bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibitors of N‐linked oligosaccharide processing glucosidases interfere with oligodendrocyte differentiation in culture
Authors: Bhat N. R. , Zhang P.
Published: January 2005

Previous studies have demonstrated that inhibitors of glycoprotein processing glucosidases interfere with the development of oligodendrocyte properties in primary cultures of embryonic rat brain cells (Bhat, J Neurosci Res 20:158–164, 1988). The present study examines the effect of castanospermine, an inhibitor of the processing glucosidases, on the development and differentiation of isolated oligodendrocyte progenitor cells. Treatment of oligodendrocyte progenitors with castanospermine did not affect the developmental progression of the precursors to become committed oligodendrocytes as revealed by comparable increases in the percentages of cells positive for galactocerebroside (a surface marker for terminally differentiated oligodendrocytes) in control and drugtreated cultures. On the other hand, there was an impairment of the expression of differentiated properties of oligodendrocytes [i.e., sulfolipid synthesis, myelin basic protein (MBP)] and 2′3′‐cyclic nucleotide 3′‐phosphohydrolase in the drug‐treated cultures. Immunocytochemical analysis with anti‐MBP antibodies revealed a reduced number of MBP‐positive cells in inhibitor‐treated cultures. Furthermore, a majority of MBP‐positive cells in such cultures displayed immunoreactive MBP in their cell body and not the processes, unlike in control cultures where both cell body and the processes of oligodendrocytes stained intensely for MBP. The strong inhibitory effect of castanospermine on the expression of oligodendrocyte‐specific activities was contrasted with a relatively smaller effect of swainsonine, a mannosidase inhibitor on oligodendrocyte differentiation. Both castanospermine and swainsonine, however, effectively blocked the formation of complex‐type oligosaccharides, suggesting thereby a lack of correlation between the inhibition of the formation of complex‐type oligosaccharides and oligodendrocyte differentiation. It is suggested, therefore, that early trimming reactions involving the removal of glucose residues from the high mannose oligosaccharides in the endoplasmic reticulum may be essential for the cell surface localization and function of glycoproteins critically involved in surface interactions of oligodendrocytes with each other and/or with the substratum. Copyright © 1994 Wiley‐Liss, Inc.
Dopamine transporter mediated release of dopamine: Role of chloride
Authors: Sitges M. , Reyes A. , Chiu L. M.
Published: January 2005

Using a rapid (0.5 ml/min) flow rate superfusion system, the dopamine (DA) transporter mediated release of DA is further explored, and compared to the depolarization evoked release of DA in rat striatal synaptosomes preloaded with radioactive DA (3H‐DA). In this system external DA in the low μM range efficaciously releases the preloaded transmitter, the maximal response being reached at 3 μM DA. The external DA stimulated release is Ca2+‐independent, Cl−‐dependent, and blocked by both bupropion and nomifensine. The atypical antidepressant bupropion inhibits 3H‐DA accumulation to rat striatal synaptosomes with a calculated IC50 of 1.3 × 10−6M. Among DA uptake blockers some are known to act as DA releasing agents. Here we found that the DA uptake blocker nomifensine (30 μM) is unable to modify the baseline release of 3H‐DA, whereas bupropion (10 μM) clearly elevates the baseline release of 3H‐DA in a Ca2+‐independent and Cl−‐dependent manner. The non releasing agent nomifensine blocks the release of 3H‐DA induced by bupropion. The Ca2+‐dependent, high K+ depolarization evoked release of 3H‐DA is not modified by nomifensine and does not depend on the external Cl− concentration. When the depolarizing medium contains DA the carrier mediated release of 3H‐DA induced by the external DA is additive to the high K+ induced response. A drastic drop in the external Cl− concentration induces 3H‐DA release. This release of 3H‐DA induced by low external Cl− levels is completely blocked by nomifensine, which only slightly diminished the release of 3H‐DA induced by the absence of external Na+. On the basis of these results, it is concluded that: (1) Rapid perfusion flow rates eliminate DA reuptake. (2) DA uptake inhibitors either with or without DA releasing capabilities block the release of DA induced by μM levels of external DA. (3) By preventing translocation of the DA transporter mobile moiety, nomifensine may inhibit the release of DA induced by external DA or bupropion and by drastic drops in the external Cl−w concentration. (4) In the absence of nomifensine, the DA transporter works under both resting and depolarized conditions, but in contrast to the GABA transporter (Sitges et al.: Neurochem Res 18:1081–1087, 1993), the DA transporter does not contribute to the amount of the DA released by depolarization. (5) Reversal of the DA uptake carrier is favored by conditions increasing the internal DA levels. (6) Cl− rather than Na+ is a major determinant in 3H‐DA movements through the DA transporter. Copyright © 1994 Wiley‐Liss, Inc.
Immortalization of immature and mature mouse astrocytes with SV40 T antigen
Authors: Frisa P. S. , Goodman M. N. , Smith G. M. , Silver J. , Jacobberger J. W.
Published: January 2005

