bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitotic potential of adult rat oligodendrocytes in culture
Authors: Vick R. S. , DeVries G. H.
Published: January 2005

The proliferation of adult oligodendrocytes (OLGs) was examined in response to membrane bound and soluble mitogens. OLGs were isolated according to Vick, et al., J Neurosci Res 25:524–534, 1990, and co‐cultured with dorsal root ganglia (DRGs). Less than 5% of the total cells incorporated a 48 hr pulse of 3H thymidine during the first 4 days of co‐culture. From day 4 to day 6 there was a dramatic increase in proliferation which reached a plateau at 40% and gradually decreased to 25% from days 10 to 20 of co‐culture. Axolemma‐enriched fractions (AEF) were weak mitogens for OLGs (less than 5% proliferation after 7 days of stimulation), however, heparin extracts of AEF were five‐fold more mitogenic than the AEF from which they were derived. Basic and acidic fibroblast growth factor were effective mitogens for the adult OLGs (labelling indices of 28% and 12%, respectively) provided that the cells were treated for 7 days with the growth factor and that the cells had been in culture for at least 14 days. Other soluble growth factors (IL‐2 and PDGF) gave no mitotic response. We conclude that adult OLGs are mitotically responsive to mitogens provided that (1) the adult OLG has been cultured for sufficient time (14 days, acidic or basic fibroblast growth factor); (2) the axonal mitogen is allowed to interact with the OLG for a sufficient time (neuritic mitogen); and (3) the axonal mitogen is presented to the OLG in an activated form (AEF‐heparin extract). © 1992 Wiley‐Liss, Inc.
Identification of mouse type‐2‐like astrocytes: Demonstration of glutamate and GABA transmitter activated responses
Authors: Magoski N. S. , Walz W. , Juurlink B. H. J.
Published: January 2005

We have identified mouse type‐2‐like astrocytes and examined some of their electrophysiological properties. Cultures were prepared from P4 mouse neopallia. We demonstrate that mouse type‐2‐like astrocytes can be identified using the following criteria: presence of glial fibrillary acidic protein (GFAP), presence of chondroitin sulfate polysaccharide, and presence of γ‐aminobutyric acid (GABA). A2B5‐binding is not a sufficient criterion to identify O2A lineage cells in mouse neopallial glial cultures since the monoclonal antibody A2B5 binds not only to O2A lineage cells but also to a subpopulation of large, flat type‐1‐like astrocytes. Mouse type‐2‐like astrocytes have resting membrane potentials of −76.2 ± 2.1 mV—i.e., similar to that of mouse type‐1‐like astrocytes. The input resistance of 44.2 ± 0.5 MΩ is an order of magnitude greater than that of type‐1‐like astrocytes suggesting the type‐2‐like astrocytes are not extensively electrically coupled either to each other or to type‐1‐like astrocytes. Glutamate application caused an 8.8 ± 1.7 mV depolarization of type‐2‐like astrocytes. Application of glutamate to barium treated astrocytes caused a fast depolarization with a peak amplitude of 21.4 ± 1.8 mV; the cells repolarized from this peak by about 10 mV and upon removal of glutamate returned to its pre‐glutamate value. Application of GABA caused a transient depolarization of 14.0 ± 1.7 mV. The presence of barium resulted in a steady‐state GABA‐induced depolarization of 10.3 ± 2.0 mV. Neither SITS nor β‐alanine interfered with the amplitude of the glutamate and GABA responses. © 1992 Wiley‐Liss, Inc.
Cloning of a novel TGF‐β related cytokine, the vgr, from rat brain: Cloning of and comparison to homologous human cytokines
Authors: Sauermann U. , Meyermann R. , Schluesener H. J.
Published: January 2005

Here we describe cloning of a TGF‐β related cytokine from a rat brain cDNA library. This novel cytokine, the vgr (vegetal related), is homologous to the vegetal (Vg1) gene of Xenopus (DL Weeks and DA Melton, Cell, 51:861–867, 1987).

In rat brain mRNA a single 3.5 kb RNA could be detected by Northern blot analysis. Thus, this new cytokine is constitutively expressed in the central nervous system.

A monoclonal antibody reactive with a synthetic peptide of vgr revealed a faint vgr‐like immunoreactivity throughout the CNS, with more pronounced staining of hippocampal neurons, ependymal cells, cells of the choroid plexus, and hypophysis.

Using the rat cDNA, two homologous human cytokine cDNAs encoding the human vgr and op‐1 were cloned. © 1992 Wiley‐Liss, Inc.
β/A4 domain of APP: Antigenic differences between cell lines
Authors: Wolozin B. , Zheng B. , Loren D. , Lesch K. P. , Lebovics R. S. , Lieberburg I. , Sunderland T.
Published: January 2005

The expression of amyloid precursor protein (APP) in olfactory neuroblasts has been examined with a panel of antibodies directed against varied regions of the APP molecule. The pattern of reactivity was compared to that in the transformed human glial cell line SVG, human cortical brain tissue, and in kidney epithelial 293 cells containing stably transfected and overexpressed human APP751. Antibodies directed against the C‐terminus and extracellular domains of amyloid precursor protein (APP) react more strongly on immunoblot with transfected 293 cells and brain tissue than with olfactory neuroblasts (ON) or SVG cells. Antibodies directed against the β/A4 region of APP show a contrasting pattern of reactivity, yielding greater reactivity with ON and SVG cells than with transfected 293 cells or brain tissue. Analysis of the APP transcripts using polymerase chain reaction indicates that ON and SVG both make predominantly APP770 and 751, as does the transfected 293 cell line. In the absence of any differences in APP transcripts among the cell lines, the difference in availability of the β/A4 region appears likely to be due to posttranslational modification. These data therfore indicate that processing of APP varies among cell lines and thus may vary from tissue to tissue. © Wiley‐Liss, Inc.
Growth control of cultured microglia
Authors: Ganter S. , Northoff H. , Männel D. , Gebicke‐Härter P. J.
Published: January 2005

