bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of 5‐hydroxytryptamine2 receptors by radioligand binding, northern blot analysis, and Ca2+ responses in rat primary astrocyte cultures
Authors: Deecher D. C. , Wilcox B. D. , Dave V. , Rossman P. A. , Kimelberg H. K.
Published: January 2005

Radioligand binding, Northern blot analysis, and changes in [Ca2+]i were used to study serotonin [5‐hydroxytryptamine (5HT)] receptor subtypes in primary cultures of astrocytes from neonatal rat cerebral cortex. Radioligand binding studies revealed the presence of 5HT2, but not the 5HT1 or 5HT3 receptor subtypes. Radioligand binding was also used to show the presence of serotonin uptake sites, which had previously been shown to be present by [3H]‐5HT uptake, and also α1‐adrenergic receptors as has previously been reported by binding studies. Northern blot analysis of cortical astrocyte mRNA demonstrated the presence of transcripts for 5HT2 receptors, but failed to identify mRNA for 5HT1α or 5HT1c receptors. Thus, results from Northern blot analysis correlated with the radioligand binding data which showed only 5HT2 receptors. Equilibrium saturation studies, using 125[I]‐LSD to label 5HT2 receptors, yielded a KD of 9 nM and a Bmax of 177 fmol/mg protein. Radioligand binding studies or primary astrocyte cultures prepared from other brain regions also showed the presence of α1‐ adrenergic, 5HT2 receptor, and 5HT‐uptake sites, but no detectable 5HT1a receptors, which were the only 5HT1 receptors studied. Studies demonstrating 5HT‐induced, spiperone‐ and ketanserin‐sensitive increases in free [Ca2+]i as measured by FURA‐2, showed that the 5HT2 receptors were functional in these cells. These data provide clear evidence for the existence of both 5HT2 receptors and 5HT‐uptake sites in the same primary astrocyte cultures from neonatal rat cerebral cortex, with no detectable evidence of 5HT1a or 5HT1c subtypes. © 1993 Wiley‐Liss, Inc.
Expression of heterologous proteins in cultured rat hippocampal neurons using the semliki forest virus vector
Authors: Olkkonen V. M. , Liljeström P. , Garoff H. , Simons K. , Dotti C. G.
Published: January 2005

The Semliki Forest virus expression vector (Liljeström and Garoff: Bio/Technology 9:1356‐1361, 1991) was tested in cultured rat hippocampal neurons using two Madin‐Darby canine kidney (MDCK) cell membrane‐associated proteins as reporters: rab8, a small GTP ase involved in post‐Golgi vesicle transport, and VIP21, an integral membrane protein of caveolae, trans‐Golgi network, and post‐Golgi vesicles. Expression of the c‐myc epitope‐tagged proteins was visualized by immunofluorescence microscopy. The proteins were first detected in neurons after 3–4 hr infection by the recombinant viruses. The infection efficiency on neurons was high: after 6 hr infection at a multiplicity of one, 50–60% of the cells expressed the reporter proteins. The neurons tolerated the in fection well up to 8 hr. Their polarized organization was not disturbed, as judged from morphology and from distribution of the dendritic MAP2 and axonal synaptophysin marker proteins. The Semliki Forest virus vector thus seems suitable for short‐term expression of proteins in cultured neurons. © 1993 Wiley‐Liss, Inc.
Striatal degeneration induced by mitochondrial blockade is prevented by biologically delivered NGF
Authors: Frim D. M. , Simpson J. , Uhler T. A. , Short M. P. , Bossi S. R. , Breakefield X. O. , Isacson O.
Published: January 2005

Consistent with the notion that a defect in cellular energy metabolism is a cause of human neurodegenerative disease, systemic treatment with the mitochondrial complex II inhibitor 3‐nitropropionic acid (3‐NPA) can model the striatal neurodegeneration seen in Huntington's disease. Previously, we have found that nerve growth factor (NGF), delivered biologically by the implantation of a genetically altered fibroblast cell‐line, can protect locally against striatal degeneration induced by infusions of high doses of glutamate receptor agonists. We now report that implantation of NGF‐secreting fibroblasts reduces the size of adjacent striatal 3‐NPA lesions by an average of 64%. We conclude that biologically delivered NGF protects neurons against excitotoxicity and mitochondrial blockade—both energy‐depleting processes—implying that appropriate neurotrophic support in the adult brain could protect against neurodegenerative diseases caused in part by energy depletion. © 1993 Wiley‐Liss, Inc.
Specific regulation of immediate early genes by patterned neuronal activity
Authors: Sheng H. Z. , Fields R. D. , Nelson P. G.
Published: January 2005

Electrical activity shapes development of the nervous system, presumably in part by regulating gene expression. A set of regulatory genes, immediate early genes (IEGs), which are responsive to a number of extrinsic cellular stimuli have been proposed to play a role in coupling such activity to gene expression. Using a semiquantitative polymerase chain reaction assay, we show that in dissociated mouse dorsal root ganglion neurons the expression of two IEGs, c‐fos and nur/77, is differentially sensitive to patterns of electrical stimulation. Differences in c‐fos activation did not correlate with the peak intracellular calcium [Ca++]i produced by the different stimulation patterns or with residual [Ca++]i following stimulation. However, the net increase in [Ca++]i (calcium time integral) was greater for the pulsed stimulus that activated c‐fos (6 impulses/min), compared to the ineffective stimulus (12 impulses/2 min). This system of genes seems suited to mediating the coupling between electrical activity and other functional genes. © 1993 Wiley‐Liss, Inc.
Optimized survival of hippocampal neurons in B27‐supplemented neurobasal™, a new serum‐free medium combination
Authors: Brewer G. J. , Torricelli J. R. , Evege E. K. , Price P. J.
Published: January 2005

