bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered genetic response to β‐adrenergic receptor activation in late passage C6 glioma cells
Authors: Gubits R. M. , Yu H. , Casey G. , Munell F. , Vitek M. P.
Published: January 2005

Previous studies have demonstrated variability in the phenotype of rat C6 glioma cells. In the present study, we compared morphology, growth rate, and β‐adrenergic regulation of gene expression in early (P39–47) and late (P55–90) passage C6 cells. Morphological changes were observed in five independently derived, late passage populations. In four of the five, the untreated cells were more polygonal than the fibroblast‐like parental cells, and only a small fraction exhibited process outgrowth after dbcAMP treatment. Untreated cells from the fifth late passage population had longer cytoplasmic processes than parental cells and responded to dbcAMP with further process outgrowth. All late passage populations had shorter generation times than the parental cells. In early passage cells, treatment with the β‐adrenergic agonist, isoproterenol (IPR), resulted in an increase in c‐fos mRNA and a decrease in c‐jun mRNA (Gubits RM, Yu H: J Neurosci Res, 30:625–630, 1991). Both of these immediate early gene responses were irreversibly lost between P50 and P55. Additional differences in basal or IPR‐induced mRNA levels were observed for β‐APP, GFAP, NGF, and PPE, but not for a number of other mRNAs. These results are discussed in relationship to previously described differences in the ability of early and late passage C6 cells to accumulate cAMP (Mallorga P, et al.: Biochim Biophys Acta 678:221–229, 1981). © Wiley‐Liss, Inc.
Metabolic relationships between proteins of myelin and paranodally shedded, partially degraded myelin fragments in the rabbit CNS
Authors: Persson H. , Berthold C.‐H. , Rydmark M. , Fabricius C.
Published: January 2005

The “close‐to‐node” regions of myelinated nerve fibres, i.e., the paranodal end segments, are generally thought to be sites of high metabolic activity and myelin sheath turnover. Data on turnover rates of individual myelin constituents are conflicting but there exists a common belief that myelin is metabolized as independent molecules rather than as a unit. The occurrence of paranodal Marchi‐positive bodies, with morphological and biochemical properties consistent with partially degraded myelin, prompted us to examine the temporal dynamics of the incorporation of radioactive precursor label in the major proteins of myelin and the Marchi‐positive bodies. 3H‐leucine was administered intrathecally in adult rabbits. After various survival times, the spinal cord was subfractionated by ultracentrifugation in a discontinuous two‐step 0.32 M/0.85 M sucrose gradient. Myelin was collected from the interface and a floating fraction, heavily enriched in Marchi‐positive bodies, was recovered on top of the 0.32 M sucrose. By scintillation counting and by gel fluorography combined with immunoblotting, a gradual appearance with time of partially degraded peptides of myelin‐associated protein and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase was seen in the floating fraction but not in myelin. The temporal dynamics of the specific activities of these two proteins and myelin‐basic protein and proteolipid protein were consistent with a typical source‐product relationship between myelin and the material in the floating fraction. In conjunction with earlier morphological and biochemical findings, these data may suggest that Marchi‐positive bodies appear as a consequence of myelin catabolism. © Wiley‐Liss, Inc.
Targeting of gonadotropin‐releasing hormone axons from preoptic area grafts to the median eminence
Authors: Saitoh Y. , Gibson M. J. , Silverman A.‐J.
Published: January 2005

Implantation of normal GnRH neurons can reverse many of the reproductive deficiencies that characterize hypogonadal (hpg) mice. Since the GnRH axons follow a stereotyped trajectory to their target we investigated the possibility that host brain regions adjacent to the graft might provide signals that induced this directional growth. The role of the adenohypophysis in GnRH axonal outgrowth was studied in mice with co‐grafts of fetal preoptic area (POA) and pituitary and in hypophysectomized hosts. When fetal pituitaries were grafted together with the POA, immunoreactive GnRH fibers did enter the glandular tissue but they also grew into the host median eminence. Surgical removal of the pituitary of hpg hosts prior to POA graft placement was also compatible with GnRH innervation of the host median eminence although in some individuals that innervation pattern was confined to the more caudal aspects. The results of these two experiments suggest that the anterior pituitary gland may be an attractive target for GnRH axons but that this tissue is not essential for directed GnRH axonal outgrowth to its target.

