bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and phosphorylation of the mid‐sized neurofilament protein NF‐M during chick spinal cord neurogenesis
Authors: Bennett G. S. , Hollander B. A. , Laskowska D.
Published: January 2005

The middle molecular weight polypeptide of neurofilaments (NF‐M) is modified posttranslationally by extensive phosphorylation. This modification is slow in mature neurons, requiring approximately 24–48 hr for completion and probably occurs outside of the cell soma (Bennett and DiLullo: J Cell Biol 100:1799, 1985c). Thus, NF‐M synthesis and phosphorylation are separate events both temporally and spatially. Although it is known that NF‐M is among the earliest neuron‐specific gene products to be expressed during nervous system development, it is not known what the temporal relationship is between the initiation of NF‐M translation and its phosphorylation. To address this question, we have produced an antiserum against the dephosphorylated form of NF‐M (NF‐M130) and have used this antiserum, together with a previously characterized antiserum against completely phosphorylated NF‐M (NF‐M160), in an immunohistochemical examination of neurogenesis and the initial period of neuronal differentiation in chick spinal cord. We found that (1) nonphosphorylated and partially phosphorylated NF‐M cannot be detected prior to the completion of the terminal mitosis; (2) most postmitotic neuroblasts begin expressing NF‐M as they commence migration, but do not contain the completely phosphorylated polypeptide until some time after completion of migration; and (3) those precursor cells of a subpopulation of neuroblasts that begin expressing completely phosphorylated NF‐M during their terminal cell cycle (Bennett and DiLullo: Dev Biol 107:94, 1985a) contain no detectable nonphosphorylated or partially phosphorylated NF‐M. These cells probably complete the phosphorylation step more rapidly than do mature neurons.
Tissue plasminogen activator binding to mouse cerebellar granule neurons
Authors: Verrall S. , Seeds N. W.
Published: January 2005

Cultures of dissociated neonatal mouse cerebellar cells secrete primarily tissue plasminogen activator (tPA) and to a lesser extent urokinase plasminogen activator (uPA) into the culture medium. Fibrin overlays have localized plasminogen activator to granule neurons in these cultures; furthermore, this granule cell plasminogen activator activity is blocked by an antibody to tPA. Developmental studies indicate that maximal levels of soluble plasminogen activator in the culture medium preceed the peak of fibrinolytic activity by these cultures, suggesting that secreted PA may bind back to the surface of these granule neurons. Here we show that granule cell‐associated tPA can be displaced by a brief pH shock. However, incubation of these fibrinolytically inactive cultures with exogenously added mouse tPA leads to a specific binding of active tPA to granule neurons as visualized by subsequent fibrin overlay. In similar studies mouse uPA, human uPa, and human tPA fail to show fibrinolytic activity associated with the cerebellar culture, whereas mouse tPA fails to bind to cerebellar glial cell cultures. These findings suggest that granule neurons possess binding sites for tPA on their surface, where this protease can retain its functional activity and may play an important role in cell migration or other cell activities.
Corticorubral connections: Ultrastructural evidence for homotypical synaptic reinnervation after developmental deafferentation
Authors: Fisher R. S. , Sutton R. L. , Hovda D. A. , Villablanca J. R.
Published: January 2005

Corticorubral (CR) connections are reorganized after the red nucleus is deafferented by neonatal hemispherectomy. Our objective was to determine if the new decussating CR axons establish synaptic junctions with their target neurons. The ultrastructure of CR axonal terminal arbors was identified by the anterograde transport and histochemical labeling of wheat germ agglutinin lectin‐bound horseradish peroxidase injected unilaterally into the pericruciate neocortex of adult cats that received unilateral neonatal hemispherectomy (HEMI cases) and normal adult cats (control cases). The regional organization of undecussated and decussated CR arbors was nearly equivalent in the HEMI cases. The fine structure of both connections was uniform. Asymmetrical synaptic junctions were observed in frequent axodendritic and infrequent axosomatic boutons en passage and boutons terminaux. The undecussated arbors formed the principal connection in the control cases. However, the fine structures of the decussated and undecussated arbors were comparable and matched the arrangements in the HEMI cases. We conclude that developmental deafferentation redirects CR axons to provide stable synaptic afferents to new targets. The reinnervation is homotypical in terms of regional and fine structure organization. It is likely to be an important factor in chemically mediated neurotransmission that contributes to the sparing/recovery of function after brain damage.
Role of neuron‐target interactions in the development of a subpopulation of avian sensory neurons
Authors: Marusich M. F. , Weston J. A.
Published: January 2005

Subpopulations of sensory neurons appear in embryonic dorsal root ganglia during development. In this paper, we examine what role neuron‐target interactions play in this process. Previous work has shown that alterations in the environment of developing sensory neurons regulate the proportion of neurons that express a cell surface antigen identified by the monoclonal antibody SN1 (Marusich et al.: Dev. Biol. 118: 505–510, 1986). We now report that neuron‐target interactions may also act to stabilize the phenotype of sensory neurons. Thus, older SN1− neurons, which would normally remain SN1− if left undisturbed in vivo, can express SN1 immunoreactivity in vitro when they are deprived of contact with their normal peripheral targets. We also demonstrate that naive sensory neurons, i.e., those that have never made contact with peripheral targets, can be identified and maintained in culture. At least some of these naive neurons (all of which are initially SN1−) can express SN1 immunoreactivity in vitro, in the absence of contact with normal peripheral targets. We conclude that subpopulations of sensory neurons may arise from naive neurons in the absence of neuron‐target interactions but that subsequent neuron‐target interactions may act to stabilize or modulate subpopulation‐specific phenotypes.
Identification and cDNA cloning of a new member of the L2/HNK‐1 family of neural surface glycoproteins
Authors: Gennarini G. , Rougon G. , Vitiello F. , Corsi P. , Di Benedetta C. , Goridis C.
Published: January 2005