The ability of neonatal astrocytes to promote neurite outgrowth in vitro and in vivo diminishes as astrocytes mature. This property correlates with the developmental loss of the central nervous system's ability to regenerate after injury. Cell lines representative of immature and mature astrocytes would be useful for studies to determine differences between these two populations. Previous work on immortalization of bipotential neural/glial precursors and fully differentiated glial cells suggests that immortalization of astrocytes at timed intervals of culture may yield cell lines trapped in different maturation states. To test this, neonatal mouse cortical astrocytes were immortalized by retrovirus‐mediated transfer of the SV40 T antigen (Tag) gene at 2, 6 and 17 days of culture. The clonal cell lines express Tag and are contact‐inhibited. Three phenotypes that change as a function of astrocyte maturation were examined to determine the fidelity with which the cell lines represent immature and mature astrocytes. These were: (1) cell morphology, growth pattern and size, (2) level of glial fibrillary acidic protein (GFAP) expression, and (3) neurite outgrowth promotion. First, immature and mature lines resemble mortal type 1 astrocytes of corresponding ages with respect to morphology and growth pattern, and retain a quantitative difference in cell size (mature cells are larger). Second, the pattern of GFAP expression is preserved, with immature lines expressing lower levels than mature cell lines, but the overall GFAP levels are significantly lower in immortalized cell lines compared to mortal cells. Finally, promotion of neurite outgrowth from embryonic chick retinal ganglion cells on monolayers of the cell lines was examined. While all neurite outgrowth measures are significantly greater for the immortalized lines than for control 3T3 cells, they are attenuated relative to mortal astrocytes. The age‐related pattern of stronger outgrowth support on immature astrocytes is retained for neurite initiation, but not retained for mean neurite length. Thus, SV40 Tag‐immortalized astrocytes have a complex phenotype characterized by retention of age‐related differences in morphology, growth pattern and cell size, and by a marked attenuation of some astrocyte‐specific characteristics but retention of age‐related differences in the expression level of these same characteristics, and finally, loss of the ability to support neurite extension at levels characteristic of immature astrocytes. © 1994 Wiley‐Liss, Inc.
Neuronal progenitors identified by their inability to express class I histocompatibility antigens in response to interferon‐γ
Authors: Bailey K. A. , Drago J. , Bartlett P. F.
Published: January 2005

Interferon‐γ (IFN‐γ) can induce class I major histocompatibility complex (MHC) antigen (H‐2) expression on virtually all neuroepithelial cells isolated from embryonic day 9 (E9) mice. However, a subpopulation of cells become refractory to H‐2 induction (H‐2l−) by E10 and the percentage of H‐2 noninducible cells increases during development. Cell sorting, by flow cytometry or magnetic bead immunoselection, has shown that H‐2l− cells give rise exclusively to neuronal cells, and by E12, the majority of the neuronal progenitors are found within this population. It has also been found that 98% of the H‐2l− also express the neuron‐associated marker, A2B5. Cells of the glial cell lineage retain the ability to express class I antigens throughout development. From these studies, it is clear that the neuroepithelium contains cells committed to the neuronal cell lineage as early as E10 in the mouse. Copyright © 1994 Wiley‐Liss, Inc.
Responsiveness of cultured septal and hippocampal neurons to ethanol and neurotrophic substances
Authors: Heaton M. B. , Paiva M. , Swanson D. J. , Walker D. W.
Published: January 2005

Dissociated septal and hippocampal neurons from E18 fetal rats were cultured with varying concentrations of ethanol (0.6–2.4 g/dl) and in cultures containing ethanol plus nerve growth factor (NGF) or basic fibroblast growth factor (bFGF). These substances have been shown to provide neurotrophic support for these populations and to afford neuroprotection against certain toxic substances or conditions applied to some neuronal populations. Both the septal and hippocampal neurons responded to ethanol in a dosedependent manner. Survival of septal neurons was generally unaffected by initial ethanol concentrations of 0.6 and 1.2 g/dl but was considerably impaired by higher concentrations (1.8 and 2.4 g/dl), while neurite outgrowth was compromised by all ethanol concentrations except the lowest one applied. The hippocampal neurons survived ethanol concentrations up to 2.4 g/dl, although process extension was decreased in concentrations of 1.2 g/dl and higher. NGF or bFGF in the culture medium (in cultures without ethanol) did not affect neuronal survival or process outgrowth in either population, probably owing to the relatively high plating densities of the cultures. NGF did tend to have a moderate ameliorative effect on the ethanol neurotoxicity in the septal cultures, however, and was slightly effective in this regard in hippocampal cultures at intermediate ethanol concentrations (1.8 g/dl). High concentrations of ethanol (2.4 g/dl) reduced the proportion of cholinergic cells in the septal preparations by approximately 50%. This neuronal loss could be reversed by inclusion of high concentrations of NGF in the culture medium (100 ng/ml) but not by a lower concentration (20 ng/ml). bFGF provided some protection against ethanol cytotoxicity with respect to both populations. The implications of these results for studies of fetal alcohol effects are discussed, as well as their relation to prior reports of trophic factor neuroprotection. © 1994 Wiley‐Liss, Inc.
S100β protein expression in Alzheimer disease: Potential role in the pathogenesis of neuritic plaques
Authors: Sheng J. G. , Mrak R. E. , Griffin W. S. T.
Published: January 2005

