP, DA concentrations showed only an insignificant (12%) reduction. No differences in DA concentrations between sides infused with bovine serum albumin versus PBS or from olfactory tubercle samples were obtained in these animals. These results demonstrate that monoamine‐activated α2M is capable of producing significant degeneration of the nigrostriatal dopaminergic system in vivo and suggest that this factor may play a role in age‐related neurodegenerative disorders such as Parkinson's disease. © 1994 Wiley‐Liss, Inc.
The interleukin‐1‐induced increase of substance P in sympathetic ganglia is not mediated by ciliary neurotrophic factor
Authors: Ding M. , Hart R. P. , Shadiack A. M. , Jonakait G. M.
Published: January 2005

Interleukin‐1 (IL‐1) induction of substance P (SP) in cultured sympathetic ganglia requires a soluble intermediate molecule that is present in IL‐1 conditioned medium (IL‐1CM). One of the required intermediates is leukemia inhibitory factor (LIF; Shadiack et al., J Neurosci 13:2601–2609, 1993). In the present study we have examined the possibility that ciliary neurotrophic factor (CNTF) is another intermediate involved in the IL‐1 induction of sympathetic SP. CNTF mimics the action of IL‐1CM by raising both SP and choline acetyltransferase activity‐‐‐actions that are blocked by a specific neutralizing antiserum for CNTF. However, IL‐1CM and CNTF differ in their response to depolarizing agents: while KCl (40 mM) blocks the action of IL‐1CM (and LIF), it enhances the action of CNTF. Furthermore, neither CNTF bioactivity nor CNTF protein is detected in IL‐1CM. Neutralizing antiserum to CNTF fails to block the action of either IL‐1 or IL‐1CM, suggesting that neither a soluble nor a membrane‐bound form of the molecule is active in direct response to IL‐1 action. While Northern blots confirm the presence of both CNTF and CNTF receptor mRNA in neonatal ganglia, neither culturing nor IL‐1 treatment alters these mRNA levels. These data taken together suggest that while CNTF is present and possibly active in sympathetic ganglia, it is not a mediator of the IL‐1 induction of SP. © 1994 Wiley‐Liss, Inc.
Poly(ADP‐ribose) polymerase: Early involvement in glutamate‐induced neurotoxicity in cultured cerebellar granule cells
Authors: Cosi C. , Suzuki H. , Milani D. , Facci L. , Menegazzi M. , Vantini G. , Kanai Y. , Skaper S. D.
Published: January 2005

Glutamate neurotoxicity is correlated with an increase of cytosolic free Ca2+. In some cell systems, activation of Ca2+ dependent endonucleases or formation of free radicals can damage DNA and activate the chromatin bound enzyme poly(ADP‐ribose) polymerase (pADPRP). We have investigated whether pADPRP may be involved in glutamate neurotoxicity in vitro. Cerebellar granule cells at 12 days in culture when treated with a toxic dose of glutamate (100 μM) showed a rapid and transient increase of poly ADP‐ribose immunoreactivity. Cellular immunostaining was heterogeneous and returned to control levels after washout of glutamate. In the same cell preparations glutamate elicited a marked increase in enzyme protein immunoreactivity which persisted at later times. Non‐toxic doses of glutamate did not affect immunostaining. In another set of experiments, pADPRP mRNA was increased 30 min after glutamate. In order to investigate the role of pADPRP in glutamate‐mediated neurotoxicity, structurally different inhibitors of pADPRP (3‐aminobenzamide, benzamide, 3‐aminophthalhydrazide) and their inactive analogues (benzoic acid and phthalimide) were tested in this model. Addition of the inhibitors to cultures 60 min before and during the 30 min of glutamate treatment prevented neuronal death by 60–100%, assessed 24 hr later. Glutamate‐induced Ca2+ influx was not affected. Inactive analogues failed to afford neuroprotection. These data indicate that not only is pADPRP activated by the early, possibly Ca2+ ‐mediated mechanisms initiated by glutamate, but that it might also actively contribute to the subsequent neuronal death. © 1994 Wiley‐Liss, Inc.
Affinity isolation of neuron‐reactive antibodies in MRL/lpr mice
Authors: Moore P. M. , Joshi I. , Ghanekar S. A.
Published: January 2005