Rabbit antibodies raised against a 135‐ to 140‐kD glycoprotein isolated from the culture medium of mouse forebrain explants were used for the identification and cloning of a complex of mouse neural cell surface glycoproteins. The antibodies recognized a 135‐kD surface protein which shared the L2/HNK‐1 epitope with several neural cell adhesion molecules. Three homologous complementary deoxyribonucleic acid (cDNA) clones were isolated from a mouse brain cDNA library prepared in the expression vector lambda gt11, one of which was sequenced and found to lack sequence homologies with known proteins. In Northern blots, this clone hybridized with a single 6.3 kb messenger ribonucleic acid (mRNA). In immunoblots of mouse brain extracts, antibodies raised in rabbits against the fusion protein encoded by it stained two glycoproteins of 135 and 90 kD, which we designated F3.135 and F3.90. In the developing mouse cerebellum, F3 antigenic sites were found predominantly on parallel fibers and on postmitotic neurons. In fetal brain cell cultures, F3 antigen was detected at the surface of cells with neuronal morphology, but the antibodies also stained some non‐neuronal cells in a pattern characteristic of matrix components. Because all proteins carrying the L2/HNK‐1 epitope identified so far have a role in cell adhesion, it can be anticipated that the F3 surface proteins also are involved in cellinteraction phenomena.
A Soluble brain molecule related to epidermal growth factor receptor is a mitogen inhibitor for astrocytes
Authors: Nieto‐Sampedro M. , Broderick J. T.
Published: January 2005

The astrocyte mitogenic activity of normal and injured rat brain extracts was greatly enhanced by antibodies to epidermal growth factor receptor (EGFR). The antibodies appear to act by removing from the extracts inhibitory molecules immunologically related to EGFR. Three molecular species recognized by anti‐EGFR antibody in brain extracts (molecular weights 41, 52, and 69 kilodaltons) did not seem to originate from EGFR proteolysis. The increase in astrocyte mitogenic activity in brain tissue following injury correlated with a reduction in the levels of soluble EFGR‐cross‐reacting material and a decrease in mitogen inhibitory activity. The decrease in EGFR‐related mitogen inhibitor also correlated with a large increase in astrocyte membrane EGFR immunoreactivity, and intracerebral injection of antibodies to EGFR caused the appearance at the injection site of numerous EGFR‐positive reactive astrocytes. Invasion of brain tissue by EGF/EGFR‐related blood components may be the signal that initiates astrocyte activation. EGFR‐related immunoreactive molecules are also present in extracts of other tissues and may have a general role in the control of cell division.
Enzyme translocation in the course of regeneration of central primary afferent terminals in the substantia gelatinosa of the adult rodent spinal cord
Authors: Knyihár‐Csillik E. , Kreutzberg G. W. , Csillik B.
Published: January 2005

Synaptic circuitry in the upper dorsal horn, disorganized due to transganglionic degenerative atrophy (evoked by blockade of retrograde axoplasmic transport in the related peripheral nerve), begins to be reestablished by a process of regenerative synaptoneogenesis as soon as retrograde transport is resumed. Central axons of type C primary sensory neurons, terminating in substantia gelatinosa Rolandi, as well as their cells of origin in dorsal root ganglia are specifically and selectively labeled by the enzyme thiamine monophosphatase (TMPase). Under normal conditions, TMPase is localized in axolemmal membranes at the electron histochemical level. In axonal growth cones of regenerating central terminals, only negligible TMPase reaction was found. In axonal filopodia and young nerve sprouts, there appears an increasing number of intraaxonal grains of the reaction product. Vanicous swellings (beads) of regenerating axonal sprouts are transformed into scalloped (sinusoid) en passant terminals. TMPase reaction end product, present initially in the axoplasm of beads and scalloped terminals, is successively translocated to the axolemmal membrane in the course of cytochemical maturation. Structural regeneration and cytochemical maturation of central terminals of primary sensory neurons are completed 60 days after crush injury to the related peripheral axons, i.e., about 7 weeks after the peripheral nerve has regenerated.
Reversibility and prevention of intraspinal peptidergic loss caused by sciatic nerve lesions
Authors: Di Giulio A. M. , Tenconi B. , Mantegazza P. , Gorio A.
Published: January 2005

We have investigated the reversibility and prevention of peptidergic losses in the lumbar spinal cord caused by permanent resection of sciatic nerve. The lesion triggers a series of degenerative events involving the substance P sensory imput as well as met‐enkephalin interneurons of the substantia gelatinosa. The degenerative processes are evident 10 days after sciatic nerve lesion and are gradually reversible as shown by radioimmunoassay and quantitative immunocytochemistry. Recovery begins 30 days postlesioning and full restoration is observed at 90 days. Serotonin (5‐HT) turnover is markedly affected by sciatic nerve lesion as soon as 24 hr postlesioning, when 5‐HT metabolism is enhanced, returning to control levels just preceding the peptide alterations; 5‐HT metabolism then undergoes a transient period of hypoactivity which correlates with the beginning of the peptidergic restorative processes. Altogether these results, with previous observations showing that 5‐HT depletion prevents metenkephalin interneurons degeneration triggered by the lesion (Di Giulio et al.: J Neurosci Res 18:443–448, 1987), suggest a role for 5‐HT in the synaptic plasticity of the substantia gelatinosa of the spinal cord. The administration of gangliosides (10 or 50 mg/kg) to sciatic‐nerve‐lesioned animals fully prevented the metenkephalinergic degeneration without affecting the degenerative atrophy of the lesioned substance P sensory imput.
Interleukin‐1 Interaction with neuroregulatory systems: Selective enhancement by recombinant human and mouse interleukin‐1 of in vitro opioid peptide receptor binding in rat brain
Authors: Wiedermann C. J.
Published: January 2005