Increased synthesis and release of S100β protein from activated astrocytes has been implicated in the overgrowth of dystrophic neurites in neuritic plaques in Alzheimer disease (AD). To evaluate the quantitative relationships between tissues levels of S100β and the numbers of neuritic plaques, by Tau‐2 immunoreactive (Tau‐2+) labeling, in tissue sections of hippocampus and adjacent temporal cortex and measured the levels of S100β protein, by Western immunoblot labeling, in samples of analagous regions from contralateral hemisphere of the same patients. In AD, tissue levels of S100β (two‐ to fivefold that of controls) were significantly correlated with the number of Tau‐2+ plaques (R = 0.82, P < .01). Dual‐label immunohis‐tochemical analysis showed that most S100β+ cells were activated GFAP+ astrocytes. These results were substantiated by a significant correlation between S100β and GFAP tissue levels (R = 0.81, P < .05). Many of the S100β+ astrocytes were clustered around and within Tau‐2+ plaques. Indeed, no Tau‐2+ plaques were found without associated activated S100β+ astrocytes. Our findings provide further evidence of a role for S100β protein in dysregulation of neurons that leads to apparently nonsensical growth of imperfect neurites in AD, a potential key element in early stages of neuritic plaque pathogeneis. © 1994 Wiley‐Liss, Inc.
Presence and regulation of transforming growth factor beta mRNA and protein in the normal and lesioned rat sciatic nerve
Authors: Rufer M. , Flanders K. , Unsicker K.
Published: January 2005

The transforming growth factors beta (TGF‐β), a family of regulatory polypeptides, are involved in numerous vital processes including inflammation and wound healing. Since repair of a peripheral nerve lesion includes a series of well‐defined steps of cellular actions possibly controlled by TGF‐βs, and since TGF‐β mRNA and immunoreactivity have been found in the normal peripheral nerve, we have examined TGF‐β mRNA regulation and protein expression in the lesioned peripheral nerve. Sciatic nerves of adult rats were either crushed (allowing axonal regenration) or transected (to prevent axonal regeneration and to induce Wallerian degeneration in the distal stump). After intervals of 6 hours, 2 and 6 days post‐lesion, the rats were sacrificed and each nerve was cut into four segments, two proximal and two distal to the lesion site. TGF‐β 1‐3 mRNA were determined for each segment. We demonstrate that TGF‐ß1 mRNA levels are higher than those of TGF‐ß3; the amplitude of mRNA regulation depends on time, type of lesion and localization relative to the lesion site. TGF‐ß2 mRNA could not be detected. For TGF‐ß1‐3 immunocytochemistry, animals were sacrificed 12, 24, 48, 72 hours and 7 and 14 days after surgery. TGF‐β immunoreactivity (IR) was observed for all isoforms in lesioned and unlesioned nerves. In the segment directly adjacent to the lesion at its proximal side, an increase of TGF‐β‐IR became apparent as soon as 12 hours after surgery; it remained elevated during the whole period observed in both models. In the segment adjoining the distal side of the lesion, an increase of TGF‐β‐IR was observed after 48 hours, which was still present after 14 days. At day 7 after crush or transection, an increase of TGF‐β‐IR was detected in the most distal segments, which reached its highest levels at the end of our observation period. Our results suggest that the presence of axonal contact might induce an enhancement of TGF‐β expression by Schwann cells in the distal stump of a lesioned and regenerating peripheral nerve. Since we demonstrate an increase of TGF‐β mRNA and protein expression also in the distal stump of transected nerves where Schwann cells are not able to contact sprouting axons from the proximal part, other regulatory pathways must exist. The acquisition of a “reactive” Schwann cell phenotype after peripheral nerve lesion might involve an upregulation of TGF‐β expression. © 1994 Wiley‐Liss, Inc.
Expression and distribution of functional intergrins in rat CNS glia
Authors: Tawil N. J. , Wilson P. , Carbonetto S.
Published: January 2005

In previous studies, we have reported on the expression of β1 intergrins in type 1 astrocytes and their function in cell‐substratum attachment (Tawil et al., J Cell Biol 120:261–271, 1993). Here we extend those findings by providing evidence that type 1 astrocytes express integrins of the β3 and possibly β4 subclasses and that the former (αvβ3) functions in attachment by recognizing the peptide, Arg‐Gly‐Asp, in vitronectin. In addition, we have examined immunocyto‐chemically the expression of β1 integrins on type 2 astrocytes and oligodendrocytes. The pattern of expression of integrins on these two cell types is distinct from type 1 astrocytes; most notably type 1 astrocytes but not oligodendrocytes or type 2 astrocytes express α1β1 heterodimers. Since type 2 astrocytes and oligodendrocytes originate from a common precursor (0‐2A), the α1β1 heterodimer may be a functional marker which distinguishes O‐2A‐derived cells from those of the type 1 astrocyte lineage. © 1994 Wiley‐Liss, Inc.
Formation of new sensory cells in deafferented tuberous organs of the gymnotid fish Eigenmannia virescens
Authors: Bensouilah M. , Denizot J. P.
Published: January 2005

The cutaneous electroreceptor “tuberous organs” of the lateral line system of Eigenmannia virescens were studied at light and electron microscopic levels with immunohistochemical and autoradiographic techniques after sectioning of the posterior branch of the lateral line nerve. After deafferentation total degeneration of the sensory cells was observed. The accessory cells of the basal platform, however, remain intact and undergo a process of differentiation. The cytoplasms and nuclei of these cells increase in volume, and the nuclei incorporate tritiated thymidine. In control tuberous organs with intact innervation, tritiated thymidine is absorbed by the nuclei of the elongated epidermal cells surrounding the sensory cavity. The newly formed sensory cells, but not those of the intact organs, are substance P immunoreactive. They have synaptic bars surrounded by vesicles and their free membrane surface is covered with microvilli. The new sensory cells are fully differentiated 35 days after the lateral line was cut. These results demonstrate that in the tuberous organs of E. virescens new sensory cells are formed in the absence of an innervation. © 1994 Wiley‐Liss, Inc.
Insertional mutagenesis inducing hypomyelination in transgenic mice
Authors: Orian J. M. , Mitchell A. W. S. , Marshman W. E. , Webb G. C. , Ayers M. M. , Grail D. , Ford J. H. , Kaye A. H. , Gonzales M. F.
Published: January 2005

