bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the rat light neurofilament (NF‐L) gene promoter and identification of NGF and cAMP responsive regions
Authors: Reeben Mati , Neuman T. , Palgi J. , Palm K. , Paalme V. , Saarma M.
Published: January 2005

We have isolated a genomic DNA clone covering the coding and 14 kb upstream region of the rat light neurofilament (NF‐L) gene and sequenced 2.3 kb of its promoter. DNase I hypersensitive sites have been mapped in PC12 cells. For functional analysis of the NF‐L promoter, constructs carrying 38, 97, 407, 564, 650, 1,099, 1,660, 2,003 base pairs (bp) upstream region in front of the chloramphenicol acetyltransferase (CAT) reporter gene were tested for their capability to direct CAT expression after transient transfection into various cell lines. Similar CAT activities were recorded both in rat pheochromocytoma (PC12) and mouse neuroblastoma N115 cells and also in several nonneural cell lines (HeLa, C127, NIH 3T3). Regions responsible for the basic promoter activity were located between −407 and +75 bp from the transcription initiation site. The NGF‐responsive element was located between −38 and +75 bp, and sequence −97 to −38 was found to contain a functional cAMP‐responsive element. In PC12 cells in which nerve growth factor (NGF) induces neurite outgrowth and NF‐L transcription, NF‐L promoter‐driven CAT expression was stimulated up to 12‐fold within three days of NGF treatment, whereas epidermal growth factor (EGF) had no effect. Rat NF‐L promoter contained Sp1, AP‐2 and CGCCCCCGC elements. In PC12 cells, NGF transiently induced the binding of transcription factors to the deoxyoligonucleotide probes containing the binding sites of these elements. The role of these factors in NF‐L gene transcriptional induction by NGF in PC12 cells is discussed. © 1995 Wiley‐Liss, Inc.
Tenotomy prevents the functional improvement of a muscle reinnervated with a chronically severed nerve
Authors: Carobi Clara , Brunetti O.
Published: January 2005

During the early stages of nerve implantation, we followed the dynamic properties of the lateral gastrocnemius muscle of the rat, reinnervated with an acutely or chronically severed peroneal nerve. The aim of this study was to ascertain whether (1) the better functional recovery of a muscle reinnervated by a chronically severed foreign nerve is present from the onset of reinnervation, and (2) whether such functional improvement is due to the conditioning lesion effect. Our results indicate that better functional recovery is already apparent one week after nerve implantation, and it is due to the conditioning lesion effect, since tenotomy prevents such improvement. The tenotomy effect underlines the fact that some environmental factors concerning the target tissue, and not only the predegenerated nerve, are involved in the conditioning effect. © 1995 Wiley‐Liss, Inc.
Modulation of Schwann cell P0 glycoprotein and galactocerebroside by the surface organization of axolemma
Authors: Calderon Reyna O. , Maggio B. , Neuberger T. J. , Devries G. H.
Published: January 2005

The nature of the axon signal for the induction of proliferation and differentiation of peripheral glial cells is still unknown. Besides the existence of interactions among surface molecules the cellular responses can also be regulated by physicochemical parameters of the membrane. We have previously reported that planar axolemma monolayers coated on glass coverslips at different defined surface molecular packing affected the Schwann cell (SC) morphology and their proliferative response (Calderon et al.: J Neurosci Res 34:206–218, 1993). In this paper we report that relative to SC cultured on uncoated coverslips, the level of expression of both glycoprotein P0 and galactocerebroside (GC) (as revealed by immunofluorescence) was increased 2–4 times in SC cultured on axolemma monolayers with either high or low molecular packing. However, the cellular distribution of these antigens was profoundly influenced by the molecular packing density of the axolemma monolayer. SC cultured on an axolemma monolayer at high molecular packing showed preferential expression of P0 at the SC surface whereas GC was concentrated intracellularly. On the other hand, SC grown on an axolemma monolayer at low molecular density GC showed preferential expression at the cell surface whereas P0 was concentrated intracellularly. © 1995 Wiley‐Liss, Inc.
Expression and distribution of phosphorylated MAP1B in growing axons of cultured hippocampal neurons
Authors: Boyne L. J. , Martin K. , Hockfield S. , Fischer Itzhak
Published: January 2005

Microtubule associated proteins (MAPs) interact with tubulin to modulate neurite stability and growth during development. The phosphorylated form of one of these MAPs, MAP1B (MAP1B‐P) is hypothesized to be of particular importance for the regulation of neurite outgrowth. To investigate the mechanisms by which MAP1B and MAP1B‐P contribute to this regulation, we used a new antibody against an isoform of MAP1B‐P to determine its pattern of expression during neuronal development in vitro. We examined cultured hippocampal neurons because these provide a well‐established system to evaluate the development of axons and dendrites. MAP1B, MAP1B‐P and MAP2 colocalized to the cell bodies and minor processes during the first 24 hours of culture, but MAP1B‐P also extended well into the growth cones. As neurite outgrowth and differentiation proceeded, MAP1B and MAP1B‐P became localized to the cell bodies and axons, and MAP2 to the cell bodies and dendrites. After 3 days, MAP1B‐P declined in the cell body and was segregated to the distal axon; MAP1B remained in the cell body, but was also concentrated in the distal axon. Over 5–9 days in culture, MAP1B‐P levels decreased and became undetectable; MAP1B levels decreased later (19–23 days). MAP2 levels, however, remained high through the entire culture period in cell bodies and dendrites. These results are consistent with the hypothesis that MAP1B‐P plays an important role in the initiation and elongation of axons by regulating the dynamics of microtubules near the growth cone: MAP1B‐P expression is greatest during the period of active neurite extension, is particularly prominent in growth cones where axon outgrowth is most active, and decreases along with the decline in active axon extension. © 1995 Wiley‐Liss, Inc.
Cortical astroglial conditioned medium induces in vitro radial‐like forms
Authors: Colombo J. A. , Napp M. , Puissant V.
Published: January 2005