Microglia, the resident macrophages of the brain, typically react to injuries or chronic diseases with proliferation and expression of differentiated features, such as production of cytokines associated with inflammatory events. Regulation and control of microglial cytokine expression, therefore, is a major focus of scientific interest. It has been shown that GMCSF and Il‐3 are potent mitogens for microglia. Moreover, Il‐3 and other cytokines are products of microglia. It is shown here that interleukin‐1 (Il‐1) as well as tumor necrosis factor (TNFα) increased microglial proliferation in mixed astrocyte‐microglial cultures but had no mitogenic effects on isolated microglia. © Wiley‐Liss, Inc.

Lipopolysaccharide (LPS), the bacterial endotoxin, irreversibly inhibited microglial cell division in both mixed astrocyte‐microglial cultures and in isolated microglial cultures. By contrast, the corticosteroids hydrocortisone and aldosterone and the synthetic glucocorticoid dexamethasone reversibly inhibited microglial proliferation. They also antagonized the stimulatory effects of Il‐3 and granulocyte macrophage colony‐stimulating factor (GMCSF). Estradiol and progesterone had no significant effects on mixed cultures but inhibited microglial proliferation in isolated cultures. Conditioned media from mixed cultures, isolated cultures, from the WEHI‐2B cell line, or from fresh (serum‐supplemented) media stimulated microglial proliferation to various extents.

In summary, cytokine‐mediated microglial proliferation can be down‐regulated by a variety of steroid hormones. Along with their unimpaired access to brain cells in general, corticosteroids likely maintain an inhibitory tonus on microglial proliferation. It is hypothesized that this inhibition is overcome locally and temporally in brain injury and repair. © Wiley‐Liss, Inc.
Isolation of activated adult schwann cells and a spontaneously immortal schwann cell clone
Authors: Bolin L. M. , Iismaa T. P. , Shooter E. M.
Published: January 2005

Successful mammalian peripheral nerve regeneration is dependent on activated Schwann cells. Schwann cells facilitate neuronal regrowth through the production of tropic cell membrane molecules, neurotrophins, and extracellular matrix components. To better understand Schwann cell function in the regenerating nerve, we have designed a method of isolating proliferating adult Schwann cells from the injured rat sciatic nerve. Relying on the mitotic signal that is present after a crush injury, we can obtain sufficient numbers of dividing Schwann cells within one week of initial culture. A spontaneously immortal Schwann cell clone (iSC) was observed in and isolated from one of these primary cultures. These cells were transformed at a time of maximal Schwann cell activation in response to injury. Both the primary Schwann cells and the iSC have been characterized as Schwann cells by morphology, immunohistochemistry and gene expression. © Wiley‐Liss, Inc.
Proliferation and differentiation of O4+ oligodendrocytes in postnatal rat cerebellum: Analysis in unfixed tissue slices using anti‐glycolipid antibodies
Authors: Warrington A. E. , Pfeiffer S. E.
Published: January 2005

We report the study of the in vivo morphology, differentiation, and proliferation of oligodendrocytes (OLs) and their progenitors identified by the antiglycolipid antibodies O4, R‐mAb, and O1 in postnatal rat cerebellum, using a novel immunocytochemical staining protocol which allows the analysis of the expression of OL‐specific glycolipids in live, unfixed brain slices. An analysis of the individual cells identified in double label immunocytochemistry indicated that the order of antigen expression in OLs during in vivo development is, first, antigens recognized by O4, second, antigens recognized R‐mAb, and third, antigens recognized by O1. This order of antigen expression is correlated with increasing morphological complexity and is a pattern mimicked in many culture systems. In vivo O4 identified 3 distinct stages of the OL lineage: (1) morphologically simple proligoden‐drocyte antigen+ (POA+) R‐mAb− blast cells localized at the leading edge of myelinogenesis; (2) morphologically more complex R‐mAb+O1− cells; and (3) actively myelinating O1+ [i.e., galactocerebroside+ (GalC)] OLs residing within the white matter. Only the POA+R‐mAb− cells incorporated BrdU in animals that were prelabeled 3 hr before immunocyto‐chemistry. We have demonstrated in vivo the subdivision of pre‐GalC+ OL progenitors into shorter, biologically noteworthy, stages of maturation. A spatial comparison of the cell populations identified by O4, R‐mAb, and O1 demonstrated a progressive wave of OL maturation from the base of the cerebellum toward the folia. The data are consistent with the hypothesis that multiprocessed O4+GalC− progenitors are the most mature stage of the OL lineage with significant proliferative capacity and the first postmigratory stage in normal development. © Wiley‐Liss, Inc.
3H‐D‐aspartate release from cerebellar granule neurons is differentially regulated by glutamate‐ and K+‐stimulation
Authors: Belhage B. , Rehder V. , Hansen G. H. , Kater S. B. , Schousboe A.
Published: January 2005