We have systematically optimized the concentrations of 20 components of a previously published serumfree medium (Brewer and Cotman, Brain Res 494: 65‐74, 1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum‐freemedium Supplement, B27, produced neuron survival above 60% independent of plating density above 160 plated cells/mm2 For isolated cells (<100 cells/mm2), survival at 4 days was still above 45%, but could be rescued to the 60% level at 40 cells/mm2 by simply applying a coverslip on top of the cells. This suggests a need for additional trophic factors. High survival was achieved with osmolarity lower than found in Dulbecco's Modified Eagle's Medium (DMEM), and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrnous sulphate found in DME/F12. Neurobasal is a new medium that incorporates these modifications to DMEM. In B27/ Neurobasal, glial growth is reduced to less than 0.5% of the nearly pure neuronal population, as judged by immunocytochemistry for glial fibrillary acidic protein and neuron‐specific enolase. Excellent long‐term viability is achieved after 4 weeks in culture with greater than 90%viability for cells plated at 640/mm2 and greater than 50 viability for cells plated at 160/mm2 Since the medium also supports the growth of neurons from embryonic rat striatum, substantia nigra, septum, and cortex, and neonatal dentate gyrus and cerebellum (Brewer, in preparation), support for other neuron types is likely. B27/Neurobasal should be useful for in vitro studies of neuronal toxicology, pharmacology, electrophysiology, gene expression, development, and effects of growth factors and hormones. © 1993 Wiley‐Liss, Inc.
B‐50 (GAP‐43) in the spinal cord caudal to hemisection: Indication for lack of intraspinal sprouting in dorsal root axons
Authors: Nacimiento W. , Mautes A. , Töpper R. , Oestreicher A. B. , Gispen W. H. , Nacimiento A. C. , Noth J. , Kreutzberg G. W.
Published: January 2005

Sprouting of dorsal root axons has been suggested to occur in the mature cat spinal cord caudal to a hemisection at a low thoracic level sparing the dorsal columns. The lesion interrupts supraspinal descending projections, while leaving ascending collaterals of dorsal root axons intact. This hypothesis was re‐evaluated by comparing the light and electron microscopic immunoreactivity of B‐50 (GAP‐43) on both sides of the postulated target regions for sprouting, the intermediate gray and the dorsal horn. The neural‐specific phosphoprotein B‐50 is involved in regenerative and developmental axonal outgrowth and synaptic plasticity. The light microscopic distribution pattern and density of B‐50 immunostaining, measured by quantitative densitometry, were bilaterally symmetrical in all segments below the hemisection 3.5, 8, 14, 21, and 56 days postoperatively, as they were in the intact animal. Ultrastructurally, growth cone‐like profiles were not detectable during putative periods of sprouting in regions of interest. After removal of degenerated axon terminals, vacated postsynaptic places appeared to be covered by astrocytic processes. These results indicate that, under the present experimental conditions, sprouting of primary afferents in adult cats is unlikely to be involved in functional plasticity after removal of descending pathways. © 1993 Wiley‐Liss, Inc.
Age‐related loss of knee joint afferents in mice
Authors: Salo P. T. , Tatton W. G.
Published: January 2005

Previous work in our laboratory revealed markedly different rates of age‐related death of four monoaminergic neuronal populations in the C57BL/6 mouse. Although dorsal root ganglion neurons (DRGns) have been reported not to suffer similar age‐related death in rodents, we determined if there is age‐related death of the subpopulation of DRGns innervating the knee joints of C57BL/6 mice, which are known to develop degenerative arthritis with aging. The somata of dorsal root ganglion neurons innervating the mouse knee joint (KJ‐DRGns) were identified by retrograde tracing with Fluoro‐Gold (FG). Lumbar ganglia were serially sectioned and the numbers of FG‐labelled KJ‐DRGns counted at five ages encompassing the animal's life span. Changes in size of the total population of lumbar DRGns (L‐DRGns) were estimated by counting nucleated somata from every fifth toluidine blue‐stained serial section from the L3 and L4 lumbar ganglia at three different ages. Using a computer‐assisted video morphometric technique somal areas were measured from random sections to determine the distribution of sizes of neurons in the KJ‐DRGn and general lumbar DRGn populations at different ages. Counts of FG‐labelled joint afferents were 238.5 ± 80.3 (mean SD) KJ‐DRGns per knee at 2 months of age, declining to 103.2 ± 20.1 by 24 months, representing a 57% loss over the average life span of the C57 mice. The loss occurred in two phases, with a rapid rate over the first 8 months of life and a more moderate rate of loss over the remaining months. L‐DRGn numbers revealed a slower overall rate of loss in comparison to the KJ‐DRGn population with an average 33.7% loss over the life span of this mouse. Somal size measurements revealed that the larger sizes of KJ‐DRGns were lost over the first 8 months of life, with little change in the distribution of somal sizes thereafter. The distributions of sizes of the L‐DRGn population did not change significantly over the life spans of the mice. The data provides evidence that the age‐related loss of KJ‐DRGns is significantly greater than DRGns in general, and may be particularly apparent in the population of largersized presumed mechanoreceptor neurons. The loss of the KJ‐DRGns is approximately reciprocal to the incidence rate of knee joint osteoarthritis reported for the C57BL/6 mice. © 1993 Wiley‐Liss, Inc.
Expression of the metabotropic glutamate receptor mGluR1α and the ionotropic glutamate receptor GluR1 in the brain during the postnatal development of normal mouse and in the cerebellum from mutant mice
Authors: Ryo Y. , Miyawaki A. , Furuichi T. , Mikoshiba K.
Published: January 2005