Cerebral cortex and striatal cell dissociates obtained from rat fetuses (E17) were subcultured and enriched in astroglial cells before being grown in regional (cerebral cortex, striatum) astroglial conditioned media (CM) or defined basal medium. Incidence of radial‐like astroglia (vimentin + or glial fibrillary acid protein, GFAP + ) and length of processes in cortical cell subcultures showed a greater increase when exposed to cerebral cortex CM than to striatal CM or basal medium. Stellate (GFAP + ) forms prevailed in subcultures grown in basal medium while striatal cells exposed to CM of either origin remained undifferentiated. Additionally, cultures were treated with various concentrations of cAMP (0.25 and 0.5 mM) and calcitonin gene related peptide (CGRP) (0.1, 0.5, and 1.0 μ). Under these conditions CM‐exposed cultures (with predominant “radial‐like” forms) did not increase stellate glial numbers, while fetal calf serum (FCS)‐exposed cultures (morphologically undifferentiated) underwent significant degrees of stellate transformation. When CM‐exposed cultures were shifted to FCS supplemented basal medium for 24–48 hr and then to basal medium alone prior to treatment, cAMP and CGRP were effective in transforming flat astroglia into stellate morphology. Results are indicative of the existence of astroglial diffusible factors affecting the in vitro expression of astroglial morphotypes from the cerebral cortex. Previous exposure to CM interferes with cytoskeletal astrocytic changes induced by cAMP and CGRP. It is speculated that astroglial factors could act in vivo to maintain the expression of radial‐like cells during early developmental stages of the cerebral cortex, but it would not be effective in E 17 striatum. It is also suggested that transformation of radial glia into astrocytes may require previous dedifferentiation of the cytoskeleton. © 1995 Wiley‐Liss, Inc.
Adult rat optic nerve oligodendrocyte progenitor cells express a distinct repertoire of voltage‐ and ligand‐gated ion channels
Authors: Borges K. , Wolswijk G. , Ohlemeyer C. , Kettenmann H.
Published: January 2005

Cultured oligodendrocyte progenitor cells derived from the developing central nervous system (CNS) express a pattern of ion channels that is distinct from mature oligodendrocytes and other cell types of the CNS. In the present study, we used the whole‐cell patch‐clamp technique and the fura‐2‐based Ca++ imaging system to study the ion channel expression of oligodendrocyte progenitor cells derived from the optic nerves of adult rats. We found that the adult oligodendrocyte progenitor cell membrane is dominated by K+ currents, both delayed outward and inward rectifying. The inwardly rectifying K+ currents were often as large as the outward delayed rectifying K+ currents. The delayed rectifying outward currents were partially blocked by 50 mM tetraethylammonium or 1 mM 4‐aminopyridine, but not by 2 or 5 mM BaCl2. This suggests that the delayed rectifier channels expressed by adult progenitor cells are different from those expressed by perinatal cells. Most adult oligodendrocyte progenitor cells showed no or only small A‐type K+ currents. Both Ca++ and Na+ channels were also detected in these cells. Furthermore, adult progenitor cells responded to the neurotransmitters GABA and kainate and the pharmacology of these responses indicated that these cells express GABAA receptors and kainate receptors that are Ca++ ‐permeable. Our study suggests that adult oligodendrocyte progenitor cells are electrophysiologically distinct and that these cells share electrophysiological characteristics with both perinatal progenitor cells and immature oligodendrocytes. © 1995 Wiley‐Liss, Inc.
Selection of stably transfected cells expressing a high level of fetal muscle nicotinic receptors
Authors: Chen Q. , Fletcher G. H. , Steinbach J. H.
Published: January 2005

We had earlier found that the numbers of mouse muscle nicotinic receptors expressed on the surface of individual cells of a stably transfected clonal line of quail fibroblasts varied from cell to cell (Kopta and Steinbach: J Neurosci 14:3922–3933, 1994). We have now used repeated selective passages of these clonal cells to produce a population of cells which expresses a greater and more uniform number of surface receptors per cell. The increased level is stable over many cell divisions, and over many half‐lives for the metabolic degradation of the surface receptors. Selection was performed by adhesion to a surface coated with a monoclonal antibody to a surface epitope on the muscle receptor, followed by expansion of the most tightly attached population of cells. Studies of the selected cells show that the surface receptors contain all four subunits of the muscle nicotinic receptor, and the functional properties of the receptors appear normal. The metabolic stability of the surface receptors is not altered, while the amount of mRNA for the subunits is increased in the selected population of cells. These observations indicate that the more likely reason for increased expression is a transcriptional effect, and that translational or posttranslational changes are unlikely. © 1995 Wiley‐Liss, Inc.
Differential expression of neurotensin receptor mRNA in the dopaminergic cell groups of the rat diencephalon and mesencephalon
Authors: Nicot A. , Rostène W. , Bérod A.
Published: January 2005