Neurotransmitter release in response to either 55 mM K+ or 25 μM glutamate as well as its dependency on Ca2+ from different sources was compared in cultured glutamatergic cerebellar granule cells from rat brain. The intracellular Ca+ concentration was monitored at the single cell level in neurites as well as cell bodies employing the fluorescent Ca2+ indicator fura‐2. Transmitter release was assayed using 3H‐D‐aspartate to label the exogenously accessable glutamate pools, which in these neurons is believed to also include the transmitter pool. In an attempt to distinguish whether transmitter release was dependent on an intact cytoskeleton or not, the colchicine‐like drug Nocodazole, which also blocks transport of vesicles, was used. K+‐stimulated transmitter release consisted for the major part (around 70%) of a Ca2+ dependent, Nocodazole sensitive release component and this K+‐induced release appeared to be almost exclusively dependent on N‐type Ca2+ channels. In contrast, 50% of the glutamate‐induced Ca2+‐dependent release was triggered by Ca2+ from a Dantrolene sensitive intracellular Ca2+ pool. Since these neurons undergo a pronounced maturational change in which neurotransmitter vesicles become increasingly prominent, the Ca2+ responses and transmitter release evoked by the two different stimuli were investigated as a function of the culture period. K+ and glutamate were found to increase intracellular [Ca2+] differentlly. In 1‐day‐old cultures K+ elicited a small albeit significant increase in [Ca2+]i while glutamate was completely without effect. In 7‐day‐old neurons both agents induced a large increase in [Ca2+]i. However, K+ induced equal Ca2+ responses in cell bodies and neurites, whereas glutamate induced an almost 4 times lower Ca2+ response in neurites compared to cell bodies.

These findings suggest that K+ and glutamate may recruit transmitter and release‐triggering‐Ca2+ from different pools. This could be of pivotal importance for the understanding of glutamate homeostasis during physiological as well as pathological conditions. © 1992 Wiley‐Liss, Inc.
The role of microtubule‐associated protein 2 (MAP‐2) in neuronal growth, plasticity, and degeneration
Authors: Johnson G. V. W. , Jope R. S.
Published: January 2005

Microtubule associated protein 2 (MAP‐2) historically has been perceived primarily as a static, structural protein, necessary along with other cytoskeletal proteins to maintain neuroarchitecture but somewhat removed from the “mainstream” of neuronal response mechanisms. Quite to the contrary, MAP‐2 is exquisitely sensitive to many inputs and recent investigations have revealed dynamic functions for MAP‐2 in the growth, differentiation, and plasticity of neurons, with key roles in neuronal responses to growth factors, neurotransmitters, synaptic activity, and neurotoxins. These discoveries indicate that modification and rearrangement of MAP‐2 is an early obligatory step in many processes which modify neuronal function. © 1992 Wiley‐Liss, Inc.
The gp120 glycoprotein of HIV‐1 binds to sulfatide and to the myelin associated glycoprotein
Authors: Van Berg L. H. Den , Sadiq S. A. , Lederman S. , Latov N.
Published: January 2005

We investigated the binding of the gp120 glycoprotein of the human immunodeficiency virus (HIV‐1) to neural glycolipids and glycoproteins by ELISA. The gp120 protein bound to sulfatide (GalS), a sulfated glycolipid autoantigen implicated in sensory neuritis, and to the myelin associated glycoprotein (MAG), an autoantigen in demyelinating neuropathy. Binding of gp120 to MAG was inhibited by the HNK‐1 antibody, which recognizes a sulfated glucuronic acid epitope, suggesting that the interaction involves carbohydrate determinants. Sulfatide and MAG are potential receptors for gp120 in peripheral nerve and may have a role in the neuropathy associated with HIV‐1 infection. © 1992 Wiley‐Liss, Inc.
Developmental expression of neuronal calmodulin mRNA species in the rat brain analyzed by in situ hybridization
Authors: Ni B. , Landry C. F. , Brown I. R.
Published: January 2005

The temporal and spatial distribution of calmodulin mRNAs which are preferentially expressed in neurons was determined during postnatal development of rat central nervous system. Expression of these mRNAs was strongly detected in the developing neocortex, hippocampus, and cerebellum. Differences in the pattern of expression of a 1.8 and 4.0 kb neuronal calmodulin mRNA species were identified in the developing cerebellum. Expression of the smaller mRNA appeared to correlate with proliferating and developing cerebellar granule neurons while the larger mRNA was present in the mature granule neuron population. A transient elevation in the neuronal calmodulin mRNA species was observed in the suprior and inferior colliculus and in the thalamus at postnatal days 5 and 10. © 1992 Wiley‐Liss, Inc.
In search of the central respiratory neurons: I. Dissociated cell cultures of respiratory areas from the upper medulla
Authors: Fitzgerald S. C. , Willis M. A. , Yu C. , Rigatto H.
Published: January 2005

Dissociated cells from the areas of the nucleus ambiguus and the nucleus tractus solitarius obtained by tissue punch or block dissection from coronal slices of the medulla at the level of the obex were cultured from fetal rats at 18 to 21 days gestation. The dissociated neurons were plated either directly in vitrogen‐coated 35 mm tissue culture dishes or in such dishes which had been seeded with subcultures of cortex‐ or medulla‐derived astrocytes. After the astrocytes reached confluency and were treated with an antimitotic agent, dissociated nucleus ambiguus or nucleus tractus solitarius was plated at 0.5–1.0 × 106 cells per dish. Neurons grew well on monolayers of medullary or cortical astrocytes, but survived poorly on vitrogen‐coated dishes without a cellular substrate. Rat medulla was preferred as the source of astrocytes. Tissue dissociation with papain rather than trypsin produced less cellular debris, and the neuronal yield from the tissue was higher. The neuronal population was heterogenous in morphology including small and large bipolar, pyramidal, and multipolar cells. Neurons sensitive to CO2 and/or low pH (Rigatto et al., J Neurosci Res 33:590–597, 1992) did not appear to have any definitive morphologic characteristics, but most were multipolar. These neurons stained well with antibodies to neuron‐specific enolase and Fragment C of tetanus toxin, but not to choline acetyltransferase (ChAT). These findings suggest that neurons possibly responsible for the central regulation of respiration can be maintained for several weeks in dissociated cell culture, providing a system for neurotransmitter, electrophysiological, and morphological studies. © 1992 Wiley‐Liss, Inc.
Elevation of extracellular potassium facilitates the induction of hippocampal long‐term potentiation
Authors: Ballyk B. A. , Goh J. W.
Published: January 2005