Investigations of myelin disorders, in particular multiple sclerosis (MS), have concentrated on immunemediated damage to formed myelin, while there has been less emphasis on the molecular genetics of myelin formation. We have generated a transgenic mouse mutant (designated 2‐50) which carries an insertional mutation in a locus regulating myelination. These mice carry a transgene comprising 1.3 Kb of the mouse myelin basic protein (BMP) promoter conjugated to a fragment containing exons 2 and 3 of the human c‐myc gene. Positive mice show a significant reduction in myelin compared to controls and a shivering phenotype. Unlike other myelin mutants, all 2‐50 mice lose the shivering phenotype and breed normally. Expression of c‐myc is detectable in only 65% of transgene‐carrying mice, and when present occurs at extremely low levels. This shows that the phenotype is caused by insertional inactivation of a gene necessary for myelination rather than ectopic expression of the transgene. The transgene was found by in situ hybridization to be inserted into a single site which is very distally located on chromosome 9. The 2‐50 mice represent a unique model which will be ideal for investigating the molecular basis of myelin assembly and for developing gene therapy to promote remyelination in conditions such as MS. © 1994 Wiley‐Liss, Inc.
Methylation of the rat glial fibrillary acidic protein gene shows tissue‐specific domains
Authors: Teter B. , Osterburg H. H. , Anderson C. P. , Finch C. E.
Published: January 2005

The gene for glial fibrillary acidic protein (GFAP) was compared for CpG sites that are potential locations of methylated cytosine (mC). GFAP sequences in the 5′‐upstream promoter and in exon 1 of rat, mouse, and human showed extensive similarity in the locations of CpG sites in the promoter and in exon 1, implying conservation. The methylation of mC at 9 CpG sites in the promoter and 10 sites in exon 1 was analyzed in F344 male rats by a quantitative application of ligation‐mediated polymerase chain reaction (LMPCR). CpG sites with varying mC in different tissues were found in the GFAP promoter and in a CpG island in exon 1. In the brain, the promoter had about 40% less mC than in testis and liver. The degree of methylation varied strikingly between adjacent sites within and between tissues. Testis GFAP exon 1 had a gradient of mC from 5′ to 3′ across the exon that was absent in liver, brain, and cultured neurons and astrocytes. Among brain regions, the hippocampus had 10–40% less mC at 12 CpG sites than in hypothalamus; the other sites (7/19) showed smaller differences between these brain regions. In DNA from primary cultures, astrocytes has slightly less mC than neurons at all sites. Because neuron‐rich hippocampal subregions and primary neuron cultures had less methylation than nonneural tissues, we hypothesize that neuroectodermal derivatives tend to be less methylated, whether or not GFAP is expressed. Four domains of methylated CpG sites are proposed on the basis of tissue and cell‐type distribution: (I) a constitutively methylated domain in the mid‐upstream promoter; (II) a testis‐specific gradient of methylation in exon 1; (III) a hypomethylated domain found in neuroectodermal derivatives; and (IV) subsets of sites in the promoter and in exon 1 that have the least methylation in astrocytes, and therefore may be astrocytespecific domains. © 1994 Wiley‐Liss, Inc.
Domain‐specific antibodies against the B2 chain of laminin inhibit neuronal migration in the neonatal rat cerebellum
Authors: Liesi Päivi , Hager G. , Dodt H.‐U. , Seppälä I. , Zieglgänsberger W.
Published: January 2005

Although the spatial and temporal patterns of neuronal migration have been analyzed in great detail, little direct evidence is available as to what extracellular matrix molecules are involved. Because there is indirect evidence implicating the extracellular matrix protein laminin in neuronal migration, we investigated the effects of antibodies against a synthetic peptide derived from a neurite outgrowth domain of the B2 chain of laminin on neuronal migration in living cerebellar slices. We show by using infrared video microscopy that divalent Fab2 fragments of these antibodies inhibit granule neuronal movement in living slices of (P8) rat cerebellum. This inhibition of neuronal movement manifests itself by cessation of both radial and horizontal translocations of nuclei inside the granule neuronal processes. Fab2 fragments of antibodies against the intact (native) laminin molecule or Fab2 fragments from the preimmune serum do not affect nuclear translocation. Immunocytochemistry shows binding of the divalent Fab2 fragments of the B2 chain‐specific antibodies to the Purkinje and Bergmann glial cell areas, and as punctate deposits in between the cells of the external granule cell layer. Native laminin antibodies bind to the basement membranes, and binding of the Fab2 fragments from the preimmune sera cannot be demonstrated. These results indicate that neuronal migration in the postnatal rat cerebellum in vivo involves nuclear translocation that can be inhibited by antibodies against a neurite outgrowth domain of the B2 chain of laminin. Thus, migration of cerebellar granule neurons may depend on the interaction between a neurite outgrowth domain of the B2 chain of laminin and neuronal cytoskeleton involved in nuclear movement. © 1995 Wiley‐Liss, Inc.
Novel forms of neuronal migration in the rat cerebellum
Authors: Hager G. , Dodt H.‐U. , Zieglgänsberger W. , Liesi P.
Published: January 2005