Several lines of evidence support interactions between neurotensin (NT) and dopaminergic (DAergic) neurons in the brain. In order to obtain further knowledge about the anatomical substrate for such interactions, the distribution of cells expressing the cloned neurotensin receptor (NTR) mRNA was examined in relation to tyrosine hydroxylase (TH) mRNA‐expressing cells within different subnuclei of the diencephalon and ventral mesencephalon of the male rat. In situ hybridization was performed on consecutive sections labeled with 33P‐labeled oligonucleotide probes. In the hypothalamus, NTR mRNA signals were mostly found in the suprachiasmatic, dorsomedial, dorsal premammillary, and supramammillary nuclei. On the other hand, DAergic cells of the periventricular nucleus of the hypothalamus and dorsal aspect of the arcuate nucleus, revealed by TH in situ hybridization, did not exhibit NTR mRNA even though dense NT binding sites have been previously described in this nuclei. In the zona incerta, TH mRNA‐containing cells were concentrated in the medial part, with little overlap with NTR mRNA‐expressing cells located mainly in its mediolateral extent. In contrast, the distribution of both markers was superimposable within the different subdivisions of the ventral tegmental area and substantia nigra, as previously suggested, but also in the retrorubral field. These anatomical data further support the NT‐dopamine interactions on both mesocorticolimbic and nigrostriatal DAergic systems. Moreover, the results suggest that diencephalic DAergic neurons do not synthesize the cloned NTR mRNA or express it at considerably lower levels than DAergic mesencephalic cells. © 1995 Wiley‐Liss, Inc.
Effect of diet on tissue protease activity
Authors: Benuck Myron , Banay‐Schwartz M. , Deguzman T. , Vizi E. S. , Kekes‐Szabo A. , Lajtha A.
Published: January 2005

Rats 1,3,12, and 24 months old were fed diets low in protein (8% casein), and proteolytic activity in tissue from brain, liver, and lung was determined. After a low‐protein diet was fed for 4 weeks to 1‐month‐old rats, there was a significant increase in cathepsin D activity in liver, and calpain activity was increased in lung. Little change was seen in proteolytic activity in brain. In 12‐month‐old rats, there was an increase in cathepsin D activity in brain and liver. In 24‐month‐old rats, cathepsin D activity in the liver and calpain activity in lung were increased. There was no change in proteolytic activity in the brain. When animals were fed diets supplemented with fatty acids or antioxidants for 2 months, in 3‐month‐old rats calpain activity was increased in brain but decreased in lung. Cathepsin D activity was significantly increased in young and adult animals in brain and in liver. These observations suggest that diet changes result in significant alteration in tissue calpain and cathepsin D levels, and possibly activity, in vivo. Generally, changes are greater for cathepsin D than for calpain, and are smaller in brain than in other tissues. © 1995 Wiley‐Liss, Inc.
Establishment and characterization of a multipotential neural cell line that can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro
Authors: Nakafuku Masato , Nakamura S.
Published: January 2005

In the mammalian central nervous system (CNS), multipotential neural stem cells in the neuroepithelium generate the three major types of neural cells, namely, neurons, astrocytes, and oligodendrocytes. To explore the molecular mechanisms underlying proliferation and differentiation of these neural stem cells, we established a cell line named MNS‐57 from the embryonic day 12 rat neuroepithelium by introducing the mycer fusion gene, in which c‐myc can be conditionally activated by adding oestrogen to the culture medium. MNS‐57 cells expressed nestin, vimentin, and the RC1 antigen, which are potential markers for neural stem cells. We show that under particular culture conditions, MNS‐57 cells can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro, indicating that they are likely to originate from multipotential neural stem cells. Incubating MNS‐57 cells with either oestrogen, which activates mycer, or growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulated their growth, and the combination of oestrogen and bFGF (or EGF) had a synergistically stronger mitogenic effect than the single factors. Furthermore, both c‐myc activation and bFGF appeared to be necessary for the differentiation of MNS‐57 cells, and only when stimulated by both signals simultaneously, the cells committed to generating multiple neural cell types. Thus, the property of the cell line is unique in that its differentiation into neurons and glia can be conditionally manipulated invitro in an exogenous signal‐dependent manner. We propose that the cell line described here will provide an useful in vitro model to understand genetic and environmental mechanisms that control the generation of neural cell diversity in the CNS. © 1995 Wiley‐Liss, Inc.
Excitatory amino acid‐induced AP‐1 DNA binding activity in müller glia
Authors: López‐Colomé A. M. , Murbartián J. , Ortega Arturo
Published: January 2005

The effect of L‐glutamate (L‐Glu) and its structural analogs N‐methyl‐D‐aspartate (NMDA), quisqualate (QA), and kainate (KA) on the DNA binding activity of the Activator Protein 1 (AP‐(1) and the Ca2+/cAMP Responsive Element Binding Protein (CREB) families of transcription factors was examined in cultured chick retinal Müller glia cells. L‐Glu, NMDA, and KA evoked a dose and time dependent increase in AP‐1 DNA binding activity and had no effect on CREB binding. The order of potency for stimulating AP‐1 DNA binding was NMDA≥Glu>KA>>QA. L‐Glu responses were partially blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and by 3‐[RS)‐2‐carboxypiperazin‐4‐yl)]‐propyl‐l‐phosphonate (CPP) indicating that the increase in DNA binding is mediated both by an a‐amino‐3‐hydroxy5‐methyl‐4‐isoxazolepropionate (AMPA)/low affinity KA and a NMDA subtypes of L‐Glu receptors. Since Müller glia L‐Glu receptors are probably mediators of the efficacy of the excitatory transmission in the retina, the present findings suggest that a stimulustranscription coupling triggered by L‐Glu in the glial cells might have a role in the long‐term modulation of these synapses. © 1995 Wiley‐Liss, Inc.
In vitro glial responses to nerve growth factor
Authors: Hutton L. A. , Perez‐Polo J. R.
Published: January 2005