Expression of the metabotropic glutamate receptor type 1α (mGluR1α) and the non‐N‐methyl‐D‐aspartate (NMDA) ionotropic glutamate receptor type 1 (GluR1) in mouse brain was investigated using the antibodies raised against the synthetic peptides corresponding to their C‐terminal amino acid sequences. Both receptor proteins are glycosylated predominantly in an asparagine‐linked manner, and are abundant in post‐synaptic membranes. We showed that mGluR1α and GluR1 expression within the first 3 postnatal weeks undergoes dramatic changes in time and space, i.e., in the hippocampus and cerebellum. These spatio‐temporal expression patterns appear to be correlated with the postnatal ontogenesis and establishment of the glutamatergic neurotransmission system in the hippocampus and cerebellum, cell migration, dendritic and axonal growth, spine formation, and synaptogenesis. In the adult cerebellum, mGluR1α is intensely expressed in Purkinje neurons and GluR1 in Bergmann glial cells. Both receptors are expressed to a fair degree in weaver mutant cerebellum despite granule cell degeneration. However, the intrinsic expression levels of both mGluR1α and GluR1 are markedly reduced in the cerebellum of the Purkinje cell‐deficient and underdeveloped mutant mice, Purkinje‐cell‐degeneration, Lurcher, and staggerer, suggesting that GluR1 expression in Bergmann glia cells may be correlated with the sustained interaction with adjacent Purkinje neurons. © 1993 Wiley‐Liss, Inc.
Purification and characterization of NCAD90, a Soluble endogenous form of N‐cadherin, which is generated by proteolysis during retinal development and retains adhesive and neurite‐promoting function
Authors: Paradies N. E. , Grunwald G. B.
Published: January 2005

The cadherins are calcium‐dependent cell adhesion molecules which regulate cell–cell interactions during morphogenesis. During development, cadherin expression is subject to dynamic patterns of regulation. We have previously demonstrated that expression of N‐cadherin, the predominant cadherin of neural tissues, is sharply down‐regulated during development of the retina and brain during later stages of histogenesis (Lagunowich and Grunwald, Dev Biol 135:158–171, 1989; Lagunowich et al., J Neurosci Res32;202–208, 1992), and that this down‐regulation is due to multiple factors, including decreased mRNA levels and turnover apparently mediated by endogenous metalloproteolytic activity (Roark et al., Development 114:973–984, 1992). In the present study, we describe metabolic studies which provide direct biochemical evidence for turnover of 130‐kDa N‐cadherin in embryonic retina tissues, yielding a soluble 90‐kDa N‐terminal fragment. We demonstrate that this form of N‐cadherin, which we refer to as NCAD90, accumulates in vivo during development. We further demonstrate that purified NCAD90, obtained from embryonic vitreous humor, retains biological function and promotes cell adhesion and neurite growth in a dose‐dependent fashion among chick embryo neural retina cells when present in a substrate‐bound form. The morphology of retinal cells and neurites grown on a substrate of NCAD90 differs strikingly from that seen on a laminin substrate, in a manner similar to that described for intact 130‐kDa N‐cadherin. We conclude that proteolysis of N‐cadherin at the cell surface during embryonic retinal histogenesis is an endogenous mechanism for regulating N‐cadherin expression which generates a novel and functional from of the protein. The results further indicate that an intact cytoplasmic domain is not essential for all cadherin functions. © 1993 Wiley‐Liss, Inc.
Microtubule‐associated protein 2 appears in axons of cultured dorsal root ganglia and spinal cord neurons after rotavirus infection
Authors: Weclewicz K. , Svensson L. , Billger M. , Holmberg K. , Wallin M. , Kristensson K.
Published: January 2005

The immunohistochemical distribution of microtubule‐associated protein 2 (MAP2), being normally restricted to nerve cell bodies and dendrites, became altered in rat dorsal root ganglia and spinal cord neurons in cultures infected with rhesus rotavirus. MAP2 appeared in axons of both sources of neurons as displayed with monoclonal antibodies to MAP2a+b and MAP2a+b+c at 48 hr post‐infection (p.i.). Other cytoskeletal elements, i.e., tau, MAP1, MAP5, neurofilament, actin, and tubulin, did not reveal any alterations in the rotavirus‐infected neurons. One of the rotavirus cytosolic proteins, the inner capsid protein vp6, was expressed in axons at 48 hr p.i. simultaneously with the appearance of MAP2, while two other viral proteins, vp4 and NS28, remained in the nerve cell bodies. By quantitative enzyme‐linked immunosorbent assay (ELISA) a binding of single‐shelled rotaviruses, which express vp6 on their surfaces, to purified MAP2 was found. There was no binding of these viral particles to tau or tubulin proteins. This study indicates that a selective interaction between certain viral and neuronal cytoskeletal proteins can occur and that a non‐cytolytic viral infection can cause alterations in the polarized sorting of neuronal proteins. © 1993 Wiley‐Liss, Inc.
Atypical localization of the oligodendrocytic isoform (PI) of glutathione‐S‐transferase in astrocytes during cuprizone intoxication
Authors: Cammer W. , Zhang H.
Published: January 2005