Interleukin‐1 (IL‐1) exerts a wide variety of biological effects on various cell types and may be regarded as a pleiotropic peptide hormone. Biological evidence suggests that IL‐1 participates in the modulation of central nervous system physiology and behaviour in a fashion characteristic of neuroendocrine hormones. In this investigation, recombinant (r) human (h) IL‐1 and r mouse (m) IL‐1 were examined for their modulation of opioid peptide receptor binding in vitro. Experiments were performed on frozen sections of rat brain. Receptor binding of radiolabeled substance P and of radiolabeled neurotensin were not significantly affected by the presence of rIL‐1s. Recombinant IL‐1s, however, significantly enhanced specific binding of 125I‐beta‐endorphin (125I‐beta‐END) and of D‐ala2‐(tyrosyl‐3,5‐3H)enkephalin‐(5‐D‐leucine) (3H‐D‐ALA), equipotently and in a concentration‐dependent manner with maximal activity occurring at a concentration of 10 LAF units/ml. The increased binding of 125I‐β‐END and 3H‐D‐ALA was blocked steroselectively by (−)‐naloxone and by etorphine, suggesting detection of opiate receptors. In addition, brain distribution patterns of receptors labeled in the presence of rIL‐1s corresponded to patterns previously published for opiate receptors. Autoradiographic visualization of receptors revealed that rIL‐1s in the different areas of the brain exert their effect on opioid binding with comparable potencies. The data suggest that certain central nervous system effects of IL‐1s may be mediated by their selective interaction with opiatergic systems at the receptor level.
Potassium‐stimulated calcium uptake in astrocytes and its potent inhibition by nimodipine
Authors: Hertz L. , Bender A. S. , Woodbury D. M. , White H. S.
Published: January 2005

Elevation of the extracellular potassium concentration above its “resting” level of 5.4 mM stimulated uptake of 45Ca2+ in primary cultures of astrocytes. This effect was only observed when cells were exposed to excess potassium shortly after their exposure to 45Ca2+ and was potently inhibited (IC50 ⋍ 3 nM) by the calcium channel blocker nimodipine. In contrast, nimodipine exerted little effect on unstimulated basal uptake of 45Ca2+. These findings suggest that the therapeutic benefit of calcium channel blockers in epilepsy may result in part from the ability of these drugs to prevent calcium entry into astrocytes during seizures when the extracellular potassium is elevated four‐ to fivefold above normal.
Pharmacological characterization of the glutamate receptor in cultured astrocytes
Authors: Backus K. H. , Kettenmann H. , Schachner M.
Published: January 2005

Cultured astrocytes from neonatal rat cerebral hemispheres are depolarized by the excitatory neurotransmitter glutamate. In this study we have used selective agonists of different neuronal glutamate receptor subtypes, namely, the N‐methyl‐D‐aspartate (NMDA), kainate, and quisqualate type, to characterize pharmacologically the glutamate receptor in astrocytes. The agonists of the neuronal quisqualate receptor, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐4‐propionic acid (AMPA) and quisqualate, depolarized the membrane. Kainate, an agonist of the neuronal kainate receptor, depolarized astrocytes more effectively than quisqualate. Combined application of kainate and quisqualate depolarized astrocytes to a level which was intermediate to that evoked by quisqualate and kainate individually. Agonists activating the neuronal NMDA receptor, namely NMDA and quinolinate, were ineffective. Application of NMDA did not alter the membrane potential even in combination with glycine or in Mg2+‐free solution, conditions under which neuronal NMDA receptor activation is facilitated. The nonselective agonists L‐cysteate, L‐homocysteate, and β‐N‐oxalylamino‐L‐alanine (BOAA) mimicked the effect of glutamate. Dihydrokainate, a blocker of glutamate uptake, did not, and several antagonists of neuronal glutamate receptors only slightly affect the glutamate response. These findings suggest that astrocytes express one type of glutamate receptor which is activated by both kainate and quisqualate, lending further support to the notion that cultured astrocytes express excitatory amino acid receptors which have some pharmacological similarities to their neuronal counterparts.
Superoxide dismutase and cytochrome oxidase activities in light and heavy synaptic mitochondria from rat cerebral cortex during aging
Authors: Vanella A. , Villa R. F. , Gorini A. , Campisi A. , Giuffrida‐Stella A. M.
Published: January 2005

The specific activities of superoxide dismutase (SOD) and cytochrome oxidase (COX) in light and heavy synaptic mitochondria from rat cerebral cortex at different ages (4, 8, 12, 16, 20, and 24 months) have been measured. The specific activity of COX was significantly higher in light synaptic mitochondria than in heavy ones at all ages examined. However, no significant difference during aging was shown. A marked decrease of SOD in light and heavy synaptic mitochondria during aging was observed.
Expression of nerve growth factor receptors by human peripheral blood mononuclear cells
Authors: Morgan B. , Thorpe L. W. , Marchetti D. , Perez‐Polo J. R.
Published: January 2005