To determine if the median eminence itself could direct the growth of GnRH axons, co‐grafts of POA and a fetal medial basal hypothalamic (MBH) block, which was predominantly median eminence, were made. Immunocytochemistry showed that an intragraft mini‐median eminence was formed with a highly organized and robust GnRH innervation. Ultrastructural analysis indicated that these axons terminated near fenestrated capillaries. However, even under these conditions some GnRH axons exited into the host median eminence. It now seems likely that a cellular component of the median eminence can provide a signal to attract GnRH axons. Whether this signal is produced by the specialized ependymal cells, by the endothelia, or by meningeal (pial) components must now be tested. © 1992 Wiley‐Liss, Inc.
Amygdala neurons in vitro: Neurite growth and effects of estradiol
Authors: Lorenzo A. , Díaz H. , Carrer H. , Cáceres A.
Published: January 2005

Dissociated cell cultures from embryonic rat medial amygdala were studied using sequential photography and immunocytochemical staining for cytoskeletal proteins and substance P (sP). Cultures were seeded with cells taken from fetuses grouped by sex; experimental cultures were raised in medium containing 17‐beta‐estradiol (E2). Forty‐eight hours after plating a few neurons begin to define their morphological polarity by the differentiation of an axon‐like process; at 5 days in vitro (DIV) almost all neurons had developed an axon. Tapering, daughter branch ratio and branch power coefficient coincided with identification of dendrites which could be confirmed by retrospective analysis of immunocytochemically stained cultures: at 5 DIV MAP‐2 was restricted to dendrites whereas Tau immunoreactivity was differentially localized with a clear predominance in the axon. At 21 DIV neuronal shape parameters were strikingly like those of amygdaloid neurons in vivo. It was demonstrated in living neurons that E2 increased total dendritic length and that this is due to increased ramification of third or higher order dendritic segments whose individual lengths are not different from controls. Densitometric measurement of MAP‐2 stained neurons showed a highly significant increase of immunoreactive material in cells grown in the presence of E2; readings for alpha‐tubulin were not different between controls and E2 treated cultures. The effect of E2 on dendritic length was just as manifest in sP‐positive as in sP‐negative neurons. No sexual differences in morphological parameters, growth characteristics or effects of E2 were found in neurons taken from female fetuses versus neurons from male fetuses. The significance of these results for the generation of sexual differences in the amygdala in vivo is discussed and contrasted with reported results on the effects of E2 in cultures of different neural regions. © 1992 Wiley‐Liss, Inc.
Astrogliosis in culture: I. The model and the effect of antisense oligonucleotides on glial fibrillary acidic protein synthesis
Authors: Yu Albert C. H. , Lee Y. L. , Eng L. F.
Published: January 2005

Astrogliosis is a predictable response of astrocytes to various types of injury caused by physical, chemical, and pathological trauma. It is characterized by hyperplasia, hypertrophy, and an increase in immunodetectable glial fibrillary acidic protein (GFAP). As GFAP accumulation is one of the prominent features of astrogliosis, inhibition or delay in GFAP synthesis in damaged and reactive astrocytes might affect astrogliosis and delay scar formation. The aim of this study is to investigate the possibility of utilizing antisense oligonucleotides in controlling the response of astrocytes after mechanically induced injury. We scratched primary astrocyte cultures prepared from newborn rat cerebral cortex with a plastic pipette tip as an injury model and studied the astrogliotic responses in culture. Injured astrocytes became hyperplastic, hypertrophic, and had an increased GFAP content. These observations demonstrate that injured astrocytes in culture are capable of becoming reactive and exhibit gliotic behaviors in culture without neurons. The increase in GFAP content in injured astrocytes could be inhibited by incubating the scratched culture with commerically available liposome complexed with 3′ or 5′ antisense oligonucleotides (20 nt) in the coding region of mouse GFAP. The scratch model provides a simple system to examine in more detail the mechanisms involved in triggering glial reactivity and many of the cellular dynamics associated with scar formation. Antisense oligonucleotide treatment could inhibit the GFAP synthesis in injured astrocytes, hence it may be applicable in modifying scar formation in CNS injury in vivo. © 1993 Wiley‐Liss, Inc.
Serotonin‐activated α2‐macroglobulin inhibits neurite outgrowth and survival of embryonic sensory and cerebral cortical neurons
Authors: Liebl D. J. , Koo P. H.
Published: January 2005