Nerve growth factor (NGF) stimulates expression of the low affinity neurotrophin receptor p75NGFR mRNA in primary cultures of neonatal rat cortical type I astrocytes. Nerve growth factor treatment altered glial morphology in glial fibrillary acidic protein positive (GFAP+) cell cultures derived from newborn (P0) and 3‐day‐old (P3) rat pups. When P0‐ or P3‐derived primary glial cultures were serum‐deprived, in the presence of 200 pM NGF for 5 days, the flat polygonal glia present in culture assumed a fibrous morphology, an effect not seen in the untreated serum‐deprived controls. The NGF effect on astrocytic morphology was blocked by continuous serum treatment. Nerve growth factor did not stimulate astrocytic proliferation under these culture conditions, as assayed by cell cycle analysis using 3H thymidine autoradiography. P0‐derived primary glial cultures expressed the signal transducing neurotrophin receptors p145trkB and p140trka as determined by reverse transcription‐polymerase chain reaction (RT‐PCR). RT‐PCR products were identified by sequencing or restriction enzyme analysis. Astrocytes internalized 125I‐NGF at 37°C but not at 4°C, consistent with energy requirements for internalization. Also, internalization of 125I‐NGF was abolished by the addition of a 300‐1,000‐fold excess of unlabeled NGF. Thus, astroglial cells in culture internalize NGF through a specific receptor‐mediated process, express trkA and full‐length trkB mRNAs at low levels, and respond to exogenous NGF by expressing a fibrous morphology under serum‐free culture conditions. © 1995 Wiley‐Liss, Inc.
A morphometric analysis of bipolar and multipolar TH‐IR neurons treated with the neurotoxin MPP+ in co‐cultures from mesencephalon and striatum of embryonic C57BL/6 mice
Authors: Koutsilieri E. , Chen T.‐S. , Kruzik P. , Rausch W.‐D.
Published: January 2005

Mesencephalic cultures contain two morphologically different tyrosine hydroxylase (TH)‐immunoreactive (IR) neurons, fusiform bipolar, and pyramidal multipolar, which project to different anatomical structures (ventral striatum and neostriatum). The possibility of functional difference of these cells in Parkinson's disease led us study the effect of 1‐methyl‐4‐phenylpyridinium (MPP+) on them. Survival and morphology of the two groups was studied in dissociated co‐cultures from mesencephalon and striatum of embryonic C57BL/6 mice. Cells were grown at first in serum containing medium and then in serum free medium supplemented with hormones. Cultures were exposed to different concentrations of MPP+ on day 9 and 13 for 24 hr. They were fixed and stained, with an anti‐TH antibody. 0.1‐1.0μM MPP+ caused a dramatic reduction of the total area of TH‐IR neurons. At 0.1 μM MPP+ some area was reduced, at 0.5 μM it appeared similar to controls, and decreased further at 1.0 μM. The relation of soma to total area showed that the decrease of the neuronal size was mainly due to the degeneration of the neuronal processes. The length of neuronal tree as well as the number of terminal segments were reduced dose dependently when cells were treated with the toxin. Similar results were obtained for bipolar and multipolar neurons. A significant difference in the decrease in total area was observed between the two age groups when cells were treated with MPP+, as older cells appeared to be more sensitive. When other parameters were checked no apparent difference was present. © 1995 Wiley‐Liss, Inc.
Correlates between nuclear magnetic resonance spectroscopy, diffusion weighted imaging and CA1 morphometry following chronic brain ischemia
Authors: de la Torre J. C. , Butler K. , Kozlowski P. , Fortin T. , Saunders J. K.
Published: January 2005

Chronic brain ischemia (CBI) was induced in aging (13 month) rats by ligating the left subclavian artery and placing temporary occluders in each common carotid artery [three‐vessel occlusion (3‐VO)]. Carotid artery occluders were removed after 1, 2, or 3 weeks following brain ischemia or maintained for 9 weeks. Two rats were kept with their occluders in place for 25 weeks. On weeks 3 and 9 after CBI, 31 P‐/1H‐nuclear magnetic resonance (NMR) spectroscopy and high resolution diffusion weighted imaging were performed in vivo, non‐invasively for detection of hippocampal high energy phosphates, lactate, intracellular pH, N‐acetyl‐aspartate, choline, glutamate, creatine, and structural alterations of the brain following CBI. Brains were histologically processed for morphometry of glial fibrillary acidic protein (GFAP) and CA1 damaged neurons 9 weeks after CBI. 31P‐/1H‐NMR spectroscopy showed that high energy substrates remained normal in ischemic animals when compared to non‐ischemic controls except for an elevation of phosphomonoesters in the hippocampal region. Rats deoccluded 1 and 2 weeks after initiation of CBI had no NMR spectroscopic or imaging changes. Rats kept ischemic for 9 weeks showed high signal intensities in the parietal cortex detected by diffusion weighted imaging as well as CAI damage and increased GFAP density but no cortical atrophy or neuronal damage could be detected histologically. Rats kept ischemic for 25 weeks showed extensive cortical atrophy which corresponded to the high, signal intensity observed with diffusion weighted imaging in the group kept ischemic for 9 weeks. These findings indicate that NMR spectroscopy and difussion weighted imaging can be used to follow the progress of neuronal injury in vivo and non‐invasively. Moreover, diffusion weighted imaging may be an excellent predictor of cerebral damage and atrophy prior to histopatholog ical detection of such damage. Our present and previous findings using our 3‐VO model indicate that this aging rat model may be useful in screening potential therapy for neurodegenerative disorders associated with abnormal aging and memory impairment. © 1995 Wiley‐Liss, Inc.
Signal transduction alterations in peripheral nerves from streptozotocin‐induced diabetic rats
Authors: Goraya T. Y. , Wilkins P. , Douglas J. G. , Zhou J. , Berti‐Mattera L. N.
Published: January 2005