Autoantibodies from the MRL/lpr mice react with numerous proteins on neuronal cell surfaces. The purpose of this study was to isolate and characterize a population of autoantibodies reactive preferentially or exclusively with nervous system tissue. Using a purified plasma membrane preparation from brain cortex of balb/c mice coupled to diaminopropylamine agarose gel, we affinity‐isolated antineuronal antibodies from pooled MRL/lpr immunoglobulins. The isolated immunoglobulins reacted with brain cortex plasma membranes and neuroblastoma cells (but not liver, kidney, or fibroblasts) by Western blot and indirect immunofluorescence with confocal microscopy. By Western blot, the epitopes in the brain cortex were proteins of apparent molecular weights 101, 63, 53, 43, 39, and 33, kd; the epitopes in the neuroblastoma cells were 63, 57, and 53 kd. Lectin column isolation revealed that the 101 and 63 kd epitopes were glycosylated. Indirect immunofluorescence revealed that the antibodies bound to the cell soma more intensely than to the cell processes of viable cultured neuroblastoma cells. The cell surface localization of this binding was confirmed by confocal microscopy. Within the central nervous system the antibodies bound more intensely to primary cultures of isolated neurons from fetal cortex than to hippocampal or neostriatal cells. With these antibodies we can begin studies of their potential pathogenic effects. © 1994 Wiley‐Liss, Inc.
Patterns of reactivity with anti‐glycolipid antibodies in human primary brain tumors
Authors: Li J. , Pearl D. K. , Pfeiffer S. E. , Yates A. J.
Published: January 2005

Antibodies against carbohydrates of three glycolipids were used to determine patterns of immunohistochemical reactivity of histologically identifiable cell subpopulations in 101 human primary brain tumors. For all tumor types fibrillary cells, polar cells, and gemistocytes (commonly seen in astrocytomas and ependymomas) stained more frequently for galactosylcerebroside with mAbO1 than small tumor cells and macrophages. Frequency of staining for sulfatide with mAbO4 was fibrillary > polar > small cells = macrophages. Gemistocytes stained more frequently with mAbO4 than polar cells in all tumors except low grade astrocytomas. These data indicate that tumors classified on histological grounds as astrocytic are often stained with antibodies that recognize oligodendrocytes and their progenitors. Thus, anti‐glycolipid antibodies used in the study of developmental lineage may offer useful tools for classification of human brain tumors. Staining of fibrillary cells, polar cells, and gemistocytes for paragloboside directly with mAb F1H11 was much less common than with mAbO1, but this increased by pretreatment of the tissues with neuraminidase (F1H11 + N). Of particular note was the finding that small tumor cells frequently stained with F1H11 + N. Evidence that these were not macrophages was obtained using double immunostaining with F1H11 + N and anti‐macrophage antibodies. In astrocytomas the frequency of small tumor cells immunostained with F1H11 + N was high grade > anaplastic > low grade, demonstrating a correlation of this tumor cell population with more F1H11 + N may be of value in identifying small, ana‐plastic tumor cells, especially in small biopsies or tissue taken adjacent to the main tumor mass. Copyright © 1994 Wiley‐Liss, Inc.
Developmental expression of protein kinase C isozymes in oligodendrocytes and their differentail modulation by 4β‐phorbol‐12, 13‐dibutyrate
Authors: Asotra K. , MacKlin W. B.
Published: January 2005

Myelin gene expression in normal oligodendrocytes (OLG) depends on developmentally regulated protein kinase C (PKC) enzyme activity (Asotra and Macklin: J Neurosci Res 34:571–588, 1993). We studied the developmental expression of the Ca++‐dependent PKC‐α, ‐βI, ‐βII and ‐γ isozymes, and the Ca++‐independent PKC‐δ, ‐ϵ, ‐ζ and ‐η isozymes in enriched rat brain OLG cultures. In A2B5+ O‐2A progenitors, only PKC‐δ, PKC‐ϵ and PKC‐ζ were detected immunocytochemically. In 04+ proligondendrocytes, PKC‐βI, ‐δ and ‐ζ were expressed moderately and low levels of PKC‐α and ‐ϵ were detected. GD3+ OLG, GC+ OLG and MBP+ OLG showed increased levels of PKC‐α, ‐βI, ‐δ and ‐ζ isozymes. PKC‐βII, ‐γ and ‐η were poorly expressed in OLG. On immunoblots, PKC‐α was present early and increased continually up to 18 days but PKC‐βI increased until 12 days in cultured OLG. High levels of PKC‐δ, PKC‐ϵ and PKC‐η, the most abundant PKC isozymes in OLG, were maintained up to 12 days and were then slightly reduced. Interestingly, relatively high levels of PKC‐α, PKC‐βI, PKC‐βII, PKC‐γ and PKC‐ϵ isozymes were detected in purified myelin membrane although greater levels of PKC‐δ were found in OLG than in purified myelin. Thus, most of the PKC isozymes found in cultured OLG were also present in myelin, although at different levels. Treatment with 50 nM 4β‐phorbol‐12,13‐dibutyrate (PDB) caused a delayed downregulation of PKC‐δ levels after 8 hr without modulating the expression of other PKC isozymes in 1‐day OLG; in the 3‐day‐old and 6‐day‐old OLG, PDB downmodulated PKC‐βI, ‐δ and ϵ isozymes with only a minor effect on PKC‐α and no reduction in PKC‐ζ. Induction or downmodulation of individual PKC isozymes by phorbol esters appears to depend on the differentiation state of OLG. These data suggest that PKC‐βI, ‐δ and ‐ϵ isozymes have an important function in different cellular events of OLG differentiation. We conclude that the PKC‐dependent modulation of myelin gene expression in OLG results predominantly from the Ca++‐dependent PKC‐βI isozyme activity and the Ca++‐independent PKC‐δ and PKC‐ϵ activities in a cell differentiation state‐dependent manner. Because of continuous presence of PKC‐ζ isozyme in all the OLG phenotypes and its regulation by a mechanism unlike that for the regulation of other PKC isozymes, PKC‐ζ may have a unique role in OLG function. © 1994 Wiley‐Liss, Inc.
Deafferentation removes calretinin immunopositive terminals, but does not induce degeneration of calbindin D‐28k and parvalbumin expressing neurons in the hippocampus of adult rats
Authors: Beck K. D. , Hefti F. , Widmer H. R.
Published: January 2005