Hippocampal long‐term potentiation (LTP) is widely believed to be a cellular substrate for learning and memory. A likely physiological stimulus for initiating LTP is repetitive neuronal activity, which also results in K+ accumulation extracellularly. Therefore, the involvement of elevated extracellular K+ concentrations in the induction of LTP of the stratum radiatum‐CA1 neuronal synapse was investigated in the hippocampal slice preparation. Increasing the K+ content in extracellular perfusing medium from 3.1 to 15 mM resulted in facilitation of LTP induction in weak excitatory postsynaptic potentials (EPSPs). Since changes that occur to generate LTP are thought to be localized to synaptic regions, it would be relevant to selectively increase synaptic K+ levels. To this end, the following experiments were conducted: (i) baclofen, a GABAB receptor agonist which, in addition to having a disinhibitory presynaptic action, activates a K+ conductance in CA1 neuronal dendrites, was applied to the slice; (ii) K+ was directly applied by iontophoresis. At a concentration of 5 μM baclofen, as well as with K+ iontophoresis (200–300 nA), LTP of weak EPSPs was facilitated. The present data suggest that an increase in synaptic K+ levels can fulfill the condition of cooperativity for LTP induction, raising the possibility that an elevation of this monovalent ion plays a physiological role in triggering LTP. © 1992 Wiley‐Liss, Inc.
Haloperidol prevents induction of the hsp70 heat shock gene in neurons injured by phencyclidine (PCP), MK801, and ketamine
Authors: Sharp F. R. , Butman M. , Wang S. , Koistinaho J. , Graham S. H. , Sagar S. M. , Noble L. , Berger P. , Longo F. M.
Published: January 2005

The non‐competitive NMDA receptor antagonists, PCP (phencyclidine), MK801, and ketamine produce psychosis in humans and abnormal vacuoles in posterior cingulate and retrosplenial rat cortical neurons. We show that PCP (≥5 mg/kg), MK801 (≥0.1 mg/kg), and ketamine (>20 mg/kg) induce hsp70 mRNA and HSP70 heat shock protein in these vacuolated, injured neurons, and PCP also induces hsp70 in injured neocortical, piriform, and amygdala neurons. The PCP, MK801, and ketamine drug induced injury occurs in 30 day and older rats, but not in 0–20 day old rats, and is prevented by prior administration of the antipsychotic drugs haloperidol and rimcazole. Since haloperidol and rimcazole block dopamine and sigma receptors, and since M1 muscarinic cholinergic receptor antagonists also prevent the injury produced by PCP, MK801, and ketamine, future studies will be needed to determine whether dopamine, sigma, M1, or other receptors mediate the injury. © 1992 Wiley‐Liss, Inc.
Neurotoxicity of HIV coat protein gp120, NMDA receptors, and protein kinase C: A study with rat cerebellar granule cell cultures
Authors: Savio T. , Levi G.
Published: January 2005

The neuronal loss observed in AIDS patients may be partly due to the neurotoxicity of HIV coat protein gp120, whose mechanism of action has been suggested to involve an interaction with voltage‐dependent Ca2+ channels and NMDA receptors (Lipton, Trends Neurosci 15:75–79, 1992). In the present investigation we analyzed the acute neurotoxicity of gp120 on a purified neuronal population (rat cerebellar granule cell cultures) amply used for studies on glutamate toxicity. Cultures of 7–8 days were exposed for 15 min to a buffered Locke's solution containing the substances under study, washed, and cultured for another 24 hr in their original medium. The cells were stained with the nuclear dyes propidium iodide (for dead cells) and Hoechst 33258 (for total cells) and counted. Average cell death in controls was 8%. gp120 (1 pM‐10 nM) caused an increase of cell death of about 80%. The effect was totally antagonized by NMDA antagonists (1 mM APV and 10 μM MK‐801), by 1 üM nifedipine, and by anti‐gp120 antibodies. At a concentration of 100 μM glutamate caused an average 130% increase of cell death, which was totally antagonized by APV. The effect of gp120 or glutamate did not appear to be mediated by the secretion of neurotoxins by nonneuronal cells present in a low proportion in the cultures nor to be due to the inactivation of (or competition with) neurotrophic factors present in the medium. The simultaneous administration of gp120 and glutamate (in various combinations of concentrations) had an effect that was less than additive. The combined effect of the two substances was partially antagonized by anti‐gp120 antibodies and totally prevented by the combined action of anti‐gp120 antibodies and NMDA antagonists. Surprisingly, however, NMDA antagonists prevented only partially the neuronal death elicited by gp120 + glutamate. In order to determine whether gp120 affected one of the events potentially involved in NMDA‐mediated neurotoxicity [protein kinase C (PKC) translocation to the plasma membrane], we studied the effect of gp120 and glutamate on the binding of 4β‐[3H]phorbol‐12,13‐dibutyrate, a specific ligand for membrane PKC. While glutamate caused a dose‐dependent, APV‐sensitive increase in binding, gp120 determined a decrease in basal binding, suggesting that its mechanism of action does not involve PKC translocation. © 1993 Wiley‐Liss, Inc.
Regulation of NCAM by growth factors in serum‐free myotube cultures
Authors: Lyles Joan M. , Amin W. , Bock E. , Weill C. L.
Published: January 2005