In the rat, nerve growth factor (NGF) has been shown to affect immune reactivity by binding to cell surface receptors on a subpopulation of splenic mononuclear cells. This binding occurs in a specific and saturable fashion to what appear to be low‐affinity (type II) NGF receptors (NGFR). Immunofluorescence studies here showed that NGFR are also present on a proportion of human peripheral blood mononuclear cells (PBMC). Equilibrium binding studies demonstrated that the binding of NGF to its receptors on PBMC occurs with a single equilibrium binding constant (mean) of 2.11 × 10−9 M. The number of receptors per cell was determined to be approximately 6.94 × 103 receptors/cell. These results would suggest a role for NGF in the regulation of immune function in man, as well as in animals.
Isolation of a Thy‐1‐like glycoprotein from cat brain: Distribution in retina and brain defined by monoclonal antibodies
Authors: Saleh M. , Bartlett P. F.
Published: January 2005

Thy‐1 is a glycoprotein primarily expressed in the nervous system of a large number of vertebrate species. A concanavalin‐A binding glycoprotein with a molecular weight of 25, 000 daltons has been isolated from cat brain, which corresponds to the Thy‐1 molecule isolated in other species. Monoclonal antibodies were generated against this cat Thy‐1 molecule, and immunohistochemical staining on adult cat brain frozen sections demonstrated a localisation of the molecule to regions rich in synapses. In the cerebellum, the immunoperoxidase staining with the antibodies was intense in the molecular layer, the fibres of the granular layer, and the Purkinje cell bodies. This distribution of Thy‐1 is consistent with that seen with the rodent and chicken Thy‐1 molecule. In the retina, one antibody (WEHY‐MS‐1) detected the cat Thy‐1 molecule in the ganglion cell layer and on fibres of the inner plexiform and inner nuclear layers. Another antibody (WEHY‐MS‐2) produced a similar retinal staining pattern except that the ganglion cell layer was devoid of Thy‐1. Although the Thy‐1 molecule in the rodent and chicken retina have been reported in the ganglion cell and inner plexiform layers, there have been no consistent reports of Thy‐1 in the inner nuclear layer. This suggests that this putative cat Thy‐1 molecule may have a retinal distribution pattern different from that seen in other species.
Purification and culture of adult rat dorsal root ganglia neurons
Authors: Delree P. , Leprince P. , Schoenen J. , Moonen G.
Published: January 2005

To study the trophic requirements of adult rat dorsal root ganglia neurons (DRG) in vitro, we developed a purification procedure that yields highly enriched neuronal cultures. Forty to fifty ganglia are dissected from the spinal column of an adult rat. After enzymatic and mechanical dissociation of the ganglia, myelin debris are eliminated by centrifugation on a Percoll gradient. The resulting cell suspension is layered onto a nylon mesh with a pore size of 10 μm. Most of the neurons, the diameter of which ranged from 17 μm to > 100 μm, are retained on the upper surface of the sieve; most of the non‐neuronal cells with a caliber of < 10 μm after trypsinization go through it. Recovery of neurons is achieved by reversing the mesh onto a Petri dish containing culture medium. Neurons to non‐neurons ratio is 1 to 10 in the initial cell suspension and 1 to 1 after separation. When these purified neurons are seeded at a density of 3, 000 neurons/cm2 in 6 mm polyornithine–‐laminin (PORN‐LAM) coated wells, neuronal survival (assessed by the ability to extend neurites), measured after 48 hr of culture, is very low (from 0 to 16%). Addition of nerve growth factor (NGF) does not improve neuronal survival. However, when neurons are cultured in the presence of medium conditioned (CM) by astrocytes or Schwann cells, 60–80% of the seeded, dye‐excluding neurons survive. So, purified adult DRG neurons require for their short‐term survival and regeneration in culture, a trophic support that is present in conditioned medium from PNS or CNS glia. As NGF has no survival‐promoting effect on these purified adult neurons, the trophic effect of CMs is likely to be mediated by non‐NGF molecule(s).
TIS gene expression in cultured rat astrocytes: Induction by mitogens and stellation agents
Authors: Arenander A. T. , Lim R. W. , Varnum B. C. , Cole R. , de Vellis J. , Herschman H. R.
Published: January 2005

The expression of a number of TIS genes (Lim et al.:Oncogene 1:263–270, 1987) was examined in secondary cultures of rat neocortical astrocytes treated with mitogens and stellation agents, to study the early nuclear events which accompany the induction of glial proliferation and/or differentiation. Tetradecanoyl phorbol acetate (TPA), epidermal growth factor, and firoblast growth factor, three mitogens for astrocytes, stimulated marked, rapid, and transient increases in TIS gene mRNAS. TIS10, which is not expressed in rat PC12 pheochromocytoma cells, could be induced by these mitogens in rat astrocytes. Dibutyryl cyclic adenosine monophosphate and forskolin, which induce rapid stellation in astrocytes, and ganglioside GM1, a potent mitogen as well as an antagonist of the induction and maintenance of stellation, all induced TIS gene expression. Thus, a broad range of agents which elicit both proliferative and differentiation responses in astrocytes are capable of inducing a family of genes that may play a role in the early events of signal transduction.
UDP galactose: Ceramide galactosyltransferase, CDP choline:1, 2‐diacyl‐sn‐glycerol phosphocholine transferase, and microsomal reductases in major regions of the developing rat brain in nutritional stress
Authors: Padmini S. , Rao P. Srinivasa
Published: January 2005