Unilateral combined transections of the fimbriafornix and angular bundle in adult Fischer 344 rats were used to study the effects of deafferentation on hippocampal expression of calretinin, calbindin D‐28k, and parvalbumin. Reflecting the widespread degeneration of synaptic contacts, immunostaining for glial fibrillary acidic protein 6 days after the lesions was increased in lacunosum‐molecular and oriens layers of CA1, 2, and 3 in ipsi‐ and contralateral hippocampus and in the ipsilateral dentate gyrus outer molecular layer. At 21 days the immunoreactivity had decreased to control levels except for a still slightly increased signal in the oriens layer of CA1‐3. At 6 and 21 days after the combined lesions the numbers of hippocampal neurons containing calretinin, parvalbumin, and calbindin D‐28k was unaltered. The combined lesions abolished calretinin containing terminals in the dentate gyrus inner molecular layer on the deafferentated side. This could be reproduced by single unilateral fimbria‐fornix transections, suggesting that the axons of these calretinin positive terminals project to the hippocampus through the fimbria‐fornix. The most likely origin of the calretinin positive terminals are neurons in the supramammillary hypothalamic nucleus. Our findings demonstrate that the extensive lesion‐induced synaptic rearrangements in the adult hippocampus do not induce degeneration of hippocampal neurons expressing calretinin, calbindin D‐28k, and parvalbumin, but do remove calretinin containing terminals which reach their targets in the hippocampus through the fimbria‐fornix. © 1994 Wiley‐Liss, Inc.
Normal distribution of alpha 2‐adrenoceptors in the rat spinal cord and its modification after noradrenergic denervation: A quantitative autoradiographic study
Authors: Roudet C. , Mouchet P. , Feuerstein C. , Savasta M.
Published: January 2005

The distribution of alpha 2 (α2)‐adrenoceptors along cervical, thoracic, lumbar, and sacral segments of the spinal cord of normal rats has been studied by quantitative autoradiography using the specific α2‐antagonist [3H]rauwolscine as a ligand. In addition, the influence of noradrenergic (NA) denervation [obtained either by complete transection of the spinal cord at vertebrae level T8–T9 or by selective lesion of NA spinal cord system carried out by intracisternal injection of 6‐hydroxydopamine (6‐OHDA)] on eventual variations of α2‐adrenoceptor density at spinal cord target cells was studied in parallel.

In control rats, the quantitative analysis of α2‐adrenoceptor densities revealed the presence of these receptors throughout the whole gray matter with a preferential location in the superficial dorsal horn. This pattern was the same at all rostro‐caudal levels of the cord and appeared very well correlated with the distribution of NA terminals revealed by immunohistochemistry, particularly in the supeficial layers of the dorsal horn.

After total transection of the spinal cord (caudally to the section) and 6‐OHDA‐induced lesion, an increase of α2‐adrenoceptor density was mainly observed within the distal dorsal horn thus evidencing supersensitivity in this area, while modifications were not detectable in other regions of the spinal gray matter, except at the lumbar level where other dorsal, central, and intermediate zones were significantly enriched. © 1994 Wiley‐Liss, Inc.
Isoprenylation of brain 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase modulates cell morphology
Authors: De Angelis D. A. , Braun P. E.
Published: January 2005