Infrared video microscopy of neonatal rat cerebellum (P0‐P14) was used to directly visualize migrating granule neurons in relation to other cerebellar cells in a brain slice for up to 24 hr. Initially (P0‐P5), granule neurons move along radial migration pathways of other neuronal fibers. These pathways are probably established by the bipolar granule neurons that attach to the external basement membrane via one process and extend another process toward the Purkinje cell layer. At P5‐P8, a substantial number of granule neurons move horizontally and extend long parallel fibers. Both radially and horizontally migrating granule neurons move by nuclear translocation inside their preformed processes with a speed that varies between 6 and 120 μ/hr. In P10‐P12 animals, the horizontally oriented granule neurons start to migrate radially. They move into the internal granule cell layer either along the radial pathways of other neuronal fibers or in contact with the matured glial processes. The radial neuronal migration pathways disappear by P14 whereas the glial cell processes are maintained and reach the basal lamina. These results describe novel radial and horizontal modes of neuronal migration that proceed independently of the physical glial guidance. © 1995 Wiley‐Liss, Inc.
Microglia in colony‐stimulating factor 1‐deficient op/op mice
Authors: Blevins G. , Fedoroff S.
Published: January 2005

Mice that are homozygous for the autosomal recessive mutation osteopetrosis (op) suffer from a general skeletal sclerosis, and the numbers of macrophages in various tissues are significantly decreased. We report that microglia in op/op mice are not affected by the mutation. They have normal morphology and are present in the CNS in normal frequency.

In cultures, disaggregated cells of neopallia can form microglia, but such cells from neopallia of op/op mice form microglia only when colony‐stimulating factor 1 (CSF‐1) is added to the culture medium. The addition of granulocyte/macrophage (GM)‐CSF or interleukin (IL)‐3 to the culture medium does not stimulate production of microglia. Microglia that form in op/op neopallial cell cultures, in the presence of CSF‐1, are capable of Fc‐receptor‐mediated phagocytosis.

Based on our experiments, it seems that microglia are CSF‐1 dependent but in op/op mice (in which CSF‐1 is absent) microglia may use other locally produced factors. © 1995 Wiley‐Liss, Inc.
Long, interlaminar astroglial cell processes in the cortex of adult monkeys
Authors: Colombo Jorge A. , Yáñez A. , Puissant V. , Lipina S.
Published: January 2005

At variance with current descriptions stressing the stellate geometry of cortical astrocytes in the brain of adult mammals, GFAP‐immunoreactive astrocytes from prefrontal and rostral cingulate cortices in two adult New World monkey species, Cebus apella and Saimiri sciureus, were found to have long cellular processes traversing several cortical lamina. These unreported features of cortical astroglial cells in adult nonhuman primates pose new issues for the understanding of iso‐ and allocortical organization and processing in higher mammals. © 1995 Wiley‐Liss, Inc.
Myelinogenic potential of an immortalized oligodendrocyte cell line
Authors: Newman S. L. , Weikle A. A. , Neuberger T. J. , Bigbee J. W.
Published: January 2005

The myelinogenic potential of an oligodendrocyte cell line (N20.1) immortalized by transformation with a temperature‐sensitive retrovirus (Verity et al., J Neurochem 60:577–587, 1993) has been evaluated in a co‐culture system utilizing dorsal root ganglion neurons. When N20.1 cells were placed in co‐culture with dorsal root ganglion neurons at 39°C, the temperature at which TAg expression is decreased relative to that in cells maintained at 34°C, there was a dramatic decrease in the N20.1 proliferation rate compared to cells maintained in the absence of neurons at either temperature. This decrease in proliferation was observed within 3 days of co‐culture and appeared to precede a further decrease in TAg expression that occurred with time in response to the neurons. In co‐cultures the immunoreactivity of N20.1 cells for galactocerebroside increased with time, and the cells appeared to establish contact with neurites and initiate formation of membranous sheets. When the duration of co‐culture was extended to 52 days, myelin‐like figures were noted by electron microscopy. Thus, the extent of N20.1 differentiation is dependent on the presence of neurons and the duration of co‐culture. This culture system represents a potentially powerful tool for the study of neuronal‐glial interactions influencing myelinogenesis and remyelination. © 1995 Wiley‐Liss, Inc.
Transected axons of adult hypothalamo‐neurohypophysial neurons regenerate along tanycytic processes
Authors: Chauvet N. , Parmentier M. L. , Alonso G.
Published: January 2005