Immunocytochemical staining for the Pi and Mu isoforms of glutathione‐S‐transferase was used to investigate changes in the glial cells in the mouse forebrain. During early development in mouse forebrains the localizations of carbonic anhydrase, Pi and Mu were similar to the respective cellular localizations that had been observed in neonatal rat brain. That is, Pi was found in oligodendrocyte precursors, Mu in astrocytes, and carbonic anhydrase in both oligodendrocyte precursors and astrocytes. In forebrains of 6‐week‐old mice the neurotoxicant, cuprizone, induced oligodendrocyte degeneration, gliosis, and partial demyelinatin. Degeneration of oligodendrocytes, and astrocytosis, began during the initial week of cuprizone feeding, and by the end of the eighth week some demyelination was observed. After mice were fed cuprizone for 4 to 7 weeks, Pi appeared in some of the reactive astrocytes, and Pi‐positive astrocytes were present for at least 7 additional weeks. Normally, Pi appeared only in oligodendrocyts. Very few Pi‐positive oligodendrocyes remained after the second week. During the eighth week healthy‐looking carbonic anhydrase‐positive oligodendrocytes reappeared and began to accumulate, and a few small patches of Pi‐positive oligodendrocytes were also observed. In summary, some novel findings about glial cells were the observation of an enzyme (Pi) that is lost earlier from oligodendrocytes than is carbonic anhydrase, the apparently unique shift in Pi expression from oligodendrocytes to astrocytes and the greater temporal dissociation between loss of oligodendrocytes and demyelination in the older mice. © 1993 Wiley‐Liss, Inc.
Characterization of the neuroimmune protein F5: Localization to the dendrites and perikarya of mature neurons and the basal aspect of choroid plexus epithelial cells
Authors: Arai M. , Cohen J. A.
Published: January 2005

F5 was identified originally as an interleukin‐2‐regulated gene in L2 cells, a murine helper T‐lymphocyte clone. In adult mouse, F5 mRNA was expressed at a modest level in lymphoid tissues, at a high level in mature neurons in the nervous system, but not in other tissues. Although the F5 sequence is highly conserved over evolution, the function of the F5 protein is unknown. In the present studies, the putative F5 protein coding region was translated in vitro using a reticulocyte lysate system and in Escherichia coli, yielding a protein with the predicted molecular weight of 42 kDa. Polyclonal rabbit anti‐F5 antibody specifically recognized recombinant F5 protein. Western blot studies demonstrated a strongly‐reactive 42‐kDa band and a faint 39‐kDa band in extracts of adult mouse brain regions, the levels of which paralleled F5 mRNA expression. Immunoperoxidase studies of adult mouse brain demonstrated F5 immunoreactivity in neuronal perikarya and dendrites but not axons. Neurons expressing the highest levels of F5 protein corresponded to those with the highest levels of F5 mRNA. Choroid plexus epithelial cells also exhibited strong reactivity localized to their basal aspect. These observations suggest that the F5 protein, expression of which appears to be regulated predominantly at the RNA level, may be involved in the maintenance of the functional or anatomic polarity of neurons and choroid plexux epithelial cells. © 1993 Wiley‐Liss, Inc.
cDNA cloning and expression of stress‐inducible rat hsp70 normal and injured rat brain
Authors: Longo F. M. , Wang S. , Narasimhan P. , Zhang J. S. , Chen J. , Massa S. M. , Sharp F. R.
Published: January 2005

A reverse transcriptase‐polymerase chain reaction(RT‐PCR) product obtained from ischemic rat brain RNA was used to screen a rat ischemic forebrain cDNA library for a cDNA clone containing the entire open reading frame for the inducible hsp70. The coding sequence for the rat hsp70 cDNA demonstrated significant similarities with the human hsp70 of Hunt and Morimoto (Proc Natl Acad Sci82:6455–6459, 1985) and the mouse hsp70 of Hunt and calderwood (Gene87:199–204, 1990). The rat inducible hsp70 and constitutive hsc73 sequences are distinct. There was a low level of hsp70 mRNA expression in normal rat brain as is found in other tissues. hsp70 mRNA was markedly induced in rat brain 8 hours following global ischomia and kainic acid‐induced sizures. Northern blots showed a ∼2.9kb hsp70 mRNA band from control, kainic acid, and ischemic brains. RTPCR confirmed the presence of hsp70 mRNA in normal rat brain. Since there are at least five human and six mouse inducible hsp70 genes known, many other rat hsp70 genes probably exist that could function in different cells or organelles or be induced under different circumstances. © 1993 Wiley‐Liss, Inc.
Induction of primary T cell responses by human glial cells
Authors: Williams K. , Ulvestad E. , Cragg L. , Blain M. , Antel J. P.
Published: January 2005

Glial cells of the central nervous system (CNS) are postulated to function as immune accessory cells which may regulate immune reactivity occurring within the CNS, activating or alternatively inhibiting T cell responses. We have utilized surgically resected cerebral tissue derived from young adult humans to prepare dissociated cultures of glial cells (mixed astrocyte‐microglia‐oligodendrocyte cultures) and demonstrate that such cells are capable of acting as stimulators of primary T cell responses, using proliferation of T cells to allogeneic determinants on the glial cells as the test system. Studies of resected adult cerebral tissue indicated major histocompatibility complex (MHC) class II antigen expression on microglia in situ. Using a mixed lymphocyte reaction (MLR), we observed that enriched microglial cultures alone were capable of stimulating primary responses of freshly isolated T cells or the CD4+ T cell subset, a response which could be inhibited with an anti‐MHC class II blocking antibody. In agreement with previous studies using rodent‐derived astrocytes, we found that human astrocytes (fetal), could not initiate a primary T cell response even after up‐regulation of MHC class II antigen expression with interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα). Our results indicate that a primary T cell response, as well as a secondary response to a recall antigen, can occur within the CNS; our data implicate microglia as the central cell involved in the former. © 1993 Wiley‐Liss, Inc.
Cellular reaction to an acute demyelinating/remyelinating lesion of the rat brain stem: Localisation of GD3 ganglioside immunoreactivity
Authors: Reynolds R. , Wilkin G. P.
Published: January 2005