We have previously determined the presence of muscarinic receptors and the expression of several G proteins in homogenates and myelin fractions from ra sciatic nerves. In the present study we investigates whether changes in several signal transduction path ways in peripheral nerves might be responsible for some of the biochemical abnormalities (e.g., phosphoinositide metabolism) present in sciatic nerves from streptozotocin‐induced diabetic rats. Sciatic nerves from 5 week diabetic rats that were prelabelled with [3H]‐myo‐inositol displayed a significant increase in the basal release of inositol mono‐and bis‐phosphate, while carbamylcholine‐stimulated release was significantly smaller. Basal‐ and forskolinstimulated adenylyl cyclase activity was significantly decreased in sciatic nerve homogenates from diabetic animals. However, we were unable to detect any significant differences in the levels of cAMP in intact nerves or in nerve segments that were incubated in the presence or absence of forskolin. ADP‐ribosylation experiments showed that in sciatic nerves from experimentally diabetic rats there was a significant increase in the ADP‐ribosylation catalyzed by cholera and pertussis toxins. Measurements of the levels of a‐subunits of G proteins revealed that the expression of Gq/11α, Gsα, and Gi‐3α was increased by 30 to 50%. These results indicate that during the course of experimental diabetes, peripheral nerves exhibit an abnormal production of inositol phosphates and cAMP, together with an abnormal expression and/or function of G proteins. One of the consequences of such alterations is the diminished release of inositol phosphates triggered by muscarinic agonists in diabetic sciatic nerves. © 1995 Wiley‐Liss, Inc.
Neurons express ciliary neurotrophic factor mRNA in the early postnatal and adult rat brain
Authors: Seniuk‐Tatton N. A. , Henderson J. T. , Roder J. C.
Published: January 2005

The regional and subcellular localization in the central nervous system (CNS) of postnatal day 5, day 15, and adult rats of ciliary neurotrophic factor (CNTF) mRNA was examined by in situ hybridization with biotinylated riboprobes. Probe specificity was determined by Northern blot analysis of poly(A) + RNA extracted from adult rat brain using digoxigenin labeled riboprobes and chemiluminescent detection. Both a 4 kb and a 1.2 kb transcript were detected in the cortex and brainstem. In situ hybridization revealed that CNTF mRNA was widely distributed in neurons and glia throughout the CNS at each of the developmental time points. The density of the neuronal hybridization signal was found to be greater in neuronal nuclei than in their cytoplasm. In the nucleus of most neurons, CNTF mRNA distribution was concentrated in a perinucleolar fashion. Alternate sections from the same animals, which were incubated with a specific polyclonal antibody against a CNTF peptide fragment, revealed that both neurons and glia in postnatal day 5, day 15, and adult rat brain were immunoreactive for CNTF. © 1995 Wiley‐Liss, Inc.
Identication of glial cell types involved in mediating epidermal growth factor's effects on septal cholinergic neurons
Authors: Kenigsberg R. L. , Mazzoni I. E.
Published: January 2005

We found previously that epidermal growth factor (EGF) decreases choline acetyltransferase (ChAT) activity in forebrain cholinergic neurons in vitro indirectly via glia (Kenigsberg et al.: Neuroscience 50:85–97, 1992). However, which glial type(s) are implicated in this response remained to be determined. Here we report that in primary cultures from the fetal rat medial septal area the complete elimination of oligodendrocytes or partial elimination of microglia from these cultures does not change the cholinergic cell response to EGF. However, the elimination of astroglia in our cultures by α‐aminoadipic acid treatment blocks EGF's effects on the chofinergic neurons. Co‐culture experiments using pure neuronal and purified glial cells from the medial septum further demonstrate that the cholinergic cell response to EGF can be maintained in the presence of astroglia only. In addition, it appears that EGF regulates the release of soluble factors from pure astroglial cultures following their peak mitotic response to EGF that decreases ChAT enzymatic activity. This soluble cholinergic neuromodulatory activity found in conditioned media from EGF‐treated astrocytes has a molecular weight greater than or equal to 10 kD and loses potency following multiple freeze‐thaw cycles.

These results suggest that a direct glial cell response to a specific glial growth factor like EGF may have an important impact on the expression of local neurons, like the cholinergic in the forebrain. © 1995 Wiley‐Liss, Inc.
Coincidence of L‐glutamate/L‐aspartate transporter (GLAST) and glutamine synthetase (GS) immunoreactions in retinal glia: Evidence for coupling of GLAST and GS in transmitter clearance
Authors: Derouiche A. , Rauen T.
Published: January 2005

Our aim was to identify proteins that mediate the uptake and degradation of synaptically released glutamate, focusing on the rat retina with its well‐defined glutamatergic pathways. Immunoreactivity against the L‐glutamate/L‐aspartate transporter (GLAST) is present in Müller cells. Ultrastructurally, even the finest glial processes, particularly those ensheathing identified structures of glutamatergic transmission (rod spherules), are immunoreactive for GLAST. Further light and electron microscopic observations revealed that also retinal astrocytes and pigment epithelial cells are immunoreactive for GLAST. No neuronal or microglial staining was observed. This is in line with uptake of exogenous [3H]glutamate previously localized specifically in Müller cells and pigment epithelium (Ehinger and Falck: Brain Res 33:157‐172, 1971). Since endogenous glutamate can only be demonstrated in Muller cells if glutamine synthetase (GS) is inhibited (Pow and Robinson: Neuroscience 60:355‐366, 1994), the immunocytochemical localization of GS was determined. GS immunoreactivity was found in all but only those cell types immunoreactive for GLAST. The light and electron microscopic patterns of immunoreactivity were very similar, particularly in the outer plexiform layer: The three cell types containing both GS and GLAST (Müller cells, astrocytes, and retinal pigment epithelium) are related developmentally. In the light of the two references quoted the present data indicate that the proteins mediating retinal uptake and degradation of synaptically released glutamate may be GLAST and GS, respectively, and that they may operate in concert to terminate the neurotransmitter action of glutamate. © 1995 Wiley‐Liss, Inc.
Growth related changes in sugar determinants on the surface of C6 glioma cells in culture: A cytochemical lectin‐binding study
Authors: Berezovskaya O. L. , Mares V. , Skibo G. G.
Published: January 2005