CNP (2′,3′‐cyclic nucleotide 3′‐phosphodiesterase) is the earliest myelination specific polypeptide to be synthesized by oligodendrocytes (OLs). When non‐myelinating “naive” cells are transfected with the rat CNP cDNA, CNP accumulates intracellularly in a punctate manner, as well as at the plasma membrane. Filopodia and processes, like those of OLs become elongated and more numerous, and are filled with this protein. Post‐translational isoprenylation of the terminal C‐T‐I‐I sequence with either farnesyl or geranylgeranyl is essential for this phenomenon. In contrast, the non‐isoprenylated C397S mutant is homogeneously distributed throughout the cytoplasm and does not markedly affect cellular morphology. We have sythesized CNP and the C397S mutant in vitro and have shown that isoprenylation is essential for the binding of newly synthesized CNP to myelin. © 1994 Wiley‐Liss, Inc.
GRASP: A novel heparin‐binding serum glycoprotein that mediates oligodendrocyte‐substratum adhesion
Authors: Schirmer E. C. , Farooqui J. , Polak P. E. , Szuchet S.
Published: January 2005

Cell‐substratum adhesion plays a crucial part in the cascade of events that control growth or turn on and consummate a differentiation program. We are investigating the molecular basis of oligodendrocyte (OLG) cytodifferentiation, employing pure cultures of OLGs isolated from postmyelination brains. We have shown that such OLGs will regenerate in vitro and reenact the ontogenic development of myelin, but to do so they need a signal. Adherence to a polylysine surface in the presence of 20% horse serum generates such a signal. Among the events that are turned on upon OLG adhesion is the phosphorylation of myelin basis protein; no such phosphorylation takes place in the non‐adhered cell. We postulated that horse serum provides an adhesion molecule. Laminin, fibronectin, collagen and native vitronectin failed to replace horse serum. Hence, we set out to fractionate horse serum by screening with an adhesion assay. We report here the identification, purification and partial characterization of a novel, heparin‐binding horse serum glycoprotein that we have termed Glycine‐Rich Adhesion Serum Protein—GRASP—to stress the fact that this protein has a high content of glycine and functions, in vitro, as an adhesion molecule for OLGs. There is 61% similarity at the N‐terminus between GRASP and histidine‐rich glycoprotein precursor (HRGP), an α2‐glycoprotein from human plasma. However, our data suggest that GRASP is not the horse serum homolog of HRGP. First, the two Gps are functionally distinct: HRGP does not promote the adhesion of OLGs. Second, the amino acid compositions differ significantly, e.g., GRASP is not histidine‐but rather glycine‐rich. Third, the region of sequence similarity between GRASP and HRGP is conserved throughout the cystatin superfamily. Fourth, anti‐Gp55 polyclonal Abs recognize a similar set of polypeptides—save for slight differences in Mr—in human serum as in horse serum, indicating that HRGP and GRASP are two distinct but related proteins and are both present in human and horse sera. GRASP is a dimer/trimer of seemingly identical subunits of Mr ⌣︁ 55,000; the native protein has an Mr × 10−3 ⌣︁ 120–140, of which 24–27% is contributed by carbohydrate. Using GRASP as a substratum allows the growth of OLGs in serum‐free medium. GRASP is as good an effector of myelin basic protein phosphorylation as 20% horse serum. We conjecture that the mechanism of GRASP function features: (1) exposure of a cryptic sequence—after a change in conformation induced upon binding to polylysine—with affinity for an OLG signal‐transducing receptor; and (2) interaction of its heparin‐binding domain with OLG surface heparan sulfate proteoglycans and/or the aforementioned receptor. © 1994 Wiley‐Liss, Inc.
Synapsin I expression in spinal cord neurons during chick embryo development
Authors: Plateroti M. , Vignoli A. L. , Biagioni S. , di Stasi A. M. M. , Petrucci T. C. , Augusti‐Tocco G.
Published: January 2005

The cellular distribution of synapsin I in chick spinal cord has been examined during embryo development and in cultured neurons from different developmental stages. Using immunocytochemical methods we have observed that synapsin I appears lightly detectable in spinal cord of embryonic day (E)5–E8 embryos when the motor neurons have already established functional contacts with muscle fibers, and increases at E9. Until E8 synapsin I immunoreactivity appeared mainly localized in the gray matter of spinal cord; immunostaining of white matter becomes clearly evident only at E9. These observations indicate that synapsin I expression and possibly its transport to the nerve terminals may be stimulated by sequential signals. The cellular distribution of synapsin I observed in vivo is maintained in E8 and E9 spinal cord neuron cell cultures. In fact, in E8 cultured neurons, synapsin I immunostaining is observed only in the cell body, while in E9 cultured neurons both cell body and fibers are stained. The addition of muscle extracts to E8 cultures induces synapsin I decoration of fibers similar to that observed in E9 cultured neurons. Indeed Western and Northern blot analysis and in situ hybridization demonstrate an increase of synapsin I and its mRNA in spinal cord neurons kept in the presence of muscle extracts. These data suggest that synapsin I expression, as previously reported for other neuronal markers, can be modulated by soluble factors present in target cells. © 1994 Wiley‐Liss, Inc.
Role of GABAB receptors in intracellular Ca2+ homeostasis and possible interaction between GABAA and GABAB receptors in regulation of transmitter release in cerebellar granule neurons
Authors: Kardos J. , Elster L. , Damgaard I. , Krogsgaard‐Larsen P. , Schousboe A.
Published: January 2005