We describe a simple and reproducible acute demyelinating lesion of the rat brain stem induced by injection of ethidium bromide into the cisterna magna of young adult rats. Using immunofluorescence with a panel of antibodies to cell‐specific antigens we have studied the changes in cell populations that occur at various stages during lesion progression and repair. In particular we localized the expression of ganglioside GD3 immunoreactivity, a marker for oligodendroglial progenitors in developing brain. Both astroglia (GFAP+) and oligodendroglia (CNP+) were destroyed during the early response to the ethidium bromide although axons were spared. Splitting of myelin lamellae occurred as early as 4 days post‐injection (DPI), with extensive demyelination of the inferior cerebellar peduncle following by 6 DPI. Large numbers of ED1+ and OX‐42+ macrophages were present in the lesion site at this stage. Astrogliosis occurred around the perimeter of the lesions. Two populations of GD3+ cells appeared within and around the lesion sites during the demyelination. One population was identified by the phenotype GD3+ ED1+ and thus probably belonged to the macrophage/microglial lineage. In these cells both antigens appeared cytoplasmic. The second population of GD3+ cells exhibited cell membrane GD3 immunoreactivity but did not express the ED1 antigen. These cells are suggested to be oligodendroglial progenitors generated in response to the demyelination. No such cells were seen in control tissue. GD3+ cells were present within the lesion sites from 6 DPI until 10–12.

Following the clearance of myelin debris from the lesions, remyelination was a relatively rapid event with thin MBP+ myelin sheaths first seen at 11–12 DPI. Remyelination, which was extensive by 25 DPI, was predominantly oligodendroglial in origin (MBP+Po− myelin) with only small pockets of peripheral myelin (MBP+Po+ myelin) observed. The present study, in addition to identifying putative glial progenitors within a demyelinated lesion, also demonstrates the difficulties in unambiguously identifying such cells in the normal and damaged adult CNS. © 1993 Wiley‐Liss, Inc.
Myelination in cerebellar slice cultures: Development of a system amenable to biochemical analysis
Authors: Notterpek L. M. , Bullock P. N. , Malek‐Hedayat S. , Fisher R. , Rome L. H.
Published: January 2005

Myelin deposition and maintenance are critical to proper function of the mammalian nervous system. Previous investigations of myelination in the central nervous system (CNS) were hampered by the lack of an in vitro system that can faithfully reproduce in vivo events yet is amenable to biochemical investigation. We have developed a procedure, based on organotypic cultures, which permits efficient preparation of large numbers of cerebellar slice cultures that can be easily manipulated. Cultures have been examined to document myelination biochemically (by incorporation of [35S] sulfate into sulfolipids), immunohistochemically (by labeling the myelin components myelin basic protein and galactocerebroside), and morphologically (by both light and electron microscopy). We tested the effects of biologically active peptides and antibodies on myelination in the thin slices. The results indicate that the cultures provide an in vitro system that can be used to examine specific cellular events that occur during CNS myelination. © 1993 Wiley‐Liss, Inc.
Members of several gene families influence survival of rat motoneurons in vitro and in vivo
Authors: Hughes R. A. , Sendtner M. , Thoenen H.
Published: January 2005

The survival and functional maintenance of spinal motoneurons, both during the period of developmental cell death and in adulthood, have been shown to be dependent on trophic factors. In vitro experiments have previously been used to identify several survival factors for motoneurons, including CNTF, LIF, and members of the neurotrophin, FGF, and IGF gene families. Some of these factors have also been shown to be active in vivo, either on chick motoneurons during embryonic development or on lesioned facial and spinal motoneurons of the newborn rat. Here we demonstrate that lesioned newborn rat facial motoneurons can be rescued by NT‐4/5, IGF‐I and LIF. Furthermore, in contrast to chick motoneurons, the survival of isolated embryonic rat motoneurons can be maintained by the neurotrophins BDNF, NT‐3, and NT‐4/5. IGF‐I and FGF‐5 were also active in this system, each supporting more than 50% of the originally plated neurons. The responsiveness of motoneurons to multiple factors in vitro and in vivo suggests that motoneuron survival and function are regulated by the coordinated actions of members of different gene families. © 1993 Wiley‐Liss, Inc.
Trophic effects of basic fibroblast growth factor (bFGF) on differentiated oligodendroglia: A mechanism for regeneration of the oligodendroglial lineage
Authors: Grinspan J. B. , Stern J. L. , Franceschini B. , Pleasure D.
Published: January 2005

We have investigated the effect of basic fibroblast growth factor (bFGF) on the proliferation and phenotype of differentiated oligodendroglia. Using primary cell cultures enriched in oligodendrocytes but containing few O2A‐oligodendrocyte progenitor cells, we demonstrate that bFGF treatment greatly increases the proportion of 02A cells while decreasing the proportion of galactocerebroside +(GalC+), myelin basic protein +(MBP+) oligodendrocytes, and the steady state levels of MPB mRNA. Complement mediated cell lysis experiments using the A2B5 antibody to deplete existing O2A cells or the R‐Mab antibody to deplete existing oligodendroglia show that bFGF elicits a rapid increse in the number of O2A cells in cultures previously depleted of O2A cells, but does not cause an early incrase in O2A cells in cultures from which oligodendroglia had been removed, indication that the oligodendrocytes are the source of the newly recruited O2A cells. This bFGF‐mediated transition from oligodendrocyte to O2A cells occurs with a time course similar to the bFGF‐induced incrase of the proliferation rate of the GalC+ oligo‐dendrocvtes. Studies with purified, passaged cells of the oligodendroglial lineage show that bFGF augmenta oligodendroglial dedifferntiation and proliferation in chronologically adult oligodendrocytes and in the virtual absence of other cell types. We have thusdemonstrated that mature oligodendrocytes are induced by bFGF to dedifferentiate and proliferate, suggesting a mechanism for regeneration of the oligodendroglial lineage following demyelinating disease. © 1993 Wiley‐Liss, Inc.
Humoral immune recognition of proteolipid protein (PLP)‐specific encephalitogenic epitopes in the SJL/J mouse
Authors: Potter N. T. , Stephens T. S.
Published: January 2005