The cell surface sugar determinants (CSSD) were examined in C6 glioma cells in cultures at different conditions of growth by peroxidase conjugates of the lectins: peanut agglutinin (PNA), Ricinus communis agglutinin (RCA), Helix pomatia agglutinin (HPA), wheat germ agglutinin (WGA), lentil agglutinin (LCA), laburnum bork agglutinin (LABA), and lotus agglutinin (TPA). It was found that the cells bound more intensively WGA, LCA, and RCA compared to PNA, HPA; the weakest staining was provided by LABA and TPA. Binding intensity for PNA significantly increased after pretreatment of the cells with neuraminidase. This indicates that a part of the β‐D‐galactose residues on the surface membrane of C6 glioma cells is covered by sialic acid. The process of sialization was increased during the culturing of C6 glioma cells. Addition of cis‐DDP or dBcAMP to cultures growing in medium with 10% of CS increased the number of Gal residues which are not covered by sialic acid.

The expression of β‐D‐galactose (Gal), N‐acetyl‐Dgalactosamine (NAcDGal), and fucose (Fuc) residues appeared to be most responsive to changes in growth conditions and degree of cell differentiation. The expressions of N‐acetyl‐D‐glucosamine (NAcDGIc) and mannose (Man) residues were high and seems did not depend on changing of the conditions of culturing.

In C6 glioma cells cultures in which the rate of cell division, formation of the cell processes, and adhesiveness of the cells to the substratum were reduced by growing cells in MEM +, expression of β‐Gal, NAcDGa1, and Fuc was considerably reduced. The decrease of expression of R‐Gal, NAcDGaI, and Fuc on the surface of cell membrane was more pronounced in MEM + with 1% of CS than in MEM + with 10% of CS. In DbcAMP and cis‐DDP treated cultures, grown in medium with 1% serum, in which cell division was inhibited without obvious changes in cell adhesiveness to the substratum, binding of PNA and HPA was increased due to higher expression of β‐Gal and NAcDGaI. From these observations it was concluded that the pattern of expression of sugar residues on the cell surface varies according to the biological state of the cells and are easily affected by tissue culture conditions. © 1995 Wiley‐Liss, Inc.
Activation of the hypothalamo‐anterior pituitary corticotropin‐releasing hormone, adrenocorticotropin hormone and β‐endorphin systems during the estradiol 17β‐induced plasma LH surge in the ovariectomized monkey
Authors: Kerdelhué B. , Jones G. S. , Gordon K. , Seltman H. , Lenoir V. , Parsadaniantz S. Mélik , Williams R. F. , Hodgen G. D.
Published: January 2005

The present work describes time‐dependent changes in the content of corticotropin‐releasing hormone (CRH), adrenocorticotropin (ACTH), and β‐endorphin (β ‐EP) in the hypothalamus (HT) and anterior pituitary (AP) and in the concentration of ACTH and β‐EP in the plasma during the 17β estradiol (E2) benzoate (E2B)‐induced luteinizing hormone (LH) surge in ovariectomized cynomolgus monkeys. Monkeys were euthanized at 0, 30, 48, 72, and 96 hr post‐E2B. HT and AP were rapidly dissected, extracted in 2 N acetic acid containing 1 mM phenylmethane sulfonyl fluoride at 4°C, and centrifuged at 18,000g for 30 min. Peptide concentrations were measured in the supernatant by specific radioimmunoassays (RIAs). In the HT, there were significant (P < 0.05) decreases in ACTH and β ‐EP content by 30 hr post‐E2B and a significant (P < 0.05) decrease in HT CRH content 48 hr post‐E2B. Thereafter, CRH, ACTH, and β‐EP content increased up to 72 hr post‐E2B. In the AP, there was an almost linear decrease in the CRH content through 48 hr post‐E2B followed by a marked 20‐fold (P< 0.01) increase in the AP CRH content at 72 hr post‐E2B, which corresponds to the time of the descending arm of the LH surge. The patterns of ACTH and β‐EP content were very similar in the AP, while that of CRH differed markedly. In contrast, in the HT CRH, ACTH, and β‐EP profiles were very similar. Significant (P< 0.05) increases in circulating levels of ACTH, β‐EP, and cortisol were evident at 30 hr (all 3 hormones), 48 hr (β‐EP and cortisol), and 72 hr (cortisol) post‐E2B, which corresponds with the time of decreased hypothalamic content of CR1I, ACTH, and β‐EP. These results suggest that there may be a marked activation of the hypothalamo‐anterior pituitary‐adrenal axis during the negative and positive feedback phases of the E2B‐induced LH surge in the ovariectomized monkey. © 1995 Wiley‐Liss, Inc.
Transmitter release by non‐receptor activation of the α‐subunit of guanine nucleotide regulatory protein in rat striatal slices
Authors: Zelles T. , Chernaeva L. , Baranyi M. , Déri Z. , Adam‐Vizi V. , Vizi E. S.
Published: January 2005