The influence of nutritional inadequacy during the active growth phase of the developing brain, coinciding with myelinogenesis, was examined in rat pups. Developmental profiles of enzyme activities involved in biosynthesis of myelin membrane lipids, and those of associated pathways which generate precursor compounds for such biosynthetic reactions, were followed as functions of age in major anatomical regions of the brain. It was observed that while galactosyltransferase and choline phosphotransferase activities were significantly diminished, the microsomal cytochrome reductases, which contribute to the process of fatty acid elongation and desaturation, were also lowered. An attempt at adaptative increase of enzyme activity, in response to the stress, was apparent in the case of glycerol‐3‐phosphate dehydrogenase, which could possibly explain, in part, the lower susceptibility of phospholipid biosynthesis to impairments induced by nutritional insufficiency.
Evidence from neuronal heterokaryons for a trans‐acting factor suppressing THY‐1 Expression during neuronal development
Authors: Saleh M. , Bartlett P. F.
Published: January 2005

The expression of Thy‐1 on developing sensory neurons cultured in vitro is strictly regulated and follows a temporal course identical to that observed in situ. We have investigated the role of trans‐acting factors in this innate regulation by constructing heterokaryons between mature Thy‐1.1+ expressing neurons and immature Thy‐1.2− neurons. It has been observed by immunofluorescence that within 16 hr of fusion, Thy‐1.1 expression is suppressed in such heterokaryons. However, this suppression is reversible, and after 3–4 days in vitro, at the time at which Thy‐1.2 is normally expressed, there is re‐expression of the Thy‐1.1 molecule producing Thy‐1.1+/Thy‐1.2+ co‐expressing heterokaryons. As nuclear fusion does not occur, it appears that a developmentally regulated diffusible suppressor molecule is responsible for these observations.
Patterns of proto‐oncogene expression in human glioma cell lines
Authors: LaRocca R. V. , Rosenblum M. , Westermark B. , Israel M. A.
Published: January 2005

Previously reported studies have suggested that variations in the pattern of proto‐oncogene expression within a specific tumor type may denote an underlying difference in the biology and clinical behavior of those tumors. To more sensitively characterize malignant tumors of the central nervous system, we have used Northern blot hybridization analysis to determine the patterns of expression of seven proto‐oncogenes in 20 cell lines established from biopsy specimens of patients with malignant glioma. The following proto‐oncogenes are expressed at detectable levels in 30 μg of total RNA from most glioma cell lines examined: c‐myc (20/20), c‐mil/raf‐1 (18/18), neu (19/20), c‐erbB (19/20), and c‐myb (17/20). In contrast, only half of the cell lines expressed detectable c‐sis (10/20). In none of the cell lines tested was N‐myc (0/20) mRNA detected. Morphologic analysis of these 20 cell lines revealed three different growth patterns: bipolar, epithelial, and pleomorphic‐glial. Detectable levels of c‐sis mRNA typically occurred with either an epithelial or pleomorphic‐glial morphology. The pleomorphic‐glial subgroup was also characterized by the expression of glial fibrillary acidic protein.
Expression of vimentin by cultured astroglia and oligodendroglia
Authors: Meyer S. A. , Ingraham C. A. , McCarthy K. D.
Published: January 2005

The objective of this study was to determine whether vimentin expression by process‐bearing astroglia and oligodendroglia cultured from neonatal rat cerebral cortex resembled that in brain where vimentin is common in immature astroglia and a few subpopulations of mature astroglia, but is absent in oligodendroglia. Vimentin expression was detected by immunofluorescence microscopy using a monoclonal antibody (V9) against porcine lens vimentin in combination with either antiserum against the astroglial marker, glial fibrillary acidic protein (GFAP), or with antiserum against the oligodendroglial marker, galactocerebroside (GC). Specificity of the antivimentin antibody was indicated on immunoblots of process‐bearing cell proteins separated by two‐dimensional polyacrylamide gel electrophoresis. Enrichment of cultures for either GFAP+ astroglia or GC+ oligodendroglia was achieved by supplementation of the culture medium with fetal calf serum at 10% or 0.5%, respectively. Process‐bearing cells maintained in 10% serum exhibited heterogeneity in their expression of GFAP and vimentin. Approximately half of the cells were GFAP+/vimentin+ throughout the 2‐week culture period examined. GFAP+/vimentin− cells were a minor population at early times (3–4 days) in culture, but accounted for 40% of process‐bearing cells after 2 weeks. Cultures maintained in reduced (0.5%) serum and stained for GC and vimentin also exhibited heterogeneity. Both GC+/vimentin+ and GC+/vimentin− cells were observed, with vimentin+ cells composing two‐thirds and one‐half of the GC+ population after 3 and 6 days, respectively, in reduced serum. The high incidence of vimentin expression by process‐bearing astroglia and oligodendroglia suggests that these cultures contain glia in a relatively early stage of development. The decrease in vimentin expression with time in culture suggests that the specific developmental processes which lead to a reduction in vimentin in brain during maturation may be exhibited by process‐bearing glia in vitro.
Chelation of endogenous membrane calcium inhibits γ‐aminobutyric acid uptake in synaptosomes
Authors: Tapia R. , Salazar C.
Published: January 2005