SJL/J mice were immunized with human PLP as well as encephalitogenic PLP peptides 139–151 and 178–191 and the resulting antibody responses examined for immunochemical specificity employing a panel of 17 synthetic PLP peptide ligands. All animals had demonstrable circulating titers of antibodies early in the humoral immune response to their respective encephalitogens, however, there was no clear qualitative correlation between antibody responses and the induction of EAE. In the majority of PLP immunized animals, determinant‐specific antibody populations, including those against encephalitogenic centers, were not detectable in the presence of an anti‐PLP antibody response. Antiencephalitogenic peptide antibodies were present in both 139–151 and 178–191 immunized animals regardless of clinical/histologic status. Neither group produced cross‐reactive anti PLP antibodies as detected by ELISA. In animals immunized with peptide 139–151, only anti‐139–151 antibody specificities were noted. In contrast, all animals immunized with peptide 178–191 had an antibody population cross‐reactive with three other PLP peptides: 97–110, 209–217, and 215–228. As humoral immune responses can be demonstrated against PLP‐specific encephalitogenic epitopes, the significance of these B cell responses should be considered in the context of their potential role in the development, modulation, and/or potentiation of EAE. © 1994 Wiley‐Liss, Inc.
Posttranslational modification of glycine‐extended substance P by an α‐amidating enzyme in cultured sensory neurons of dorsal root ganglia
Authors: Wong M. , Jeng A. Y.
Published: January 2005

The terminal step in the biosynthesis of substance P is the conversion of its glycine‐extended precursor to the mature, amidated peptide by the α‐amidating enzyme. This posttranslational modification was demonstrated in cultured dissociated sensory neurons of dorsal root ganglia from neonatal rats. An assay was developed to quantitate both substance P and its precursor peptide in these cells. More than 90% of these two peptide was present as mature peptide in uncultured cells. In contrast, after 8 days in culture, about 85% of the peptides was the precursor, which increased 200‐fold, whereas the level of substance P itself tripled during this culturing period. Since α‐amidating enzyme requires ascorbate for activity, this reducing agent was added to the culture medium. Ascorbate induced a dose‐dependent rise in the percentage of amidated peptide, with an apparent Km of 20μM. After 5 days of culturing in the presence of 500 μ ascorbate, substance P increased 8‐fold, constituting 70% of the total. The α‐amidating enzyme also needs copper for activity. Even with 500 μM ascorbate in the culture medium, the copper chelator diethyldithiocarbamate dose‐dependently reduced substance P synthesis by the sensory neurons, with a concomitant increase in its precursor peptide. These results suggest the presence of α‐amidating enzyme in sensory neurons of dorsal root ganglia. It is likely that conversion of other glycine‐extended precursors to their mature peptides in cell cultures would also require ascorbate and copper. © 1994 Wiley‐Liss, Inc.
Glucocorticoid‐induced upregulation of proteolipid protein and myelin‐associated glycoprotein genes in C6 cells
Authors: Zhu W. , Wiggins R. C. , Konat G. W.
Published: January 2005

The effect of dexamethasone on the expression of proteolipid protein (PLP) and myelin‐associated glycoprotein (MAG) genes was investigated in rat C6 glioma cells. The steady state level of the respective mRNA was quantitated by Northern blot analysis. The treatment of cells with dexamethasone transiently upregulated the expression of both genes with peak mRNA levels of approximately 10‐fold over control levels occurring at day 3 for the PLP gene and at day 5 for the MAG gene. The effect was directly related to the drug concentration in the range from 10−9 to 10−5M. Combined exposure of the cells to dexamethasone and retinoic acid featured an additive effect on PLP gene expression, whereas MAG gene expression was depressed below detectability level. The dissimilarity in the response of the genes to dexamethasone and retinoic acid supports the contention that the genes are controlled by different mechanisms. Furthermore, the results indicate that the effects of dexamethasone and retinoic acid on the myelin genes are mediated by different regulatory pathways. © 1994 Wiley‐Liss, Inc.
Characteristics of HIV‐1 gp120 glycoprotein binding to glycolipids
Authors: McAlarney T. , Apostolski S. , Lederman S. , Latov N.
Published: January 2005

We examined the binding of the gp120 envelope glycoprotein (gp120) of the human immunodeficiency virus (HIV‐1) to sulfatide (GalS), galactocerebroside (GalC), and GMI‐ganglioside (GMI). The gp120 glycoprotein bound to GalS but not to GalC or GMI by enzyme‐linked immunosorbent assay (ELISA) and by an immunospot assay on nitrocellulose paper. However, it bound to all three glycolipids by an immunospot assay on thin layer chromotography (TLC) plates. In studies to determine whether GalS could be a receptor for gp120 on the surface of cells, gp120 bound to GalS incorporated into the plasma membrane of lymphoid cells as determined by cytofluorometric analysis and immunofluorescence microscopy. These studies indicate that GalS may function as a receptor for gp120 and HIV‐1. © 1994 Wiley‐Liss, Inc.
Delayed application of aurintricarboxylic acid reduces glutamate‐induced cortical neuronal injury
Authors: Csernansky C. A. , Canzoniero L. M. T. , Sensi S. L. , Yu S. P. , Choi D. W.
Published: January 2005