In the intact hypothalamo‐neurohypophysial system, oxytocinergic or vasopressinergic neurons project their axons throughout the internal layer of the median eminence towards the blood vessels of the hypophysial neural lobe. When transected at the level of the median eminence, these axons undergo massive sprouting towards the external layer of the organ and the underlying perivascular region containing hy'pophysial portal vessels. The present study was designed to explore the possible roles of median eminence glial cells in such a reorganization of transected neurohypophysial axons. The relationships between regenerating axons and glial cells were studied by laser scanning confocal microscopy and electron microscopy on vibratome sections immunostained with specific antibodies against neurohypophysial peptides and/or against glial markers. All along the intact median eminence, two main types of glial cells were identified: (1) tanycytes immunoreactive to vimentin and slightly immunoreactive to glial fibrillary acidic protein, and (2) classical astrocytes immunoreactive to glial fibrillary acidic protein but vimentin‐negative. In the intact median eminence, neurohypophysial axons were associated with astrocytic processes located in the internal layer. After a lesion of the hypophysial stalk, peptidergic regenerating axonal sprouts were found to project massively towards the external layer and to penetrate the underlying perivascular region in close association with tanycytic‐like processes immunoreactive to both vimentin and to glial fibrillary acidic protein. In contrast, regenerating sprouts were absent from those regions of the lesioned median eminence containing astrocytic processes immunoreactive to glial fibrillary acidic protein but vimentin‐negative. When fixed lesioned median eminences were treated by placing crystals of the lipophilic dye Dil on their ventricular surface, regenerating axons were found to be closely associated with Dil‐labelled tanycytic‐like end feet terminating in the external layer, and with connected thin processes projecting through the external vascular region. These data indicate that in the median eminence of the adult rat, lesioned neurohypophysial axons regenerate in close association with tanycytic processes. © 1995 Wiley‐Liss, Inc.
Myelin vesicles: What we know and what we do not know
Authors: Sedzik J. , Blaurock A. E.
Published: January 2005

In the following review, we address difficulties that have arisen when attempting to convert the myelin multilayers into vesicles. The emphasis is on CNS myelin of adult mammals although both central nervous system (CNS) and peripheral nervous system (PNS) myelin are considered. The ability to prepare vesicle of myelin membrane has yet not been feasible. We hope to clarify some aspect of this problem and offer some possible approaches. Special attention is paid to myelin swelling phenomena because these indicate ways in which the myelin multilayer can break down. Images of isolated myelin are reviewed with special attention to the ways in which the multilayer actually breaks down. Attempts at reproducing a procedure for vesiculating myelin are summarized, and a critique is given to account for the inability to reproduce the published results. Finally, novel approaches for vesiculating myelin are proposed, which are based on well‐characterized swelling phenomena. © 1995 Wiley‐Liss, Inc.
Oxidative damage to proteins and lipids of CNS myelin produced by in vitro generated reactive oxygen species
Authors: Bongarzone E. R. , Pasquini J. M. , Soto E. F.
Published: January 2005

Purified myelin isolated from 70‐day‐old rats was submitted to nonenzymatic peroxidative systems containing 100 μM FeC13.6H2O,100 μM ascorbic acid, and 100 μM CuSO4.6H2O 10 mM H2O2 in order to investigate the extent of damage produced by reactive oxygen species (ROS). Iron and copper catalyzing systems were selected because of the known importance of these metals in producing free radical chain reactions in biological membranes (Halliwell and Gutteridge: “Free Radicals in Biology and Medicine,” Oxford: Clarendon Press, 1989). Our findings, show that (1) although after 1 hour of peroxidation, an important level of thiobarbituric acid‐reactive substances (TBARS) was detected, polyunsaturated fatty acids (20:2; 20:4; 22:4 and 22:6) were markedly affected only after 14 hours of incubation; (2) protein thiol groups were very sensitive to the attack of ROS generated by copper but resistant to iron‐generated ROS; (3) aggregation of myelin proteins produced by peroxidation could be prevented by sulfhydryl (SH)‐reducing agents, and (4) as a consequence of these modifications, compact myelin suffered disruption of its intraperiodic line. In conclusion, our results demonstrate that this unique membrane of the central nervous system (CNS) is highly vulnerable to oxidative stress and that this susceptibility to oxidative damage could be prevented, at least partially, by the use of SH‐protective molecules. © 1995 Wiley‐Liss, Inc.
β‐Amyloid precursor protein is modified with O‐linked N‐acetylglucosamine
Authors: Griffith L. S. , Mathes M. , Schmitz B.
Published: January 2005

The β‐amyloid precursor protein (APP) has been implicated in the etiology of Alzheimer's disease (Kang et al.: Nature 325:733–736, 1987; Selkoe: Science 248:1058–1060, 1990; Selkoe: In Cowan et al. (eds): “Annual Review of Neuroscience.” Palo Alto, CA: Annual Reviews, Inc., pp 489–519, 1994) and numerous studies have shown that b̃‐amyloid is involved in amyloid plaque formation (Rumble et al.: N Engl J Med 320:1446–1452, 1989; Sisodia et al.: Science 248: 492–495, 1990). Evidence is presented that APP is modified with N‐acetylglucosamine linked to cytoplasmic serine or threonine residues (O‐GIcNAc). This is the first report of a plasma membrane protein modified with this carbohydrate. It has been postulated that this modification, which is ubiquitous in all organisms studied to date except bacteria (Haltiwanger et al.: Biochem Soc Trans 20:264–269, 1992; Dong et al.: J Biol Chem 268:16679–16687, 1993; Elliot et al.: J Neurosci 13:2424–2429, 1993; Kelly et al.: J Biol Chem 268:10416–10424, 1993), may function as an alternative to phosphorylation (Dong et al., 1993) and is involved in the multimerization of proteins (Haltiwanger et al., 1992; Dong et al., 1993). O‐GlcNAc occurs at “PEST” sequences (Rogers et al.: Science 234:364–368, 1986) and it has been suggested that this modification within such a sequence leads to increased proteolytic stability of the molecule (Dong et al., 1993). The importance of proteolytic cleavage (Kang et al., 1987; Selkoe, 1990, 1994), and the possible involvement of phosphorylation (Gandy et al.: Proc Natl Acad Sci USA 85:6218–6221, 1988; Gillespie et al.: Biochem Biophys Res Commun 187: 1285–1290, 1992; Suzuki et al.: J Neurosci 48:755–761, 1992; Suzuki et al.: EMBO J 13:1114–1122, 1994) in the regulation of APP processing suggest that this novel modification may somehow be involved in the biochemistry of APP. © 1995 Wiley‐Liss, Inc.
Inhibition of neurite outgrowth following intracellular delivery of anti‐GAP‐43 antibodies depends upon culture conditions and method of neurite induction
Authors: Shea T. B. , Benowitz L. I.
Published: January 2005