The expression of GABAB receptors in cultured mouse cerebellar granule cells was investigated in binding experiments using [3H](S, R)‐baclofen as well as in functional assessment of the ability of (R)‐baclofen to interact with depolarization (15–40 mM KCI) coupled changes in intracellular Ca2+ homeostasis and neurotransmitter release. In the latter case a possible functional coupling between GABAA and GABAB receptors was investigated. The binding studies showed that the granule cells express specific binding sites for (R)‐baclofen. The number of binding sites could be increased by exposure of the cells to the GABAA receptor agonist THIP (4,5,6,7‐tetrahy‐droisoxazolo[5,4‐c]pyridin‐3‐ol) during the culture period. Pretreatment of the neurons with pertussis toxin showed that the GABAB receptors are coupled to G‐proteins. This coupling was, however, less pronounced when the cells had been cultured in the presence of THIP. When 45Ca2+ uptake was measured or the intracellular Ca2+ concentration ([Ca2+]i) determined using the fluorescent Ca2+ chelator Fluo‐3 it could be demonstrated that culturing the neurons in THIP influences intracellular Ca2+ homeostasis. Moreover, this homeostasis was found to be functionally coupled to the GABAB receptors as (R)‐baclofen inhibited depolarization‐induced increases in 45Ca2+ uptake and [Ca2+]i. (R)‐Baclofen also inhibited K+‐induced transmitter release from the neurons as monitored by the use of [3H]D‐aspartate which labels the neurotransmitter pool of glutamate. Using the selective GABAA receptor agonist isoguvacine it could be demonstrated that the GABAB receptors are functionally coupled to GABAA receptors in the neurons leading to a disinhibitory action of GABAB receptor agonists. © 1994 Wiley‐Liss, Inc.
Partial purification of a novel mitogen for oligodendroglia
Authors: Kreider Barbara Q. , Grinspan J. B. , Waterstone M. B. , Bramblett G. T. , Ances B. , Williams M. , Stern J. , Lee V. M.‐Y. , Pleasure D.
Published: January 2005

A protein with a MWapp of 50–70 kDa isolated from the salt extract of crude membranes from neonatal rat brain increases the numbers of oligodendroglia in mixed glial cultures prepared from neonatal rat cerebral white matter. After partial purification by ion exchange and gel exclusion chromatography, and elution from an SDS‐polyacrylamide gel, this protein ( “oligodendroglial trophic factor,” OTF) elicited half‐maximal oligodendroglial recruitment at a concentration of 5 ng/mL. OTF is a mitogen for oligodendroglia, and to a lesser extent, for oligodendroglial progenitor (O2A) cells, but does not stimulate proliferation of astroglia, Schwann cells, or endoneurial fibroblasts. OTF, unlike platelet‐derived growth factor (PDGF), is not an oligodendroglial survival factor. Antibodies against PDGF and basic fibroblast growth factor (bFGF) do not interfere with the accumulation of oligodendroglia induced by OTF. When OTF is given simultaneously with either PDGF or bFGF, there is an additive increase in the numbers of cells of the oligodendroglial lineage. © 1995 Wiley‐Liss, Inc.
Transforming growth factor‐β1 inhibits the proliferation of rat astrocytes induced by serum and growth factors
Authors: Vergelli M. , Mazzanti B. , Ballerini C. , Gran B. , Amaducci L. , Massacesi Luca
Published: January 2005

A number of cytokines and growth factors may affect astrocyte proliferation and functions. Transforming growth factor‐β1 (TGF‐β1) is a pleiotropic cytokine which exerts multiple effects on growth and differentiation of different cell types. TGF‐β1 is present in low amounts in the normal brain. TGF‐β1 gene expression, however, is increased in the central nervous system (CNS) in several pathological conditions. In this study we examined the in vitro effects of TGF‐β1 on the proliferative response of rat astrocytes to serum and growth factors. Astrocyte cultures were established from the cerebellum and cortex of newborn Lewis rats. The proliferative response of these cultures to serum and growth factors [platelet‐derived growth factor (PDGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin‐like growth factor 1 (IGF‐1), IGF‐2, interleukin 1 (IL‐1)] was studied by [3H]‐thymidine incorporation test in the presence or absence of TGF‐β1. TGF‐β1 significantly inhibited the proliferative response of astrocyte cultures to both autologous and heterologous serum. In addition, a strong inhibition of bFGF‐, EGF‐, and PDGF‐induced proliferation was observed. The effect of TGF‐β1 on the proliferative response to IL‐1 was less evident but still significant. No effect was observed when TGF‐β1 was added to IGF‐1 and IGF‐2 stimulated cultures. These data confirm previous reports showing a down‐regulating activity of TGF‐β on astrocyte proliferation and suggest that this cytokine may play physiological and pharmacological roles in the regulation of reactive astrocytosis in the CNS. © 1995 Wiley‐Liss, Inc.
Synaptic vesicle‐associated glutamate decarboxylase: Identification and relationship to insulin‐dependent diabetes mellitus
Authors: Nathan B. , Floor E. , Kuo C.‐Y. , Wu Jang‐Yen
Published: January 2005