Methylamine‐modified alpha‐2‐macroglobulin (MA‐α2M) has been recently shown to inhibit the biological activity of β‐nerve growth factor (NGF) in promoting neurite outgrowth by embryonic dorsal root ganglia in culture (Koo PH, Liebl DJ, J Neurosci Res 31:678–692, 1992). The objectives of this study are to determine whether α2M can also be modified by larger aromatic biogenic amines such as 5‐hydroxytryptamine (5HT; serotonin), the nature of interaction between NGF and 5HT‐modified alpha‐2‐M (5HT‐α2M), and the effect of 5HT‐α2M on the neurite extension and the growth of embryonic sensory and cholinergic neurons in 2 disparate animal species (chicken and rats). This study demonstrates that each mole of α2M can combine with 15.2 ± 1.8 moles of 5HT, in which up to 4.5 ± 0.4 moles may be covalently bonded. As determined by gel filtration and polyacrylamide gel electrophoresis studies, both 5HT‐α2M and normal α2M combine noncovalently with NGF, but 5HT‐α2M by comparison can combine with NGF somewhat more effectively. In contrast to normal α2M, 5HT‐α2M at concentrations greater than about 0.17 μM exerts a dose‐dependent inhibition on the NGF‐stimulated neurite outgrowth by embryonic dorsal root ganglia and dissociated cells in culture, and the inhibitory effect can be overcome by higher NGF concentrations. Both 5HT‐α2M and MA‐α2M at 1.0 μM inhibit neurite extension by embryonic rat cerebral cortical cells and seriously damage these cells in culture. Such neurite‐inhibitory activity, however, can only be partially blocked by extraneously added NGF alone. Normal α2M (at 1.0 μM) and 5HT (at 188 μM), on the other hand, under the identical conditions produce very little or no effect on the normal cellular and axonal growth of these cells. We conclude that α2M can potentially interact with nucleophilic monoamines, including neurotransmitters, to form inhibitory complexes which may inhibit/regulate NGF‐promoted neurite outgrowth and neuronal survival. In addition, higher concentrations of such complexes can seriously damage certain CNS neurons which do not depend solely on NGF for survival. © 1993 Wiley‐Liss, Inc.
Glial progenitor cells of the nerve fiber layer of the olfactory bulb: Effect of astrocyte growth media
Authors: Doucette R.
Published: January 2005

There are two morphologically distinct types of glial cells (i.e., ensheathing cells and astrocytes) in the nerve fiber layer (NFL) of the adult mammalian olfactory bulb. Ensheathing cells provide ensheathment for olfactory axons, whereas astrocytes occupy the interfascicular spaces of the olfactory NFL. During embryonic development, however, only one type of glial cell is found in this layer of the olfactory bulb, namely, the ensheathing cell. Even though ensheathing cells take up residence within the CNS, they are actually derived from the olfactory placode. Far less is known about the developmental origin of interfascicular astrocytes, which arise either from the glial progenitor cells that give rise to ensheathing cells or from astrocyte precursor cells that migrate into the NFL from deeper layers of the bulb primordium. In the present study, enriched populations of ensheathing cells were grown in vitro in media known to promote the growth and differentiation of astrocytes to determine whether ensheathing cell progenitors could differentiate into astrocytes. These media failed to induce the appearance of astrocytes in the ensheathing cell cultures. It was concluded that the astrocytes of the NFL most likely arise from progenitor cells that migrate into this layer from deeper parts of the developing bulb. © 1993 Wiley‐Liss, Inc.
Protease production by cultured microglia: Substrate gel analysis and immobilized matrix degradation
Authors: Colton C. A. , Keri J. E. , Chen W.‐T. , Monsky W. L.
Published: January 2005

The production of collagen‐degrading proteases by cultured neonatal rat microglia was examined using an immobilized fibronectin‐gelatin matrix coupled to a fluorescent marker and by substrate gel analysis. When microglia were plated onto the surface of the matrix and incubated under resting (nonstimulated) conditions, a small but visible amount of immobilized matrix was degraded. Treatment with lipopolysaccharide (LPS) or interleukin‐1 (IL‐1) significantly increased the number of microglia demonstrating substrate degradation. Substrate‐SDS polyacrylamide gel electrophoresis of samples of supernatants from untreated cultured microglia indicated the presence of a 72 and a 92 kD metalloproteinase with characteristics corresponding to collagenases. Supernatants from untreated astrocyte cultures were shown to have primarily a 72 kD metalloproteinase. Proteinase activity increased on stimulation of the microglia with LPS and IL‐1 in a dose‐dependent fashion. These results indicate that cultured microglia release active proteases capable of degrading the extracellular matrix in a localized region. The production of proteases by activated microglia may have important physiological and pathophysiological consequences within the restricted extracellular matrix of the CNS. © 1993 Wiley‐Liss, Inc.
Pathways of migration of transplanted Schwann cells in the demyelinated mouse spinal cord
Authors: Evercooren A. Baron‐van , Duhamel‐Clerin E. , Boutry J. M. , Hauw J. J. , Gumpel M.
Published: January 2005