In a previous work, we have demonstrated that calcium chelators induce the release of γ‐aminobutyric acid (GABA) from synaptosomes in a Na+ ‐dependent manner and that this release is blocked by cations such as Mg2+, La3+, and ruthenium red. In the present study, we show that treatment of synaptosomes with 0.1 mM EGTA in the absence of both Ca2+ and Mg2+ inhibits the sodium‐dependent high‐affinity uptake of [3H]GABA by about 50%. This inhibition increased to about 65% with 1.5 mM EGTA, and it was completely prevented by an excess of Ca2+ or by 1.2 mM Mg2+. In contrast, when EDTA was used as a chelator, Mg2+ was unable to reverse the inhibition. The inhibitory effect of 0.1 mM EGTA was also prevented by 250 μM La3+ or by 20 μM ruthenium red. In the absence of chelators and the presence of Ca2+ and Mg2+, 50 μM and 200 μM La3+ inhibited GABA uptake by about 20 and 50%, respectively, whereas 20 μM ruthenium red produced a nonsignificant 25% inhibition and nifedipine was without effect. It is concluded that the membranesurface negative charges, probably those of the sialic acid molecules that have been implicated in the functioning of the GABA carrier, must be neutralized by endogenous Ca2+ or by another cation in order to permit the adequate function of the transporter. The inhibition by La3+ in the absence of the chelators could be explained by a binding of this cation to the Na+ sites on the GABA carrier.
Differential control of tryptophan hydroxylase expression in raphe and in pineal gland: Evidence for a role of translation efficiency
Authors: Dumas S. , Darmon M. C. , Delort J. , Mallet J.
Published: January 2005

In a previous study, we characterized two tryptophan hydroxylase mRNAs (TPH mRNAs) in the pineal gland. However, we failed to detect these species in the raphe by Northern blot experiments. Here, we report by S1 nuclease analysis and in situ hybridization that these two TPH mRNAs, as well as a third species, are expressed both in pineal gland and in raphe. In both tissues, the three mRNAs are transcribed predominantly from the same promoter. Strikingly, from the results of S1 maping analysis, it was observed that the total level of TPH mRNA per tissue is at least 150 times lower in the raphe than in the pineal gland. In contrast, TPH antigen as quantified by immunoblot experiments is about threefold more abundant per raphe than per pineal gland. TPH mRNA from one raphe and one pineal gland yield in vitro about the same amount of TPH antigen, suggesting that the discrepancy in the ratios of TPH mRNA and TPH antigen between the raphe and the pineal gland results, at least in part, from a difference in the translation efficiency of TPH mRNAs in the two structures.
Development of neurophysin‐containing neurons in primary cultures of rat hypothalami is related to the age of the embryo: Morphological study and comparison of in vivo and in vitro neurophysins, oxytocin, and vasopressin content
Authors: Di Scala‐Guenot D. , Strosser M. T. , Sarlieve L. L. , Legros J. J. , Richard P.
Published: January 2005

The morphological development of immunocytochemically identified neurophysin neurons and the evolution of neuropeptide content (neurophysins, vasopressin, and oxytocin) were studied in primary cultures of hypothalami obtained from 15‐to 19‐day‐old embryos. According to their perikaryal surface, two populations of neurons were distinguished: large and small cells. Full development (defined by the perikaryal surface) of these neurons was reached at day 21 only in cultures from 15‐ or 16‐day‐old embryos. These two types of neurons may correspond to the magnocellular and parvocellular neurons described in vivo. Total neurophysins, vasopressin, and oxytocin content were measured by specific radioimmunoassays. Ontogeny of neurophysins and vasopressin showed a good correlation between cells cultured from 15‐ to 16‐day‐old embryos and hypothalami from age‐matched rats. However, oxytocin was never detected in any of the cultures whatever the age of the embryos. Under our experimental conditions, hypothalamic primary cultures from 15‐ to 16‐day‐old embryos therefore appeared to be suitable for studying the differentiation and regulation of neurophysin‐and vasopressin‐containing neurons.
Coupling of glutamatergic receptors to changes in intracellular Ca2+ in rat cerebellar granule cells in primary culture
Authors: Holopainen I. , Louve M. , Enkvist M. O. K. , Akerman K. E. O.
Published: January 2005

Changes in cytosolic free Ca2+ concentrations, [Ca2+]i, in response to glutamate and glutamate receptor agonists were measured in rat cerebellar granule cells grown on coverlips. The intracellular Ca2+ as measured with fura‐2increased by applying kainate, N‐methyl‐D‐aspartate (NMDA), quisqualate, and (RS)‐d‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic (AMPA). When the extracellular Mg2+ was removed, the effects of NMDA and the NMDA receptor agonist cis‐(±)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid (cis‐ACPD) ON intracellular Ca2+ were augmented. Glycine potentiated the effects of NMDA and cis‐ACPD if the membrane was depolarized by increasing the extracellular K+ concentration. The NMDA receptor antagonist DL‐2‐AMONO‐5‐phosphonopentanoic acid (AP5) abolished and the antagonist 3‐([±]‐2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonic acid (CPP) greatly reduced the effect of NMDA in both the normal and the Mg‐free media. The dose‐ response curves of NMDA and, to a lesser extent, of kainate were shifted to the left, and that of quisqualate became biphasic in the Mg‐free medium. The increase in [Ca2+]i produced by high quisqualate concentrations in the Mg‐free medium was totally abolished by AP5. The results suggest that Ca2+ influx in cerebellar granule cells occurs through both NMDA‐ and non‐NMDA‐Coupled ion channels. A part of the quisqualate‐induced rise in cytosolic Ca2+ seems to be linked to the activation of NMDA recptors.
Bilateral recovery of striatal dopamine after unilateral adrenal grafting into the striatum of the 1‐methyl‐4‐(2′methylphenyl)‐1,2,3,6‐tetrahydropyridine (2′CH3‐MPTP)‐treated mouse
Authors: Bohn M. Churchill , Kanuicki M.
Published: January 2005