The effects of 5 mM NaF + 10 μM AIC13, a direct activator of guanine nucleotide‐binding proteins (G proteins), on the release of [3H]dopamine ([3H]DA), [3H]gamma‐aminobutyric acid ([3H]GABA), and [3H]acethylcholine ([3H]ACh) were investigated in slices of rat striatum. When the tissue was exposed to NaF + AIC13 the release of [3H]DA, [3H]GABA, and [3H]ACh was enhanced significantly. In a calcium free solution the release of [3H]GABA and [3H]DA was increased by NaF + A1C13 much more than in the presence of [Ca2+]. In slice preparations taken from reserpinized animals, in which the vesicular storage of [3H]DA was therefore prevented, NaF + AIC13 had no effect on [3H]DA release. HPLC analysis of the radioactivity of the perfusate showed that, in the presence of NaF + AICI3, the content of dihydroxyphenylacetic acid (DOPAC) in perfusate samples increased significantly, while in pargyliue‐treated animals only the DA content was increased. Inhibition of DA carriers by nomifensine or low temperature prevented the effect of NaF + AIC13. N‐ethylmaleimide (NEM) preincubation did not modify the effect of NaF + AICl3 on [3H]DA release. Neomycin (0.1 mM), a phospholipase C (PLC) inhibitor, significantly decreased the effect of NaF + AlC13 on [3H]DA and [3H]GABA release. The internal concentration of Ca2+ in synaptosomes was enhanced by NaF + AIC13 in normal solution. However, [Ca2+]i was not influenced by NaF + AIC13 in Ca2+ ‐free medium. It is concluded that a non‐receptor‐mediated activation, by NaF + AICl3, of the a‐subunit of a G protein, results in a [Ca2+]o,‐independent release of DA and GABA, but not that of ACh. © 1995 Wiley‐Liss, Inc.
A 110‐kD nuclear shuttling protein, nucleolin, binds to the neurite‐promoting IKVAV site of laminin‐1
Authors: Kibbey M. C. , Johnson B. , Petryshyn R. , Jucker M. , Kleinman H. K.
Published: January 2005

The basement membrane protein laminin and the IKVAV‐containing sequence from the laminin αl chain have been found to promote the differentiation of primary neurons and a variety of neural cell lines. We previously reported that a 110‐kd IKVAV‐binding protein (LBP110) isolated from brain appears to be a member of the β‐amyloid precursor protein (APP) family by immunologic and functional studies, which showed that LBP110/APP is also important in neurite outgrowth (Kibbey et al.: Proc Natl Acad Sci USA 90:10150‐10153, 1993). In the preparation of this binding protein, a contaminating IKVAV‐binding protein of identical molecular weight, nucleolin, was also identified. Here we have studied the relationship between these binding proteins. We find that nucleolin binds specifically to the IKVAV sequence independently of LBP110/APP. We have also demonstrated significant levels of nucleolin in mature brain and in differentiating neural cells, suggesting that nucleolin functions not only in cell proliferation and in ribosome biogenesis as was previously reported, but also in the differentiation and maintenance of neural tissue. Our identification of cytoplasmic and cell‐surface nucleolin, an IKVAV‐binding protein, suggests that this protein may function in signalling by extracellular matrix. © 1995 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Fever induced in rats by intrahypothalamic macrophage inflammatory protein (MIP)‐1β: Role of protein synthesis
Authors: Fernández‐Alonso A. , Miñano F. J. , Myers R. D.
Published: January 2005

The effect of macrophage inflammatory protein‐β (MIP‐1β) on body temperature, following its injection into the anterior hypothalamic pre‐optic area (AH/POA), was examined by a radiotelemetry system in the freely moving rat. The purpose of this study was to examine the action of an inhibitor of protein synthesis, anisomycin, on the pyrexia which follows intrahypothalamic injection of MIP‐1β. The microinjection of 10 to 20 pg MIP‐1β into the AH/POA induced a. dose‐dependent monophasic increase in body temperature, whereas a higher dose of 25 pg of the cytokine caused a biphasic febrile response. When MIP‐β was heated at 70°C for 30 min prior to its administration, the pyrogenic response was abolished. Pretreatment of the micro‐injection site in the AH/POA with 10 αg anisomycin did not alter the febrile response to 25 pg MIP‐1β given at the same site in the AH/POA. When 10 mg/kg anisomycin was administered subcutaneously, the febrile response to 25 pg MIP‐1β injected in the AH/POA was significantly suppressed. The present results suggest that fever caused by MIP‐β within the cells of the AH/ POA may not require the synthesis of a new protein factor; however, the de novo synthesis of a protein outside of the AH/POA presumably plays a functional role, at least in part, in the intense fever produced by this cytokine in the hypothalamus. © 1995 Wiley‐Liss, Inc.
Effects of ethanol on neuroblastoma cells in culture: Role of gangliosides in neuritogenesis and substrate adhesion
Authors: Leskawa K. C. , Maddox T. , Webster K. A.
Published: January 2005

Murine Neuro‐2A neuroblastoma cells were exposed to ethanol in culture under two experimental paradigms: (1) short‐term (24 hr or less) and low concentrations (0.05 to 0.5%; 8.5 to 86 mM) and (2) longterm (48 hr at 0.5%; 86 mM). Long‐term ethanol exposure did not affect Neuro‐2A viability, determined by DNA synthesis or the ability to exclude Trypan Blue. Similarly, long‐term ethanol treatment did not inhibit differentiation, exhibited by the extension of neurites, promoted by either dibutyryl‐cyclic‐AMP or by incubation with exogenous ganglioside GMI. The incorporation of exogenous ganglioside GM1 into plasma membranes was not influenced by varying concentrations of ethanol (up to 1.2%; 204 mM). In contrast, ethanol did influence Neuro‐2A cell attachment to collagen in a dualistic manner. During short‐term ethanol exposure, cell attachment was enhanced. However, when cells were initially exposed to ethanol for 48 hr a marked inhibition of subsequent attachment was observed. Long‐term ethanol exposure also inhibited attachment to other substrata, including laminin, fibronectin and vitronectin. Incubation of Neuro‐2A cells with either exogenous ganglioside Gull or a mixture of brain gangliosides partially reversed the inhibition of attachment to collagen. This reversal did not appear to be due to any one particular ganglioside structure, however. Mixed brain gangliosides were fractionated into three fractions, according to the number of sialic acid residues. Each of the three fractions were equally effective in partially restoring Neuro‐2A cell attachment to collagen after long‐term ethanol treatment. The results suggest that the mechanism by which these effects occur is at the level of plasma membrane fluidity, because both ethanol and glycosphingolipid content are known to influence membrane lateral mobility, although other mechanisms, such as changes in headgroup hydration, are possible. © 1995 Wiley‐Liss, Inc.
Perinatal morphine I: Effects on synapsin and neurotransmitter systems in the brain
Authors: Di Giulio A. M. , Tenconi B. , Malosio M. L. , Vergani L. , Bertelli A. , Gorio A.
Published: January 2005