The non‐specific endonuclease inhibitor, aurintricarboxylic acid (ATA), attenuated glutamate‐induced destruction of cultured cortical neurons. In part, this protective effect likely reflected the ability of ATA to produce a slowly developing block of N‐methyl‐D‐aspartate receptor‐mediated inward whole cell current or increase in intracellular free Ca2+. However, ATA also attenuated a high K+‐induced increase in intracellular free Ca2+ in the presence of D‐aminophosphonovalerate, suggesting that ATA may have a more general effect on Ca2+ homeostasis. In addition, ATA attenuated glutamate neurotoxicity even if added up to 2 hr after completion of glutamate exposure, a time when glutamate antagonists or lipid peroxidation inhibitors are no longer neuroprotective. Involvement of apoptosis in this excitotoxic death is unlikely, as Southern blotting of genomic DNA revealed no evidence of fragmentation, and death was not prevented by inhibitors of RNA or protein synthesis. Most likely, ATA interferes with some key downstream consequences of excitotoxic glutamate receptor overactivation. © 1994 Wiley‐Liss, Inc.
Neural regulation of muscle acetylcholinesterase is exerted on the level of its mRNA
Authors: Črešnar B. , Črne‐Finderle N. , Breskvar K. , Sketelj J.
Published: January 2005

In rat muscles, AChE activity drops rapidly after denervation, and the patterns of AChE molecular forms in slow and fast muscles differ considerably. Both observations imply that muscle AChE is regulated by the motor nerve. In order to obtain a better insight into the underlying mechanism, AChE regulation in rat muscles was examined on the level of its catalytic subunit mRNA using northern blot analysis. The level of two AChE transcripts (2.4 and 3.2 kb) was much higher in the fast sternomastoid (STM) than in the slow soleus muscle, which explains the difference in AChE activity between the two types of muscles. Expression of AChE mRNA in the extrajunctional region of STM muscle is fairly high so that little difference in the level of AChE mRNAs was observed in comparison to the region rich in the neuromuscular junctions. This indicates that very high AChE activity in the neuromuscular junctions is achieved by unique posttranslational modifications and cellular processing of AChE enhancing stability of the junctional in comparison to the extrajunctional AChE. Denervation as well as botulinum toxin evoked paralysis of STM muscle caused rapid decline of AChE transcripts to almost undetectable levels both in the junctional and extrajunctional regions. The low level of AChE mRNA is therefore largely responsible for low AChE activity in denervated rat muscles. It seems that either muscle activity and/or quantal ACh release enhance the level of AChE mRNA in the junctional as well as extrajunctional regions. In rat muscles, extrajunctional mRNA level of the catalytic subunit of AChE is neurally regulated in exact opposite fashion from that of acetylcholine receptor subunits. © 1994 Wiley‐Liss, Inc.
Astrocyte‐derived lipoxygenase product evokes endothelium‐dependent relaxation of the basilar artery
Authors: Murphy S. , Rich G. , Orgren K. I. , Moore S. A. , Faraci F. M.
Published: January 2005

The goal of this study was to examine the possible production of vasoactive factors by astrocytes. We consistently observe that rat astroglial cells in suspension produce marked relaxation when added to precontracted rings of intact (but not endothelium‐denuded) rabbit basilar artery. The ultimate mediator of this relaxation was endothelium‐derived nitric oxide whose synthesis is activated by an as yet unidentified factor(s) produced tonically by astrocytes. The factor is relatively stable, and is not arachidonate, or a product of cyclooxygenase or P450 metabolism. Based upon studies with selective inhibitors, the factor appears to result from 12‐ or 15‐lipoxygenase metabolism, the products of which are known to be vasoactive. In a separate series of experiments, astrocyte‐conditioned medium stimulated the production of citrulline from L‐arginine by nitric oxide synthase in bovine aortic endothelial cells. The possible significance for central nervous system (CNS) pathophysiology of an astrocyte‐derived vasodilator is discussed. © 1994 Wiley‐Liss, Inc.
Subcellular localization of the F5 protein to the neuronal membrane‐associated cytoskeleton
Authors: Arai M. , Cohen J. A.
Published: January 2005

F5 was identified originally as an interleukin‐2‐regulated gene in the murine helper T‐lymphocyte clone L2. Subsequent studies demonstrated high levels of F5 mRNA and protein in mature neurons in adult mouse central and peripheral nervous systems. The F5 protein was present in dendrites and perikarya but not in axons. In the present studies, the intracellular localization of the F5 protein in adult mouse brain was determined by subcellular fractionation and Western blotting. Although the deduced F5 sequence predicts a soluble protein, virtually no F5 immunoreactivity was found in the cytosol. The F5 protein was restricted to the P2 crude mitochondrial and P3 crude microsomal particulate fractions. Within the P2 fraction, F5 protein was enriched in the P2B synaptosomal subfraction. The results of temperature‐dependent phase separation with Triton X‐114 and alkaline extraction with sodium carbonate of the P2 and P3 fractions were consistent with the F5 protein being an extrinsic membrane‐associated protein. Although essentially all of the F5 protein in the P3 fraction was membrane‐associated, a substantial proportion of P2‐associated F5 protein and nearly all of the synaptosomal F5 protein was detergent‐insoluble. Direct isolation and subfractionation of brain cytoskeleton confirmed colocalization of F5 immunoreactivity with the membrane‐associated cytoskeleton and postsynaptic densities. These studies suggest that the F5 protein, which has a large number of potential phosphorylation sites, plays a role in membrane‐cytoskeletal interactions and in dynamic aspects of synaptic structure or function. © 1994 Wiley‐Liss, Inc.
Coexpression of alpha and gamma enolase genes in neurons of adult rat brain
Authors: Keller A. , Bérod A. , Dussaillant M. , Lamandé N. , Gros F. , Lucas M.
Published: January 2005