NB2a/dl neuroblastoma cells acquire a neuronal phenotype in response to several differentiating agents, including dibutyryl cAMP (dbcAMP) and the withdrawal of serum. As shown previously, antibodies to the growth‐associated protein, GAP‐43, introduced intracellularly using a lipid carrier, blocked the differentiation induced by dbcAMP. Antibodies to GAP43, at a low concentration, also blocked neurite out‐growth induced by serum withdrawal when cells were grown on a relatively unadhesive substrate. On more adhesive substrates such as poly‐L‐lysine and laminin, however, anti‐GAP‐43 antibodies had less of an effect on neurite outgrowth. Previous studies have shown that the increased adhesivity of laminin allows a small but significant population of neurites to grow from serum‐deprived cells, even in the presence of the microtubule‐depolymerizing drug, colchicine. The outgrowth of this population of neurites was blocked by antibodies to GAP‐43. These results are in conformity with recent studies showing that the requirement for GAP‐43 in neuritogenesis may be related to membrane adhesiveness, and may contribute to an understanding of some of the apparent discrepancies in the literature concerning the involvement of GAP‐43 in neuronal differentiation. © 1995 Wiley‐Liss, Inc.
Adaptation of C6 glioma cells to serum‐free conditions leads to the expression of a mixed astrocyte‐oligodendrocyte phenotype and increased production of neurite‐promoting activity
Authors: Coyle D. E.
Published: January 2005

C6 cells were adapted to proliferate in defined culture medium to allow the study of long‐term effects of serum‐free growth conditions on their phenotypic antigen expression and production of neurite promoting factors (NPFs). Cultures were grown in either Ham's F‐12 or supplemented Opti‐MEM‐I containing 15% heat‐inactivated horse serum and 2.5% fetal calf serum (serum‐containing) or in supplemented OptiMEM‐I alone (serum‐free). Immunocytochemical and immunofluorescence techniques were used to determine the antigenic expression of A2B5, galactocerebroside (Ga1C), and glial fibrillary acidic protein (GFAP) in passage matched and sister cultures of serum and serum‐free grown C6 cells. When C6 cells were grown under serum‐containing conditions, two populations of cells were seen: young oligodendrocytes (A2B5+, GFAP‐, GalC+ ), and mixed astrocyte‐oligodendrocyte phenotype (A2B5+, GFAP‐, Ga1C+). After adaptation of the C6 cells to serumfree conditions over 2–3 passages, only one population of cells was observed, the mixed astrocyte‐oligodendrocyte phenotype. The serum‐free conditions also resulted in, greater staining of the C6 cells. Conditioned media from the two growth conditions were fractionated by ultrafiltration into two fractions: components >50 kDa and components of 10–50 kDa. The amount of neurite promoting activity seen between the two culture conditions resulted in a 3‐fold increase in NPF activity under serum‐free conditions in the >50 kDa fraction. The 10–50 kDa fraction only expressed NPF activity if obtained from the serumgrown C6 cells. This alteration in NPF activity appears to be the result of the phenotypical alteration of the C6 cells, and may suggest that the NPF activities from the two culture conditions may not be identical. © 1995 Wiley‐Liss, Inc.
Calbindin D28K‐containing neurons, and not HSP70‐expressing neurons, are more resistant to HIV‐1 envelope (gp120) toxicity in cortical cell cultures
Authors: Diop A. G. , Lesort M. , Esclaire F. , Dumas M. , Hugon J.
Published: January 2005

HIV‐1‐associated cognitive/motor complex is one of the major neurological complications of AIDS and is associated with neuronal loss. Gp120, the HIV envelope protein, is toxic for neurons in cultures and produces a rise in intracytosolic calcium. This neurotoxicity is dose‐dependent and time‐dependent. We evaluated the selective gp120 toxicity in primary neuronal cultures for calbindin‐free and calbindin‐containing neurons with semi‐quantitative immunocytochemistry using an anti‐calbindin D28K monoclonal antibody. The number of immunolabelled neurons was inversely correlated to neuronal survival. In cultures exposed to gp120 (100 pM) for 24 hr the neuronal survival of initial platings was 19.7 ± 2.1% and the percentage of neuronal survival was 84.6 ± 4.9% in control cultures exposed to the vehicle. The corresponding percentages of immunolabelled neurons were 85.0 ± 2.1% in treated cultures and 23.6 ± 3.1% in control cultures (P < 0.001). The expression of heat shock proteins by heating cell cultures did not: protect neurons from gp120 toxicity. These results suggest that calbindin D28K‐containing neurons are more resistant to gp120‐toxicity in this cell culture system. © 1995 Wiley‐Liss, Inc.
Comparison of c‐jun, junB, and junD mRNA expression and protein in the rat dorsal root ganglia following sciatic nerve transection
Authors: De León M. , Nahin R. L. , Molina C. A. , De Léon D. D. , Ruda M. A.
Published: January 2005