A rodent model of Parkinson's disease, the 1‐methyl‐4‐(2′methylphenyl)‐1,2,3,6‐tetrahydropyridine treated mouse, was used to determine whether striatal dopamine levels recover following grafting adrenal medulla into the striatum. Four types of grafts were performed: (1) adult mouse adrenal medulla, (2) adult adrenal medulla that had been freeze‐thawed to kill viable cells, (3) postnatal day 7 adrenal medulla, and (4) sham grafts lacking tissue. At 1 month after grafting, only postnatal day 7 grafts contained surviving cells. However, all three types of tissue grafts promoted a unilateral recovery of host dopaminergic fibers on the side of the graft. In striking contrast to the unilateral recovery of the dopaminergic fibers, striatal dopamine levels were increased bilaterally in all tissue grafted mice. These observations suggest that adrenal tissue grafted into the striatum, whether it remains viable or not, has more widespread biochemical effects on the host dopaminergic system than previously recognized. Moreover, these observations bear on mechanisms that may underlie the general recovery of motor disturbances reported in human Parkinson's disease patients who have received a striatal graft of adrenal tissue.
Identification and isolation of an axonal plasma membrance enriched fraction from cerebellar granule cell neurites
Authors: Mason P. W. , Russell T. L. , DeVries G. H.
Published: January 2005

A procedure is described to isolate a fraction enriched in cerebellar granule cell neuritic membranes. Morphological markers that are specific for either the granule cell perikarya or neuritic membranes have been identified. Concanavalin A (Con A) has been shown to bind predominantly to the granule cell neurites whereas, the enzymes acetylcholinesterase (AChE) and 2′,3′, cyclic nucleotide‐3‐phosphohydrolase (CNPase) are localized predominantly in the neuronal cell bodies. The membrane fraction enriched in Con A binding has been used to generate a monoclonal antibody which morphologically recognized the cerebellar granule cell nueritic membrance. Following fractionation of the granule cells, each marker was used to identify the cellular origin of the fractions. The neuritic markers Con A and the neuritic membrane antibody MR2 bound predominately to membrances found in the 29.1% and 31.5% region of the sucrose gradient. The perikaryal markers, CNPase and AChE activity were most enriched in membrance fractions found at a sucrose concentration of 23% and 21%, respectively. Morphological examination of the neuritic enriched fraction shows that it contains predominately membranous material with few subcellular organelles. The protein profiles of the cerebellar granule cell fractions are unique when compared with the protein profiles of other neuronal and non‐neuronal fractions. The membrane fraction isolated from the cerebellar granule cells should prove useful in furthering our understanding of the axonal influence on glial development.
Neuroplasticity in the olfactory system: Differential effects of central and peripheral lesions of the primary olfactory pathway on the expression of B‐50/GAP43 and the olfactory marker protein
Authors: Verhaagen J. , Oestreicher A. B. , Grillo M. , Khew‐Goodall Y.‐S. , Gispen W. H. , Margolis F. L.
Published: January 2005

The regeneration of the olfactory neuroepithelium following olfactory bulbectomy or peripheral deafferentation was studied with mRNA probes and antibodies for B‐50/GAP43 and for olfactory marker protein (OMP). Two stages in the regeneration of the olfactory epithelium could be discerned with these reagents. The first stage occurs following either peripheral deafferentation of the olfactory epithelium with Triton X‐100 (TX‐100) or after bulbectomy and is characterized by the formation of a large population of immature olfactory receptor neurons. These newly formed neurons express B‐50/GAP43, a phosphoprotein related to neuronal growth and plasticity. During the second stage of the regeneration process the newly formed olfactory neurons mature, as evidenced by a decrease in their expression of B‐50/GAP43 and an increase in the expression of OMP. This stage is only manifested if the developing neurons have access to the target olfactory bulb. Formation of a full complement of OMP‐expressing neurons occurs only after peripheral lesion with TX‐100. In contrast, following bulbectomy the reconstituted olfactory epithelium lacks its normal target and is compromised in its ability to recover from nerve damage, as evidenced by the presence of a large number of B‐50/GAP43‐expressing neurons up to 3 months after the lesion and its failure to establish a full complement of OMP‐expressing neurons. These results demonstrate that the olfactory epithelium is capable of replacing its sensory neurons independently of the presence of its target, the olfactory bulb. However, the differential patterns of expression of B‐50/GAP43 and OMP at long times after peripheral lesion with TX‐100 or bulbectomy illustrate the profound effect the olfactory bulb has on neuronal maturation in reconstituted olfactory neuroepithelium.
Acute and persistent varicella‐zoster virus infection of human and murine neuroblastoma cell lines
Authors: Bourdon‐Wouters C. , Merville‐Louis M.‐P. , Sadzot‐Delvaux C. , Marc P. , Piette J. , Delrée P. , Moonen G. , Rentier B.
Published: January 2005

Human and murine neuroblastoma cell lines were infected in vitro with varicella‐zoster virus (VZV). Infected human neuroblastoma cells (IMR‐32) supported the synthesis of abundant viral antigens as detected by indirect immunoperoxidase labeling using human serum rich in anti‐VZV antibodies and did not survive the infection. In situ hybridization (ISH) with VZV‐cloned probes revealed a strong hybridization signal in these infected cells. During cultivation, the virus was released in the culture medium, and viral polypeptides were revealed by Western blotting of infected cells, using either a monoclonal anti‐gpI antibody or a rabbit antiserum. All these findings indicate that IMR‐32 cells support a productive and lytic infection by VZV, whether infected by cell‐free virus or by cocultivation with infected cells. Murine neuroblastoma cells (neuro‐2A) survived VZV infection and did not produce any infectious virus. No VZV‐specific proteins were detected in infected cells either by immunolabeling or by Western blotting. However, viral nucleic acids could be detected by ISH, indicating that mouse neuroblastoma cells displayed a nonproductive, nonlytic infection. Infected neuro‐2A cells have been examined by ISH using probes corresponding to immediate early (IE) genes 4, 62, and 63 and late (L) gene 31 encoding gpII. A strong hybridization signal was detected when infected cells were probed with a fragment containing the IE genes 62 and 63. Lower levels of hybridization were detected with the other probes, corresponding to IE or L genes. These systems allow comparative molecular analysis of persistent and acute infection of nerve cells by VZV.
The epileptogenic action of the taurine analogue guanidinoethane sulfonate may be caused by a blockade of GABA receptors
Authors: Herranz A. S. , Solis J. M. , Herreras O. , Menéndez N. , Ambrosio E. , Orensanz L. M. , del Rio R. Martin
Published: January 2005