We have studied the behavior of Schwann cells transplanted at a distance from an induced myelin lesion of the adult mouse spinal cord. These transplanted cells were mouse Schwann cells arising from an immortalized cell line (MSC80) which expresses several Schwann cell phenotypes including the ability to produce myelin. The behavior of MSC80 cells was compared to that of purified rat Schwann cells transplanted in the same conditions. Schwann cells were labeled in vitro with the nuclear fluorochrome Hoechst 33342 and were transplanted at distances of 2‐8 mm from a lysolecithin‐induced myelin lesion in the spinal cord of shiverer and normal mice. Our results show that transplanted MSC80 cells migrated toward the lesion, in both shiverer and normal mouse spinal cord, preferentially along the ependyma, meninges, and blood vessels. They also migrated along white matter tracts but traveled a longer distance in shiverer (8 mm) than in normal (2‐3 mm) white matter. Using these different pathways, MSC80 cells arrived within the lesion of shiverer and normal mouse spinal cord at the average speed of 166 p, m/hr (8 mm/48 hr). Migration was most efficient along the ependyma and the meninges where it attained up to 250 μm/hr. Migration was much slower in white matter tracts (95 μm/hr ± 54 in the shiverer and only 38 pm/hr ± 3 in the normal mouse). We also provide evidence for the specific attraction of MSC80 cells by the lysolecithin‐induced lesion since (1) their number increased progressively with time in the lesion, and (2) MSC80 cells left their preferential pathways of migration specifically at the level of the lesion. Finally, combining the Hoechst Schwann cell labeling method with the immunohistochemical detection of the peripheral myelin protein, PO, we show that some of the MSC80 cells which have reached the lesion participate in myelin repair in both shiverer and normal lesioned mouse spinal cord. A series of control experiments performed with rat Schwann cells indicate that the migrating behavior of transplanted MSC80 cells was identical to that of purified but non‐immortalized rat Schwann cells. © 1993 Wiley‐Liss, Inc.
Uptake, release and metabolism of alaume in neurons and astrocytes in primary cultures
Authors: Westergaard N. , Varming T. , Peng L. , Sonnewald U. , Hertz L. , Schousboe A.
Published: January 2005

The uptake, release, and metabolism of alanine were studied in primary cultures of cerebral cortical neurons or astrocytes and cerebellar granule neurons. All three cell types exhibited a saturable, sodiumdependent uptake of alanine with KM values (ULM) of 256 ± 30, 463 ± 39, and 292 ± 39, respectively, and V values (nmol/min/mg) of 15.9 ± 0.7,7.9 ± 0.01, and 17.4 ± 0.8, respectively. The corresponding val' ues < nmol/min/mg for the specific activity of alanine aminotransferase were 4.7 ± 0.4, 17.1 ± 2.5, and 4.5 ± 0.9 (all values represent the mean ± SEM). Release of alanine from the cells was rectilinear with time over a 10 hr period in case of astrocytes (40 nmol/hr/ mg) and cerebellar granule neurons (21 nmol/hr/mg). In cortical neurons the release rate declined from an initial value of 19 nmol/hr/mg during the first 3 hr to a value of less than 3 nmol/hr/mg during the subsequent 7 hr of incubation. Metabolism of [14C]alanine to 14CO2 was found to have a lag period of 15 min and subsequently the rate of CO2 production was constant over a 45 min period with a value of 0.5 nmol/min/mg in granule neurons and about 0.3 nmol/ min/mg in the other two cell types. Altogether the results show that alanine is preferentially produced in and released from astrocytes and accumulated into both GABAergic cortical neurons and gintamatergic cerebellar granule neurons. © 1993 Wiley‐Liss, Inc.
Localization of basic proteins in human myelin
Authors: McLaurin J. , Ackerley C. A. , Moscarello M. A.
Published: January 2005