The present study was designed to compare the expression of the Jun family of protooncogenes following nerve injury. Adult rats were anesthetized and the sciatic nerve transected. Dorsal root ganglia (DRG) at 1, 2, 3, and 7 days after nerve transection were collected, their total RNA extracted, and Northern blots performed using 32P‐labeled oligonucleotide probes. The constitutive expression of c‐jun mRNA was very low in DRG. Induction of c jun mRNA was observed by day 1 after nerve transection, with a sixfold peak at 3 stays and a twofold induction still present by day 7. The constitutive expression of junB mRNA was also low in the DRG, and sciatic nerve transection produced only a modest induction (1.7fold by day 3) in the DRG ipsilateral to the nerve cut. junD mRNA was constitutively expressed at high levels in the DRG, and its level of expression did not change after sciatic nerve transection. Immunocytochemistry studies demonstrated a pattern of c‐Jun, JunB, and JunD immunoreactivity (IR) associated with the cell nuclei of DRG neurons. c‐Jun IR was found at very low levels in the undamaged contralateral DRG neurons, but sciatic nerve transection dramatically increased the number of c‐Jun‐immunoreactive neurons. Dot blot immunoblotting assay confirmed that the DRG ipsilateral to the sciatic nerve cut contained a higher level of c‐Jun protein than the contralateral control DRG. Similar to c‐Jun IR, JunB IR was minimal in the undamaged contralateral DRAG. However, the DRG ipsilateral to the nerve transection did not show an increase in the number of immunoreactive neurons. JunD protein was expressed at high levels in the contralateral DRG, and this level of expression persisted after sciatic nerve transection in the ipsilateral DRG. DNA gel retardation assay experiments with an AP‐1 consensus sequence showed a single DNA‐protein complex. This complex was increased in ipsilateral as compared with contralateral DRG extracts. The amount of DNA protein complex was reduced byc‐Jun protein antiserum but was not altered when treated with a Fos antibody. We conclude that cjun, junB and junD mRNAs and proteins are differentially regulated in the DRG after sciatic nerve transection. © 1995 Wiley‐Liss, Inc.
Localization of mRNAs for adrenoleukodystrophy and the 70 kDa peroxisomal (PMP70) proteins in the rat brain during post‐natal development
Authors: Pollard H. , Moreau J. , Aubourg P.
Published: January 2005

Adrenoleukodystrophy (ALD) is a genetic demyelinating disorder caused by the mutation of a gene encoding a 75‐kDa peroxisomal protein (ALDP) that belongs to the superfamily of ATP binding casette (ABC) transporters. The PMP 70 gene codes for another peroxisomal ABC transporter that shows 38.5% amino acid identity with ALDP. ALDP and PMP70 have the structure of huff transporter and Could possibly heterodimerize to form a full transporter within the peroxisomal membrane. Using in situ hybridization histochemistry in rat brain, we demonstrate that ALD and PMP70 mRNAs have different spatial and temporal expression during postnatal development. Whereas expression of PMP 70 mRNA was low at birth and culminates between the 2nd and 3rd week in hippocampus and cerebellum, maximum expression of ALDP was found at birth in all brain areas and decreased thereafter. The absence of coordinated expression of ALD and PMP70 genes suggests therefore that ALD and PMP70 proteins are unlikely to function as exclusive and obligatory partners in the brain. © 1995 Wiley‐Liss, Inc.
Regional and temporal profiles of c‐fos and nerve growth factor mRNA expression in rat brain after lateral cortical impact injury
Authors: Yang K. , Mu X. S. , Xue J. J. , Perez‐Polo J. R. , Hayes R. L.
Published: January 2005

Lesion‐induced increases in NGF mRNA are thought to be mediated by c fos gene expression. Conversely, NGF induction of c‐fos expression has been reported following administration of exogenous NGF. However, the relationship between c‐fos and NGF gene expression after traumatic injury to the intact brain is not known. Thus, we applied in situ hybridization and semiquantitative reverse transcription‐polymerase chain reaction (RT‐PCR) methods to determine temporal profiles of c‐fos and NGF mRNA expression in rat brains after controlled impact to the exposed cortex. Using alternate sections from the same rat brains, in situ hybridization studies showed that in neocortex, c‐fos mRNA transiently increased at 30 min, 1 hr, and 3 hr after injury, while there were no increases of NGF mRNA at these postinjury time points. In the hippocampus, in situ hybridization showed that c‐fos mRNA increased at 30 min, 1 hr and 3 hr postinjury, while NGF mRNA increased at 1 hr, 3 hr but not at 30 min after injury. RT‐PCR studies in hippocampus confirmed that c‐fos mRNA increased as early as 5 min after injury, peaked at 30 min postinjury, and remained elevated 5 hr postinjury. Levels of hippocampal NGF mRNA expression increased by 1 hr after injury and plateaued until 3 and 5 hr postinjury. These data are consistent with the possible regulatory role of endogenous c‐fos on NGF expression following traumatic brain injury. © 1995 Wiley‐Liss, Inc.