Regulation of the neural cell adhesion molecule (NCAM) was examined in primary cultures of chick skeletal muscle grown in serum‐free defined medium. Relative levels of NCAM (per μg protein) increased 20–30% in myotubes grown on Matrigel, a reconstituted basement membrane preparation, compared to those grown on collagen; total NCAM levels on Matrigel were increased 40–55% due to the additional increase in total protein. A dose dependent increase in relative NCAM levels in myotubes grown on Matrigel in defined medium was observed with the addition of adsorbed horse serum, while relative NCAM levels in myotubes grown on collagen were unaffected by altering the serum concentration. Thus, extracellular matrix molecules and soluble factors exert trophic effects on myotube NCAM expression. Similar developmental changes in the expression of the different molecular size forms of NCAM occurred in myotubes grown on collagen and Matrigel: levels of 150K and 135K Mr forms decreased during development, while 125K remained prominent in older myotubes. Relative NCAM levels were specifically enhanced 11–26% by several factors: nerve growth factor, thyroxine, insulin‐like growth factor II, dibutyryl cyclic AMP, veratridine (a sodium ion channel agonist), and nisoldipine (a calcium ion channel agonist). Total protein and overall myotube development in serum‐free cultures were enhanced by fetuin, insulin‐like growth factor II, acidic fibroblast growth factor, calcitonin gene‐related peptide, dibutyryl cyclic AMP, and veratridine. Thus, changes in extracellular matrix, intracellular calcium, and sodium ions, as well as extracellular trophic factors, such as nerve growth factor, thyroxine, and insulin‐like growth factor II, may regulate muscle NCAM expression during embryonic development. © 1993 Wiley‐Liss, Inc.
Cyclic AMP has a differentiative effect on an immortalized oligodendrocyte cell line
Authors: Jensen N. A. , Smith G. M. , Garvey J. S. , Shine H. D. , Hood L.
Published: January 2005

We investigated the effects of increasing the concentration of intracellular cyclic adenosine monophosphate (cAMP) on genes associated with oligodendrocyte differentiation in an immortalized glial cell line, 6E12, derived from the spinal cord of an MBP‐SV40 large T‐antigen transgenic mouse. Raising intracellular levels of cAMP induced expression of oligodendrocyte differentiation antigens recognized by O4 and anti‐galactocerebroside antibodies, up‐regulated expression of the proteolipid protein (PLP) gene, and down‐regulated glial fibrillary acidic protein (GFAP) expression. There was no treatment effect on myelinassociated glycoprotein (MAG) expression. These phenotypic changes are consistent with oligodendrocyte differentiation. Treatment of 6E12 cells with dibutyryl cyclic AMP (DBC) down‐regulated myelin basic protein (MBP) gene expression, perhaps, because it also up‐regulated expression of a putative MBP repressor SCIP/Tst‐1. Moreover, the 6E12 cells expressed high levels of MBP mRNA but no MBP translation products were detected in the presence or absence of DBC. This immortalized glial cell line is proposed as a CNS model for cAMP‐modulated myelin gene expression and for post‐transcriptional regulation of MBP. © 1993 Wiley‐Liss, Inc.
38‐Amino acid form of pituitary adenylate cyclase activating peptide induces process outgrowth in human neuroblastoma cells
Authors: Deutsch P. J. , Schadlow V. C. , Barzilai N.
Published: January 2005

Permanent cell lines from human neuroblastoma, a sympathoadrenal malignancy, are known to exhibit a more neuronal phenotype characterized by outgrowth of long processes in response to multiple second messenger analogs. In this report we demonstrate that the 38‐amino acid form of a peptide homologous to vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), as well as the 27‐amino acid form of PACAP, induce NB‐OK human neuroblastoma cells to extrude cellular processes within 5 hr of treatment with either peptide at 10−8 M. Treatment of NB‐OK cells with PACAP38 or PACAP27 at 10−8 M for 1 hr also elevates cAMP content greater than 100‐fold and inositol lipid turnover 11‐ to 12‐fold. VIP acutely induces process outgrowth and elevates intracellular second messenger levels in NB‐OK cells only at higher concentrations, 10−6 M or greater. In contrast to the equipotency of PACAP27 and PACAP38 in stimulating the outgrowth of processes observed after 5 hr of treatment, PACAP38 is much more potent than PACAP27 when NB‐OK cells are scored for process outgrowth after 72 hr of treatment. Correlating with the extended time course over which morphologic changes are seen with PACAP38, cAMP levels remain elevated for a more prolonged time span during treatment with PACAP38 than PACAP27. After 72 hr of treatment with PACAP38 versus treatment with PACAP27, cAMP levels are elevated 10‐fold versus 3‐fold, respectively. PACAP38 at 10−8 M also induces process outgrowth in two additional human neuroblastoma lines tested, SMS‐KAN and LA‐N‐1, whereas PACAP27 and VIP at the same concentration are less effective. The effects of PACAP38 on neuroblastoma cells may be a model for neurotrophic activities of PACAP38 via G protein‐linked intracellular messengers in human sympathoadrenal cells. © 1993 Wiley‐Liss, Inc.
Pentylenetetrazol seizures increase pro‐nerve growth factor‐like immunoreactivity in the reticular thalamic nucleus and nerve growth factor mRNA in the dentate gyrus
Authors: Humpel C. , Ebendal T. , Cao Y. , Olson L.
Published: January 2005

Neurotrophins may have a neuroprotective role and are probably involved in the control of axonal sprouting and synaptic plasticity. An antibody raised against a pro‐sequence of nerve growth factor (NGF) was tested. In control undisturbed rats, a strong immunoreactivity was detected in scattered cells in and around the pyramidal and granule cell layer of the hippocampus and a moderate labeling was found in the reticular thalamic nucleus. In situ hybridization showed specific expression of NGF mRNA in a similar population of scattered cells in the hippocampal formation but not in the reticular thalamic nucleus. Acute epileptic seizures, induced by a convulsive dose of 50 mg/kg pentylenetetrazol (PTZ), strongly in creased NGF mRNA in neurons of the granular layer of the dentate gyrus 3 hr but not 6 hr after the injection. No change in pro‐NGF‐like immunoreactivity was observed in the hippocampus or reticular thalamic nucleus after acute seizures. Chemical kindling was induced by daily injections of subconvulsive doses (30 mg/kg) of PTZ for 4 weeks. This treatment significantly increased pro‐NGF‐like immunoreactivity in the reticular thalamic nucleus but did not affect NGF mRNA. These data strengthen a role for the reticular thalamic nucleus and NGF in PTZ kindling. © 1993 Wiley‐Liss, Inc.
Localization of trkB mRNA in postnatal brain development
Authors: Masana Y. , Wanaka A. , Kato H. , Asai T. , Tohyama M.
Published: January 2005