The myelin basic protein (MBPs) represent a family of proteins (charge isomers) which account for 35% of the total myelin protein. Localization studies have been inconclusive because MBP is not a single protein. Antibodies obtained by injection of MBP into animals recognized all members of the MBP family. In the studies reported here, we have fractionated the MBPs into specific components or charge isomers. One of these which contains citrulline accounts for about 20% of the total MBP. We report the localization of this single MBP to the intraperiod line of myelin by immunoelectron microscopy. For these studies several specific antibodies were used including antibodies raised against total MBP, specific MBP peptides, and against a tetracitrulline peptide. This latter antibody was specific for component 8 (C‐8) of MBP. Since C‐8 is the only MBP which contains citrulline it was used to localize this particular form of MBP principally to the intraperiod line by immunogold electron microscopy, while antibody against total MBP (consisting of all charge isomers C‐1→C‐8) labelled both the major dense line and the intraperiod line. When the anti‐citrulline antibody was used with a 3 nm gold conjugated Fab fragments prepared from the secondary antibody, 66.5% of the gold particles were localized to the intraperiod line, while 11.2% of gold particles were localized to the major dense line. On the other hand, with the monoclonal anti‐MBP antibodies reactive with residues 69–74, 59.4% of the gold particles were localized to the major dense line and 23.6% of gold particles at the intraperiod line. Other supporting evidence includes increased labelling of myelin by 125I labelled anti‐citrulline IgG when isolated myelin was swollen, a process known to take place at the intraperiod line. Gold particles were demonstrated at the intraperiod line in swollen and recompacted myelin. C‐8 was shown to associate preferentially with lipids asymmetrically localized to the intraperiod line. © 1993 Wiley‐Liss, Inc.
Reverse transcription and polymerase chain reaction technique for quantification of mRNA in Primary astrocyte cultures
Authors: Murphy G. M. , Jia X. ‐C. , Yu A. C. H. , Lee Y. L. , Tinklenberg J. R. , Eng L. F.
Published: January 2005

The reverse transcription and polymerase chain reaction technique (RT‐PCR) was assessed for the quantification of changes in mRNA levels from primary astrocyte cultures. The effects of dibutyryl cyclic AMP (dBcAMP) on glial fibrillary acidic protein (GFAP) mRNA and the effects of tumor necrosis factor‐alpha (TNF‐α), interlekin‐1 beta (IL‐1β), and lipopolysaccharide (LPS) on interleukin‐6 (IL‐6) mRNA were examined. Two quantitative PCR methods were used: one involved carrying out the reaction in the exponential phase and the other involved the coamplification of a competitive target sequence. Increased GFAP mRNA in response to chronic dBcAMP treatment and increased IL‐6 mRNA in response to TNF‐α/IL‐1β were readily detected. Both RT‐PCR techniques were found to be suitable for the detection of large as well as smaller (twofold) changes in mRNA levels. The advantages and limitations of RT‐PCR for mRNA quantification are discussed. © 1993 Wiley‐Liss, Inc.
Cell biology of neuronal endocytosis
Authors: Parton R. G. , Dotti C. G.
Published: January 2005

Endocytosis is the process by which cells take in fluid and components of the plasma membrane. In this way cells obtain nutrients and trophic factors, retrieve membrane proteins for degradation, and sample their environment. In neuronal cells endocytosis is essential for the recycling of membrane after neurotransmitter release and plays a critical role during early developmental stages. Moreover, alterations of the endocytic pathway have been attributed a crucial role in the pathophysiology of certain neurological diseases. Although well characterized at the ultrastructural level, little is known of the dynamics and molecular organization of the neuronal endocytic pathways. In this respect most of our knowledge comes from studies of non‐neuronal cells. In this review we will examine the endocytic pathways in neurons from a cell biological viewpoint by making comparisons with non‐neuronal cells and in particular with another polarized cell, the epithelial cell. © 1993 Wiley‐Liss, Inc.
Protein growth factor requirements of rat neural crest cells
Authors: Bannerman P. G. , Pleasure D.
Published: January 2005

Nascent neural crest cells derived from explanted E12 embryonic rat caudal neural tubes were used as an assay system to investigate the effects of fibroblast growth factors on neural crest cell (NCC) survival, proliferation, migration, and differentiation. In vitro and in vivo all NCC express low affinity nerve growth factor receptors (p75‐LNGFR), whereas a subpopulation of NCC expresses the carbohydrate epitope recognized by the monoclonal antibody HNK‐1 (Bannerman and Pleasure, manuscript in preparation). Both acidic and basic fibroblast growth factor (FGF) promoted the survival of proportionally greater numbers of p75‐LNGF+/HNK‐1− than P75‐LNGFR+/HNK‐1+ NCC. An as yet uncharacterized factor present in neural tube‐conditioned medium was also required for NCC survival. Mitosis was frequent in those NCC closest to the neural tube, less so as the cells migrated away. Neither basic nor acidic fibroblast growth factor (FGF) influenced rates of NCC mitosis in either of these locations, nor did these FGFs alter the rate at which nascent NCC migrated away from the neural tube. However, acidic and basic FGFs did delay the differentiation of neural crest derived neurons in the cultures. FGF is abundant in the embryonic rat neural crest outgrowth zone, and the present study strongley supports an essential role for fgf in early development of the mammalian neural crest. © 1993 Wiley‐Liss, Inc.
Demyelination in a transgenic mouse: A model for multiple sclerosis
Authors: Mastronardi F. G. , Ackerley C. A. , Arsenault L. , Roots B. I. , Moscarello M. A.
Published: January 2005