Glutamic acid decarboxylase (GAD) catalyzes the biosynthesis of the inhibitory neurotransmitter γ‐aminobutyric acid (GABA). GAD has been suggested as an autoantigen in insulin‐dependent diabetes mellitus and stiff‐man syndrome. Recently, three forms of membrane‐associated GAD (MGAD) have been characterized in porcine brain, but the subcellular localization and function of these proteins are unknown. We present evidence that GAD activity is associated with synaptic vesicles from porcine brain. These vesicles contain a 60 kDa protein recognized by serum from patients with insulin‐dependent diabetes mellitus, probably MGADII, as shown by subcellular fractionation and immunoblotting. These results raise the possibility that the association of MGADII with synaptic vesicles may be crucial for its role as an autoantigen in insulin‐dependent diabetes mellitus. © 1995 Wiley‐Liss, Inc.
Expression of bcl‐2 inhibits necrotic neural cell death
Authors: Kane D. J. , Örd T. , Anton R. , Bredesen Dale E.
Published: January 2005

Cell death has been described as either apoptotic, in which the cell actively participates, or necrotic, in which the cell is felt to be passive. The proto‐oncogene bcl‐2 has been shown to inhibit apoptosis in some hematopoietic and neural cells, by an unknown mechanism. We demonstrate that bcl‐2 inhibits the necrosis of neural cells induced by glutathione depletion. This finding demonstrates that bcl‐2 does not inhibit the cellular death program directly; rather, bcl‐2 modulates a cellular process that leads to apoptosis under some conditions but necrosis under others. © 1995 Wiley‐Liss, Inc.
Basic fibroblast growth factor down‐regulates myelin basic protein gene expression and alters myelin compaction of mature oligodendrocytes in vitro
Authors: Fressinaud C. , Vallat J. M. , Labourdette G.
Published: January 2005

The effects of basic fibroblast growth factor (bFGF) on myelin basic protein (MBP) gene expression and myelin‐like membrane formation were investigated in oligodendrocyte cultures containing mainly mature oligodendrocytes expressing MBP. These cultures were obtained by selective detachment of the cells of the oligodendrocyte lineage from 40‐day‐old mixed cultures derived from newborn rat brain. They were further purified by a 3‐day pretreatment with cytosine arabinoside (ARA‐C) in order to kill cycling cells. After withdrawal of ARA‐C, daily treatment of the cells with bFGF for 3 days induced a drastic decrease in MBP mRNA level compared to control cultures treated only with ARA‐C. Moreover, the percentage of oligodendrocytes labelled with anti‐MBP antibodies decreased by 50%, as well as that of oligodendrocytes expressing myelin oligodendrocyte glycoprotein (MOG), whereas proteolipid protein (PLP) immunolabelled cells were less affected. At the ultrastructural level, myelin‐like membranes were still abundant in the ARA‐C‐and bFGF‐treated cultures, but they were conspicuously uncompacted compared to cultures only pretreated with ARA‐C. These results bring the first evidence that bFGF is able to down‐regulate myelin protein gene expression in mature oligodendrocytes and to alter myelin structure. They imply that if bFGF is secreted after a demyelinating lesion of the central nervous system (CNS), this plasticity of mature oligodendrocytes will allow final remyelination of axons to complete only after this factor has returned to low levels. © 1995 Wiley‐Liss, Inc.
Astrogliosis in culture. IV. Effects of basic fibroblast growth factor
Authors: Hou Y.‐J. , Yu A. C. H. , Garcia J. M. R. Z. , Aotaki‐Keen A. , Lee Y.‐L. , Eng L. F. , Hjelmeland L. J. , Menon V. K.
Published: January 2005