We investigated the localization of trkB mRNA, which encodes a putative component of high‐affinity brain‐derived neurotrophic factor (BDNF) or the neurotrophin‐3 (NT‐3) receptor, in the postnatal rat brain by in situ hybridization histochemistry. At birth, trkB mRNA was strongly expressed in various regions with the thalamus and cerebral cortex showing the strongest expression. As the rat grows, expression generally persisted or declined in most regions with the exception of the hippocampus where trkB mRNA expression increased during postnatal development. In the adult brain, trkB mRNA was detected in the olfactory system, cerebral cortex, hippocampal formation, amygdala, and cerebellar cortex. These findings, together with the developmental profiles of BDNF and NT‐3 mRNA expressions, suggest that trkB product (gp145trkB) mainly transduces NT‐3 signals early in the postnatal period, and BDNF signals later in the period. © 1993 Wiley‐Liss, Inc.
The protein phosphatase inhibitor okadaic acid increases axonal neurofilaments and neurite caliber, and decreases axonal microtubules in NB2a/d1 Cells
Authors: Shea T. B. , Paskevich P. A. , Beermann M. L.
Published: January 2005

When cells were treated with dbcAMP for 3 days to induce the outgrowth of axonal neurites, the addition of the phosphatase inhibitor okadaic acid (0A; 5 nM) for the last 24 hr markedly increased neurofilament subunit immunoreactivity including phosphate‐dependent NF‐H epitopes in axonal neurites, increased axonal neurite caliber by approximately 30 %, but did not increase neurite contour length. Ultrastructural analysis demonstrated a >2‐fold increase in neurofilaments and indicated that neurofilaments were phosphorylated to a similar extent in the presence and absence of OA. Vimentin immunoreactivity, which undergoes down‐regulation during dbcAMP‐mediated differentiation, was not increased by OA. OA did not induce the precocious appearance of delayed phosphate‐dependent neurofilament epitopes suggesting that it did not induce the activation of additional neurofilament kinases. NF‐H subunits from cytoskeletons of OA‐treated cells were less susceptible to degradation by an endogenous calcium‐dependent protease, providing a possible mechanism for neuro filament accumulation during OA treatment. By contrast, OA decreased axonal neurite microtubules, and eliminated stabilized (acetylated) axonal microtubules. OA treatment at earlier times prevented and reversed neurite outgrowth. Despite increased deposition of phosphorylated neurofilaments, OA did not hasten the development of colchicine resistance to neurites, suggesting that stabilization of the axonal cytoskeletal lattice requires neurofilament‐microtubule interaction. © 1993 Wiley‐Liss, Inc.
Immunoreactivity of PMP‐22, P0, and other 19 to 28 kDa glycoprotens in peripheral nerve myelin of mammals and fish with HNK1 and related antibodies
Authors: Hammer J. A. , O'Shannessy D. J. , De Leon M. , Gould R. , Zand D. , Daune G. , Quarles R. H.
Published: January 2005

Mammalian peripheral nervous system (PNS) myelin contains several glycoproteins with molecular weights of 19 to 28 kDa, including the major 28 kDa P0 glycoprotein and a recently cloned protein called PMP22. Some glycoproteins in this Mr range in humans, cats and some other mammals react with HNK1, a mouse monoclonal antibody that identifies a carbohydrate epitope shared between the immune system and a number of adhesion proteins in the nervous system. A variety of antibodies to PO, PMP‐22, and the carbohydrate determinants reacting with HNK1 were used to characterize immunochemically these 19 to 28 kDa glycoproteins of cat PNS myelin. The HNK1‐reactive components include P0 and two slightly smaller 23 to 26 kDa proteins that are immunologically related to PO. However, HNK1 reacts most strongly with a lower molecular weight glycoprotein that does not react with the antibodies to P0 and was identified as PMP‐22. Since the carbohydrate structure reacting with HNK1 is generally expressed on adhesion molecules, this result suggests that PMP‐22 may function in cell‐cell or membranemembrane interactions. Furthermore, the related human anti‐MAG monoclonal lgM antibodies from patients with neuropathy also react strongly with PMP‐22, suggesting that it may be a target antigen in the pathogenesis of this disease. Purified PNS and CNS myelin from bony fish (toadfish and trout) were also shown to contain major glycoproteins, in the same 19 to 28 kDa Mr range, that react very strongly with HNK1. It is known that fish myelin has major proteins of this size that are immunologically and structurally related to mammalian P0, and it is demonstrated here that one of the strongly HNK1‐positive proteins reacted well with an antiserum raised to bovine P0. The presence of high levels of the adhesion‐related HNK1 epitope on these major myelin proteins of fish suggests that this carbohydrate structure may have played a role in the molecular evolution of myelin. © 1993 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
NTera 2 Cells: A human cell line which displays characteristics expected of a human committed neuronal progenitor cell
Authors: Pleasure S. J. , Lee V. M.‐Y.
Published: January 2005