We have previously shown that rat perinatal exposure to morphine causes dopaminergic and met‐enkephalin (ME) and substance P (SP) changes in the striatum during the early postnatal period (Tenconi et al.: Int J Dev Neurosci 10:517‐526, 1992); in addition it increases the susceptibility to neurotoxic lesions and impairs regenerative capacity of the serotoninergic system (Gorio et al.: J Neurosci Res 34: 462‐471, 1993). Our study shows that ME and SP levels increase postnatally in several areas of the rat brain, reaching the highest values between 30 and 60 days, after which the peptide content subsides to lower levels. Perinatal exposure to morphine increases such ME and SP levels during the early stages of postnatal life. No effect of morphine on 5‐HT and NE is observed, while the dopaminergic system is mainly affected in the mesencephalon. The pre‐ and postnatal brain expression of synapsin I mRNA is gradually and progressively localized in discrete areas of the brain. In the brain of rats perinatally exposed to morphine, the abundance of synapsin I mRNA expression is markedly reduced. Therefore, perinatal exposure to morphine affects early postnatal synaptic development in the brain as shown by the altered peptidergic and monoaminergic content and by the reduced synapsin I mRNA expression. © 1995 Wiley‐Liss, Inc.
Role of nerve growth factor in the expression of trkA mRNA in cultured embryonic rat basal forebrain cholinergic neurons
Authors: Kojima M. , Ikeuchi T. , Hatanaka H.
Published: January 2005

Using a quantitative reverse transcription‐polymerase chain reaction (RT‐PCR), we studied the regulation of trkA mRNA expression in serum‐free, cultured basal forebrain neurons from 17‐day fetal rats. Besides increasing choline acetyltransferase (ChAT) activities, nerve growth factor (NGF) strikingly induced trkA gene expression in a time‐ and NGF concentrationdependent manner. Therefore, NGF might play a critical role in trkA gene expression during the development of basal forebrain cholinergic neurons. Furthermore, to investigate whether this up‐regulation is connected with the trophic effects on basal forebrain cholinergic neurons, we examined the effects of some other neurotrophic agents (BDNF, NT‐3, bFGF, CNTF, and 40 mM KCl) upon ChAT activity and trkA gene expression. Some neurotrophic factors increased ChAT activities to the same degree as NGF, whereas they did not stimulate trkA mRNA expression so potently. NT‐3 plus K252b promotes the tyrosine phosphorylation of TrkA in PC12 cells and increases ChAT activity in cultured basal forebrain cholinergic neurons like NGF (Knusel et al., J Neurochem 59: 715–722, 1992). W e found that NT‐3 plus K252b upregulated the level of trkA mRNA. These results suggested that the expression of trkA mRNA is regulated directly by its specific ligand NGF, rather than neurotrophic effects upon basal forebrain cholinergic neurons and that the up‐regulation is connected to a molecular event initiated by the binding of NGF to the TrkA receptor. ©1995 Wiley‐Liss, Inc.
Microtubule‐associated proteins in developing oligodendrocytes: Transient expression of a MAP2c isoform in oligodendrocyte precursors
Authors: Vouyiouklis D. A. , Brophy P. J.
Published: January 2005

The morphological differentiation of oligodendrocytes is characterized by the formation of multiple, microtubule‐rich processes which endow these cells with the ability to myelinate many axons simultaneously. Since microtubule‐associated proteins (MAPs) strongly influence the structure and function of microtubules, we have investigated their expression in cultured differentiating oligodendrocytes in order to gain insights into MAP function during process formation and stabilization. MAPIB has been compared with two other structural MAPs: MAP4, which is an ubiquitously expressed protein, and MAP2, which hitherto was thought to be confined to neurons and reactive astrocytes. Immunof uorescence microscopy showed that the colocalization of MAP4 with microtubules in oligodendrocyte processes is not as extensive as found previously for MAP1B (Vouyiouklis and Brophy: J Neurosci Res 35:257–267, 1993). Nevertheless, like MAP1B, the expression of MAP4 increases during oligodendrocyte differentiation. In contrast, the expression of MAP2 is transiently elevated in preoligodendrocytes but declines precipitously at the onset of terminal differentiation. Cells of the oligodendrocyte lineage exclusively express a novel isoform of MAP2c which is primarily localized in the cell bodies of preoligodendrocytes. This suggests that MAP2c assists in the initiation of process extension rather than in the stabilization of microtubules in the cytoplasm‐filled membranous extensions of mature cells. MAP‐tau was not expressed at any developmental stage by oligodendrocytes. The distinct subcellular localizations and patterns of developmental expression of MAP1B, MAP4, and MAP2c suggest that these MAPs have different roles in the regulation of the microtubule network during the differentiation of myelin‐forming oligodendrocytes. ©1995 Wiley‐Liss, Inc.