Previous studies have shown that the mechanical wounding of 3‐week‐old cultured rat astrocytes results in cell proliferation and hypertrophy resembling astrocyte responses to a brain injury in vivo. We now report the effects of basic fibroblast growth factor (bFGF) and an anti‐bFGF antibody on astrocyte morphology, proliferation, and migration following in vitro wounding of confluent secondary cultures. Addition of bFGF (20 ng/ml) to wounded cultures induced morphological changes characteristic of differentiation in wounded and nonwounded areas of the culture. Combined treatment with bFGF and an anti‐bFGF antibody (100 μg/ml) prevented this effect. Astrocyte proliferation along the edges of a scratch wound was at maximum 24 hr after wounding in cells growing in Eagle's minimum essential medium (EMEM) containing 10% serum. Low serum concentration and treatment with dibutyryl cyclic adenosine monophosphate (dbc‐AMP) reduced injury‐associated astrocyte proliferation. Addition of bFGF to cultures in EMEM with serum increased astrocyte proliferation at 18 and 24 hr after wounding. This effect was reduced considerably by treatment of cultures with bFGF in combination with an anti‐bFGF antibody. The combined treatment and the antibody alone reduced cell division to a level lower than in control cultures. Twenty‐four hr following wounding, astrocytes along the edges of the wound exhibited extension of thick, flat processes into the wound area. At 3 and 5 days after wounding, a bodily migration of astrocytes into the wounded area was observed. Addition of bFGF significantly increased astrocyte migration 1 day after wounding, with maximum effect on day 3 and no subsequent increase on day 5. A combination of bFGF and anti‐bFGF antibody as well as the antibody alone reduced astrocyte migration to a level lower than in controls. Immunohistochemical localization and isoform pattern of bFGF in astrocytes did not change with dbc‐AMP treatment or wounding. We conclude that mechanically wounded confluent astrocytes respond to bFGF added to the culture medium by enhancing cell division, differentiation, and migration. In addition, the results of the antibody treatment also suggest a role for endogenous bFGF in astrocyte proliferation and migration elicited by wounding in vitro. These results support the notion that in vivo, both bFGF released by injury and endogenous bFGF synthesized by astrocytes, contribute to the cellular responses that lead to astrogliosis. © 1995 Wiley‐Liss, Inc.
Effects of carbonic anhydrase II (CAII) deficiency on CNS structure and function in the myelin‐deficient CAII‐deficient double mutant mouse
Authors: Cammer Wendy , Zhang H. , Tansey F. A.
Published: January 2005

In the choroid plexus carbonic anhydrase II (CAII) supports the transport of bicarbonate ions, sodium ions, and water from blood to the CSF, and in the myelin sheath CAII supports compaction of myelin by stimulating cotransport of ions and water out from between the myelin membranes. In view of the latter, it is surprising that mutant mice deficient in CAII (Car‐2n) have compact myelin. Since myelin basic protein also takes part in myelin compaction, we bred double CAII‐deficient, myelin‐deficient (MId) mutant mice, in which the adults would have some compact myelin sheaths and a partial deficiency in myelin basic protein, with a view to examining oligodendrocytes and myelin sheaths in the double mutant. Like the parent MId strain, the double mutants displayed tremors and seizures; however, the onset of seizures was delayed significantly in the double mutants, and the lifespan increased by several months. Like the brains of Car‐2n mutants, those of double mutants (MldCar‐2n) were deficient in mRNA and protein for CAII and showed upregulation of a different isozyme, CAIV. In the double mutants, oligodendrocytes were reduced in number, and the myelin sheaths and oligodendrocytes were swollen. The partial protection against seizures, which CAII deficiency conferred, suggests that acidosis in the central nervous system (CNS) of the Car‐2n and MldCar‐2n mice, due to absence of CAII from the choroid plexus, may downregulate the activity of NMDA receptors. The swelling of myelin and oligodendrocytes only in the MldCar‐2n suggests that CAIV activity is not sufficient for maintaining compact myelin when the myelin is partly deficient in myelin basic protein and severely deficient in CAII. The findings provide the first direct evidence for a role of carbonic anhydrase II in the compaction of myelin membranes. © 1995 Wiley‐Liss, Inc.
Regionally specific properties of midbrain glia: I. Interactions with midbrain neurons
Authors: Garcia‐Abreu J. , Neto Vivaldo Moura , Carvalho S. L. , Cavalcante L. A.
Published: January 2005

Regional astrocyte cultures were obtained by dissecting and dissociating medial and lateral sectors of the midbrain from 14‐day Swiss mouse embryos. Once confluent, these cultures were tested by glial fibrillary acidic protein (GFAP) immunocytochemistry to confirm their astrocyte composition and for 2′‐3′ cyclic nucleotide 3′‐phosphohydrolase (CNPase) and microtubule‐associated protein 2 (MAP2) immunocytochemistry to rule out oligodendroglial and neuronal components, respectively. In confluent astrocyte cultures from either sector, virtually all cells were GFAP‐positive elements, most of which were flat cells accompanied by smaller numbers of flat cells with processes. Confluent astrocyte cultures, derived from medial (M) or lateral (L) sectors, were used as substrata for culturing dissociated cells from medial (m) or lateral (1) sectors of 14‐day embryonic midbrains. Fixed cocultures (LI, Lm, Mm, MI) were stained with an anti‐MAP2 antibody to verify neuronal aggregation and neuritic morphology. In spite of the morphological constancy of glia substrata at plating, MAP2‐positive cells in cocultures showed differences in the aggregation of somata and in the length, caliber, and branching of neurites. These differences, which depend mostly on the sector of origin of astrocytes, suggest that the substrata may differ in adhesiveness and/or growth‐promoting vs. growth‐interfering properties. Together with evidence for sectorial heterogeneity in brainstem radial glia, the present results raise the possibility that cultured astrocytes have properties that reflect the roles played by their parent radial glia in the developing brain. © 1995 Wiley‐Liss, Inc.
Changes in c‐fos expression in the dorsal column‐medial lemniscal system following dorsal column lesions
Authors: Riley L. A. , Bernstein J. J.
Published: January 2005