We have identified a human cell line with a phenotype resembling committed CNS neuronal precursor cells. NTera 2/cl.D1 (NT2/D1) cells expressed nestin and vimentin, intermediate filament (IF) proteins expressed in neuroepithelial precursor cells, as well as MAP1b, a microtubule‐associated protein (MAP) expressed in human neuroepithelium. NT2/D1 cells also expressed the cell adhesion molecules NCAM and N‐cadherin which are thought to be important in cellcell interactions within the neuroepithelium. These NT2/D1 cells also expressed small amounts of NF‐L, α‐internexin, NF‐M, and MAP2c, indicating that they are committed to a neuronal fate. Previous studies have shown that, following RA treatment, a proportion of NT2/D1 cells terminally differentiate into neurons and that this occurs via an asymmetric stem cell mode of differentiation. In light of the identification of the neuroepithelial phenotype of NT2/D1 cells we decided to examine more closely the relationship of in vitro neurogenesis in NT2/D1 cells, during RA treatment to that of neurons in vivo. Three days after RA treatment, islands of NT2/D1 cells showed increased expression of neurofilament proteins and increased phosphorylation of NF‐M. By 10–14 days, these cells began to resemble neurons morphologically, i.e., with rounded cell bodies and processes. These neuronal cells were clustered into clumps which rested on top of a layer of progenitor cells. In this upper layer, the neurons began to express MAP2b and tau and extinguished their expression of mestin. Recently, we developed a method for obtaining pure cultures of neurons from RA treated NT2/D1 cells. The phenotype of these postmitotic neurons is clearly dissociated from that of the untreated NT2/D1 cells. Given the data obtained in this study and the characterization of the neurons derived from NT2/D1 cells, we propose that NT2/D1 cells are a committed human neuronal precursor cell line which retains some stem cell characteristics and is capable only of terminal differentiation into neurons. © 1993 Wiley‐Liss, Inc.
Early responses of PC‐12 cells to NGF and EGF: Effect of K252a and 5′‐methylthioadenosine on gene expression and membrane protein methylation
Authors: Kujubu D. A. , Stimmel J. B. , Law R. E. , Herschman H. R. , Clarke S.
Published: January 2005

Although epidermal growth factor (EGF) and nerve growth factor (NGF) have markedly different biological effects on PC‐12 cells, many of the signaling events following ligand binding are similar. Both EGF and NGF result in the induction of the primary response gene egr‐1/TIS8 and increased methylation of a variety of membrane‐associated proteins as early as 5 min after EGF or NGF treatment using a methylation assay that detects methyl esters as well as methylated arginine residues. At 20 min after stimulation with these factors, the stimulation of methylation by NGF is greater than that of EGF, especially in the polypeptides of 36–42 and 20–22 kDA. To help dissect the pathways involved in these cellular responses, the protein kinase inhibitor K252a and the methyltransferase inhibitor 5′methylthioadenosine (MTA) were used. Both K252a and MTA inhibit NGF‐, but not EGF‐mediated, primary response gene expression. In contrast, MTA, but not K252a, can block NGF‐induced membrane associated protein methylation. These data suggest a role for differential protein methylation reactions in EGF and NGF signal transduction. © 1993 Wiley‐Liss, Inc.
Protein kinases modulate the sensitivity of hippocampal neurons to nitric oxide toxicity and anoxia
Authors: Maiese K. , Boniece I. R. , Skurat K. , Wagner J. A.
Published: January 2005

Multiple processes lead to neuronal death after ischemia, but the generation of nitric oxide (NO) is a key component in this cascade of events. The mechanisms that regulate the extent of neuronal degeneration during anoxia and NO toxicity are multifactorial. Neuronal death may be modulated by the activity of signal transduction systems that influence the toxicity of NO or its metabolic products such as cGMP. The enzyme responsible for the production of NO, nitric oxide synthase (NOS), is phosphorylated by protein kinase C (PKC), the cAMP‐dependent protein kinase (PKA), and the calcium/calmodulin‐dependent protein kinase II (CaM‐II). We examined in primary cultured hippocampal neurons whether the protein kinases PKC, PKA, CaM‐II, and cGMP‐dependent protein kinase modified the toxic effects of anoxia and NO. Down‐regulation of PKC activity with PMA (1μM) increased hippocampal neuronal survival during anoxia and NO exposure from approximately 22% to 88%. Inhibitors of PKC activity 9H‐7, H‐8, sphingosine, and staurosporine also were neuroprotective. Down‐regulation of PKC activity increased survival during anoxia even in the presence of the NOS inhibitor, Nω‐methyl‐L‐arginine. Thus, although down‐regulation of PKC activity may increase neuronal survival by decreasing NOS activity, it also is likely that PKC contributes to ischemic neuronal death by mechanisms that are independent of NOS. Inhibition of the cGMP‐dependent protein kinase activity, but not the activity of the CaM‐II also was neuroprotective during NO administration. In contrast to the protective effects of inhibition of PKC and the cGMP‐dependent protein kinase, activation rather than inhibition of PKA increased hippocampal neuronal survival during NO exposure. These results indicate that neuronal survival during anoxia and NO exposure is linked to the modulation of PKC, PKA, and cGMP‐dependent protein kinase activity but is not dependent on the CaM‐II pathway. Understanding the involvement of PKC, PKA, and the cGMP‐dependent protein kinase in modulating the effect of neuronal death during ischemia and NO toxicity may help in directing future therapeutic modalities for cerebrovascular disease. © 1993 Wiley‐Liss, Inc.
Intracellular distribution of transgenic bacterial β‐galactosidase in central nervous system neurons and neuroglia
Authors: Friedrich V. L. , Holstein G. R. , Li X. , Gow A. , Kelley K. A. , Lazzarini R. A.
Published: January 2005