The aim of this paper is to clarify the mechanism through which the taurine analogue guanidinoethane sulfonate (GES) produces its epileptogenic effects. Experiments were performed in the rat hippocampus in vivo, using a brain dialysis probe also containing a recording electrode. Perfusion of 10 mM GES induced an enhancement of extracellular taurine levels probably as a result of forced efflux through the taurine uptake systems in a heteroexchange process. This taurine increase was highly reversible. GES also induced an increase of neuronal excitability and an impairment of recurrent inhibition as judged by the neuronal pattern discharge of evoked potentials. These results indicate the possible implication of GABA receptors in the epileptogenic effect of GES. Specific binding of [3H]‐GABA to P2 fractions was inhibited by both bicuculline methiodide (BMI) and GES with the same potency. Similar results were obtained using cerebral sections. Autoradiographic experiments confirm the binding results. GES and BMI completely displaced [3H]‐GABA binding. All these results suggest that the epileptogenic GES action is due to a direct antagonism on GABAA receptors.
Establishment of permanent astroglial cell lines, able to differentiate in vitro, from transgenic mice carrying the polyoma virus large T gene: An alternative approach to brain cell immortalization
Authors: Galiana E. , Borde I. , Marin P. , Rassoulzadegan M. , Cuzin F. , Gros F. , Rouget P. , Evrard C.
Published: January 2005

Permanent untransformed cell lines have been established from the cerebral cortex of transgenic mice that carry the polyoma virus large T gene. The immortalized cells described here synthesize laminin and neural cell adhesion molecules and induce primary neurons to develop neuritic processes. As shown by immunofluorescence and immunoblotting assays, they begin to synthesize the glial fibrillary acidic protein (GFAP) after confluence. Double labelling experiments indicated that GFAP expression is reversibly correlated with the arrest of cell division. The present cells also display adrenergic, serotoninergic, and high levels of muscarinic receptors coupled to the phosphatidylinositol signalling pathway. Taken together, our data show that these cell lines constitute homogeneous cell material that has retained the main differentiative, functional, and growth properties of normal astrocytes. Therefore, such clonal untransformed cell lines should be useful for further molecular studies, addressing terminal differentiation of glial cells, glioneuronal interactions, and astroglial expression of receptors for neurotransmitters. Furthermore, we suggest that this approach of cell immortalization by the use of transgenic mice carrying a non‐transforming oncogene might be extended to a variety of cell types.
Nerve growth factor and K‐252a increase catecholamine release from PC12 cells
Authors: Nikodijevic B. , Creveling C. R. , Koizumi S. , Guroff G.
Published: January 2005

PC12 cells are a nerve growth factor‐responsive clone derived from a rat pheochromocytoma. The cells contain catecholamines and secrete them in response to depolarizing stimuli and cholinergic agonists. Treatment of the cells with nerve growth factor produces a number of very rapid changes, including the structural rearrangement of the cell membrane, the generation of a number of different second messengers, and the phosphorylation of several proteins. The present studies show that nerve growth factor treatment increases the release of dopamine and norepinephrine from the cells within a few minutes and does so independently of its effect on their metabolism. The experiments indicate that this effect on nerve growth factor is dependent on the presence of extra‐cellular calcium and can be blocked by calcium channel antagonists. K‐252a, an alkaloid‐like material, usually found to inhibit the actions of nerve growth factor on PC12 cells, also increases the release of cat‐echolamines under these conditions.
Evidence for the colocalization of the axonal mitogen for Schwann cells and oligodendrocytes
Authors: Mason P. W. , Chen S. J. , De Vries G. H.
Published: January 2005

Axons that normally will encounter either CNS or PNS glia have been shown to contain a powerful mitogen for both Schwann cells and oligodendrocytes. The normally nonmyelinated, nonglial ensheathed cerebellar granule cells have been shown to possess a proliferative signal for Schwann cells, suggesting that a glial mitogen is common to all axons. To determine if a glial mitogen capable of stimulating both Schwann cells and oligodendrocytes is colocalized on all types of axons we have (1) cocultured granule cells with oligodedrocytes, (2) incubated oligodendrocytes with granule cell membranes, and (3) evaluated the ability of heparin extracts of granule cell membranes, splenic nerve microsomes, and axolemmaenriched fractions isolated from rat and bovine CNS to stimulate mitosis of cultured oligodendrocytes. Neither the intact granule cells nor the granule cell membrane fraction stimulated cultured oligodendrocytes to divide. However, heparin extracts of the granule cell membranes were significantly mitogenic to the cultured oligodendrocytes. Heparin extracts of splenic nerve microsomes were more mitogenic than the comparable extract obtained from bovine CNS axolemma‐enriched fractions. These results suggest that the neuronal mitogen for oligodendroglia is co‐localized with the neuronal mitogen for Schwann cells.