Transection of the hindlimb dorsal column fibers leads to a partial deafferentation of the neurons of the nucleus gracilis, the first relay of the ascending dorsal column‐medial lemniscal (DC‐ML) neural network. In response to the deafferentation, a synaptic renewal cycle is initiated and the neurons of the nucleus gracilis atrophy. The present study examines the molecular changes that occur in synaptic relays of the ascending DC‐ML following a hindlimb dorsal column transection. Rats were sacrificed 0.5, 1, 24, or 72 hours postlesion. Steady‐state levels of mRNA coding for c‐fos were significantly elevated only at 24 hours postlesion in caudal dorsal medulla, which contains the nucleus gracilis. The increased c‐fos is neuronal in origin since there is an increased level of c‐fos immunoreactivity in both the cluster neurons and the interneurons of the nucleus gracilis. In the third relay of the DC‐ML system, the somatomotor cortex, levels of c‐fos mRNA were significantly decreased 72 hours postlesion. These data indicate that lesions of the hindlimb dorsal column fibers have transneuronal effects on gene expression that extend to at least the third synaptic relay in the DC‐ML system. © 1995 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
CNS myelinogenesis in vitro: Time course and pattern of rat oligodendrocyte development
Authors: Asou Hiroaki , Hamada K. , Miyazaki T. , Sakota T. , Hayashi K. , Takeda Y. , Marret S. , Delpech B. , Itoh K. , Uyemura K.
Published: January 2005

Oligodendrocyte precursor cells that develop into myelin‐forming cells of the central nervous system (CNS) were cultured from newborn rat brain to study how they proliferate and differentiate in normal conditioning medium, and their cell development was characterized by scanning electron microscopy (SEM) observation and immunocytochemical studies.

We have identified A2B5‐negative pre‐O2A progenitor cells (so‐called “type‐1” oligodendrocytes) in the secondary cultures on the astrocyte feeder layer. These cells are very small (diameter: 3.5 μm), round, and glossy, and develop into the process‐bearing O2A progenitor cells (called “type‐2” oligodendrocytes), which also express myelin basic protein (MBP) both in the cell body and in their cell processes. Finally, they develop into mature oligodendrocytes (called “type‐3” oligodendrocytes). After MBP expression is elicited in these cells and MBP accumulates in the cell process in the area in contact with the axon, these cells are capable of forming the myelin sheath. Therefore, we examined the mechanism of myelin‐sheath formation of “type‐3” oligodendrocytes using video time‐lapse movies, and demonstrated that these cells initially sent out processes to search for axons several times before the onset of myelination. Then thick filopodia extended towards the axon, and at the same time, the axonal part of neuron moved forward. Finally the ruffling lamellipodial parts wrapped up the axon similarly to a transverse wave with the secured thick filopodial process on the axon acting as scaffolding. These results suggest that our experimental systems are useful in studying normal oligodendrocyte development and their cellular biochemistry, as well as investigating the mechanism of myelin formation by oligodendrocytes. © 1995 Wiley‐Liss, Inc.
Glutamate‐activated ionic currents in cultured astrocytes from trout: Evidence for the occurrence of non‐N‐methyl‐D‐aspartate receptors
Authors: Clasen T. , Jeserich G. , Krüppel T.
Published: January 2005

Glutamate‐induced currents were recorded from cultured trout astrocytes with the whole‐cell variation of the patch‐clamp technique. Ninety percent of the tested cells were directly depolarized by the amino acid neurotransmitter in a concentration‐dependent manner. The depolarizing effect was due to an inward current that reversed near 0 mV and was accompanied by a noise increase, indicating the opening of an ion channel. Ion substitution experiments revealed that the glutamate‐induced current was mainly carried by sodium ions but not chloride or calcium ions. The glutamate‐induced response could be mimicked by the neuronal glutamate receptor subtype agonists kainate and quisqualate, while N‐methyl‐D‐aspartate was without detectable effect. © 1995 Wiley‐Liss, Inc.