Bacterial β‐galactosidase is widely used as a marker for gene expression and in cell tracing experiments. In a survey of three transgenic mouse lines expressing β‐galactosidase in the central nervous system (CNS) under the control of different promotors, we find substantial variation in the intracellular distribution of the lacZ protein. In line MβP5, transgene β‐galactosidase expression is driven by a promoter/enhancer fragment from the oligodendrocyte‐specific myelin basic protein gene; however, electron microscopy of histochemically stained preparations revels transgene expression not only in oligodendrocytes but also in some neurons. Immunofluorescence and immunoperoxidase staining show the β‐galactosidase protein distributed throughout the perikaryal cytoplasm of oligodendrocytes and in processes reaching to myelin sheaths. By contrast, immunoreactive protein appears restricted in neurons to one or a few small perikaryal immunoreactive granules. The granules are visible in the electron microscope as amorphous inclusion bodies of moderate electron density and lack a limiting membrane. Histochemical staining patterns with X‐gal and Bluo‐gal echoed the protein distribution: diffuse distribution of enzyme protein yielded cells filled with substrate, while punctate enzyme distribution yielded restricted or punctate histochemical staining. Examination of two other lines using different promoter/enhancers to drive expression in the CNS showed both diffuse and punctate β‐galactosidase immunolocalization and histochemical staining. The amount of protein synthesized or other properties, yet unidentified, intrinsic to the target cells may determine the intracellular distribution of β‐galactosidase. In retroviral marking studies, clone members have been identified as those cells filled with X‐gal reaction product. This approach may underestimate both clone size and the minimum number of divisions separating the members of each clone. © 1993 Wiley‐Liss, Inc.
Neuron‐oligodendroglial interactions during central nervous system development
Authors: Hardy R. , Reynolds R.
Published: January 2005

It is well established that a variety of growth factors influence the differentiation of oligodendroglial lineage cells in culture, although little information is available concerning the role and source of these factors in vivo. Developing oligodendroglia are almost constantly in a neuronal environment and would be expected to respond to a variety of signals from neurons that affect their survival, migration, division, maturation and myelin production. However, very little is known about the specific interactions that occur between these two cell types. Here we review the experimental evidence for the influence of neurons on oligodendroglial differentiation, including studies on the effects of both soluble factors and contact dependent events. We also propose a scheme for the control of myelinogenesis via both internal and external signals. © 1993 Wiley‐Liss, Inc.
Expression of neuromodulin (GAP‐43) and its regulation by basic fibroblast growth factor during the differentiation of O‐2A progenitor cells
Authors: Deloulme J. C. , Laeng P. , Janet T. , Sensenbrenner M. , Baudier J.
Published: January 2005

In a recent work we have shown that neuromodulin (Nm, also known as GAP‐43), a protein kinase C substrate, previously believed to be expressed exclusively in neurons, is also present in glial cells. Here we investigated the expression of Nm and its mRNA in O‐2A glial progenitor cells (common precursor for oligodendrocytes and type‐2 astrocytes) during their development in secondary culture and under the influence of basic fibroblast growth factor (bFGF). The different stages of oligodendrocyte development were characterized by the expression of surface markers: A2B5, which identifies O‐2A glial precursor cells, and O4 and galactocerebroside (GC), which characterize later developmental stages. The number of cells expressing Nm (about 90% at culture initiation) decreased rapidly during the first 2 days and reached a plateau at around 30–40%. The level of Nm mRNA followed a similar kinetic. Immunocytochemistry demonstrated that at 4 days in vitro about 25–30% cells were A2B5,+, 30–40% Nm+, a high percentage (60–70%)O4 +, and 35–40% GC+. Nearly all of the morphologically immature A2B5+ cells expressed also the Nm antigen, very few of the O4+ cells still expressed Nm and almost no cells expressed both GC and Nm. Most O4+ cells developed a typical oligodendrocyte morphology and were essentially GC+. This study also showed that in the presence of serum, the A2B5+ Nm and 04+ Nm+ (GC−)xs cells retained their bipotentiality and differentiated into GFAP+ (glial fibrillary acidic protein) Nm+ type‐2 astrocytes.

The bFGF was found to stimulate the proliferation of Nm+ 0–2A precursor cells and to increase the level of Nm mRNA. At 4 days under this culture condition the predominant cell type was A2B5+ and Nm+. Only 25–35% of the cells were O4+, but 90–95% of them were Nm+. Very few GC+ cells were visible in the presence of bFGF, but 20–40% of them were Nm+. These data indicate that Nm is essentially associated to glial O‐2A precursor cells and further confirm that bFGF blocks the differentiation of these cells. It is suggested that Nm plays a role in the plasticity (developmental potential) of the bipotential 0–2A progenitor cells. © 1993 Wiley‐Liss, Inc.
Cell blotting and isoelectric focusing of neuroblastoma‐derived heparin‐binding neurotrophic activities: Detection of basic fibroblast growth factor protein and mRNA
Authors: Wewetzer K. , Janet T. , Heymann D. , Unsicker K.
Published: January 2005

Neuroblastoma cells have been shown to contain growth factors, which may be involved in the regulation of cell proliferation and/or differentiation. We have examined whether human IMR‐32 neuroblastoma cells store factors with a capacity to promote neuron survival and differentiation. Heparin affinity chromatography, cell blotting, and isoelectric focusing of IMR‐32 cell extracts revealed multiple neurotrophic activities at molecular weights of 16.8, 24.1, 39.0, 45.3, 52.3 and isoelectric points of 4.0, 5.0, 8.3, 9.0. Immunocytochemistry, immunoblotting, and radio‐immunoassay with specific antibodies suggests that one neurotrophic activity is immunologically identical with basic fibroblast growth factor (bFGF). This assumption is supported by Northern blot analysis, which shows a 6.0 kb bFGF transcript. © 1993 Wiley‐Liss, Inc.