Enolase (EC 4.2.1.11) is a glycolytic enzyme active as a dimer. In adult brain extracts, three forms, αα, αγ and γγ, have been described, with the αγ hybrid accounting for 30% of total enolase activity (Fletcher et al., Dev Biol 65:462–475, 1978; Lucas et al., Dev Neurosci 10:91–98, 1988). Previous biochemical studies strongly suggest that this hybrid is not generated artefactually during the extraction procedures (Keller et al., J Neurochem 36:1389–1397, 1981; Shimizu et al., BBA 748:278–284, 1983). Immunocytological observations have demonstrated the cell specific localization of the α subunit in astrocytes and of the γ subunit in neurons at the adult stage, but failed to identify a cell type containing both the α and γ subunits necessary for the formation of the αγ hybrid isoform (Ghandour et al., Exp Brain Res 41:271–279, 1981; Vinores et al., J Histochem Cytochem 32:1295–1302, 1984; Iwanaga et al., Arch Histol Cytol [Suppl] 52:13–24, 1989). We sought to approach this question by performing in situ hybridization studies in order to visualize the α and γ mRNAs. In agreement with the immunocytological reports, we observe a specific accumulation of the γ enolase transcripts in neurons and a high accumulation of α enolase transcripts in some glial cells such as the ependymocytes lining the ventricles.

Our observations, following hybridization with 35S labeled oligonucleotide specific probes on adjacent thin sections, demonstrate for the first time that transcription of both α and γ enolase genes occurs in many neurons of different brain regions. These results render highly probable the formation of the αγ hybrid in mature neurons.

Furthermore, we observe a differential expression of the genes encoding the α and γ enolase subunits in various neuronal populations of the brain. The implications of these observations are discussed. © 1994 Wiley‐Liss, Inc.
Fibroblast growth factor‐induced increases in calcium currents in the PC12 pheochromocytoma cell line are tyrosine phosphorylation dependent
Authors: Rane S. G. , Pollock J. D.
Published: January 2005

The PC12 rat pheochromocytoma cell line is widely used to study neuronal differentiation by growth factors. In response to nerve growth factor (NGF) and basic fibroblast growth factor (bFGF), PC12 cells differentiate into sympathetic‐like neurons and become electrically excitable. Using whole cell patch‐clamp recording, with barium as a charge carrier, we looked at the effects of bFGF on calcium channel expression as reflected by changes in barium current amplitudes normalized to cell membrane area. Similar to the effect reported for NGF, we show that 7 day treatment with bFGF increased the barium current approximately 4‐fold. The largest contributor to the increase in barium current with bFGF treatment is a 6‐fold increase in the high threshold voltage activated Ω‐conotoxin sensitive barium current. Smaller increases in current produced by bFGF treatment of PC12 cells are observed for the dihydropyridine sensitive and dihydropyridine/conotoxin insensitive currents. The bFGF‐induced increases in barium currents are dependent on tyrosine phosphorylation, since the effects of bFGF are blocked by genistein, a tyrosine kinase inhibitor. This system will ultimately be useful in understanding the signaling pathways that control calcium channel expression in response to growth factors. © 1994 Wiley‐Liss, Inc.
Expression of the intermediate filament‐associated protein related to β‐amyloid precursor protein is developmentally regulated in cultured cells
Authors: Durham H. D. , Minotti S. , Dooley N. P. , Nalbantoglu J.
Published: January 2005

It was previously reported that a monoclonal antibody to β‐amyloid precursor protein (mab22C11; Boehringer Mannheim, Indianapolis, IN) labels an intermediate filament‐associated protein (βAPP‐IFAP) in cultured human skin fibroblasts (Dooley et al.: J Neurosci Res 33:60–67, 1992). The time course of its expression and association with different classes of intermediate filaments has been assessed in neurons, Schwann cells, and astrocytes in dissociated cultures of murine brain and spinal cord‐dorsal root ganglia; in primary cultures of human muscle; and in the epithelial cell line PtK1. βAPP‐IFAP was expressed in all non‐neuronal cell types examined. Mab22C11 immunoreactivity was minimal or absent following dissociation or subculture, but gradually increased with time. In fibroblasts, myoblasts, and epithelial cells, the distribution eventually resembled that of vimentin. With the exception of glial fibrillary acidic protein (GFAP), βAPP‐IFAP was not associated with the intermediate filament proteins characteristically found in differentiated cells, i.e., desmin, the cytokeratins, and neurofilament proteins. No labeling of neurons by mab22C11 was observed at any stage of in vitro maturation. In sections of Alzheimer's brain, the antibody labeled a subpopulation of reactive astrocytes. It is suggested that βAPP‐IFAP may be the product of a member of the βAPP multigene family expressed developmentally in non‐neuronal cells. © 1994 Wiley‐Liss, Inc.
Human fetal neurons in culture: Intercellular communication and voltage‐ and ligand‐gated responses
Authors: Chiu F.‐C. , Rozental R. , Bassallo C. , Lyman W. D. , Spray D. C.
Published: January 2005

We established high density primary cultures of neural cells from dissociated second trimester human fetal brains using a novel spin seeding method. Under our culture conditions, the majority of the cells exhibited neuronal phenotypes as evidenced by morphological criteria, immunoreactivity to the 66 kDa neurofilament protein and expression of TTX‐sensitive Na+ channels and cell excitability. These cultures were enriched in glutamic acid decarboxylase (GAD), the synthetic enzyme of the neurotransmitter GABA, and responded with Ca2+ influx to acute application of glutamate. Interestingly, the human fetal neurons in culture did not express either dopaminergic or cholinergic phenotypes. In addition, the population of neurons obtained express a high incidence of gap junction‐mediated intercellular communication. These studies provide evidence that functional neuronal properties arise early during prenatal development in humans and offer the potential to evaluate pharmacological agents on primary human neurons. © 1994 Wiley‐Liss, Inc.