A transgenic mouse containing 70 copies (ND4) of the transgene encoding DM20, a myelin proteolipid protein, appeared clinically normal up to 3 months of age. By 8–10 months, it showed tremors, unsteady gait, and dies shortly thereafter. We concluded that the clinical symptoms correlated with demyelination based on the following criteria: (1) at 10 months of age only 17% of the amount of myelin obtained from normal mice was isolated from the ND4 mice; (2) astrogliosis, a prominent feature of demyelinating disease was minimal at 3 months of age but prominent by 10 months; (3) at the electron microscopic level disrupted myelin was seen at 8 months of age in the ND4 mice and ingested myelin debris was found in astrocytes; (4) lymphocytic infiltration in association with endothelial cells was observed routinely in the ND4 mice; (5) sections through optic nerves showed denuded and thinly myelinated axons in the 8 month old ND4 mice.

Although the mechanism by which demyelination takes place is not fully understood, measurements of the amounts of PLP suggest it is down‐regulated by the large amount of DM20. Since DM20 is a major proteolipid in the young but a minor one in the adult, the persistence of high levels in the adult results in improperly assembled myelin which is prone to disruption. Therefore demyelination in the ND4 mouse appears to result from the persistence of immature myelin into the adult. © 1993 Wiley‐Liss, Inc.
Synaptic and photoreceptor components in retinal pigment epithelial cell transplanted retinas of Royal College of Surgeons dystrophic rats
Authors: Sheedlo H. J. , Li L. , Barnstable C. J. , Turner J. E.
Published: January 2005

Plexiform layer synaptic and photoreceptor cell components were investigated in retinas of Royal College of Surgeons (RCS) dystrophic rats transplanted with normal retinal pigment epithelial (RPE) cells by immunocytochemistry using previously characterized monoclonal antibodies. In retinas of normal adult rats and RPE‐cell transplanted retinas of 4 month‐old RCS rats, HNK‐1, a marker for a carbohydrate of the neural cell adhesion molecule (N‐CAM), was detected immunocytochemically in the inner and outer plexiform layers and ganglion cell bodies and their axons. HNK‐1 was also detected in the inner plexiform layer of nontreated retinas of 4 month‐old RCS rats, but was reduced to scattered patches in the outer plexiform layer. In addition, immunoreactivity for the SVP‐38 antibody recognizing synaptophysin was found in both plexiform layers of normal adult rat retinas and RPE‐transplanted retinas of 4 month‐old RCS rats. Furthermore, photoreceptor cell bodies and their inner and outer segments were immunostained for the opsin monoclonal antibody RET‐P1 in retinas of normal adult rats and RPE‐cell transplanted retinas of 4 month‐old RCS rats. However, in nontreated retinas of 4 month‐old RCS rats, only immunostained debris material was detected. These results strongly suggest that normal RPE transplants not only rescue photoreceptor cells in RCS rats, but also maintain an essential functional capacity, in this case, synaptic components in the plexiform layers. © 1993 Wiley‐Liss, Inc.
Characterization of the cis‐acting elements of the mouse myelin P2 promoter
Authors: Bharucha V. A. , Peden K. W. C. , Subach B. R. , Narayanan V. , Tennekoon G. I.
Published: January 2005

Myelin P2 is a basic protein of an apparent molecular weight of 14,800. Expression of P2 has been found largely in the cytosol of Schwann cells in the peripheral nervous system. Although the function of P2 is unknown, its striking homology to a family of fatty acid binding proteins has led to the idea that P2 may function as a fatty acid transport molecule. To investigate the DNA elements that control the expression of P2, sequences 5′ to the coding region were cloned upstream of the cat reporter gene. A series of 3′ and 5′ promoter mutants was constructed and their activity determined following transfection into secondary Schwann cells and the MT4H1 Schwann cell line. Using this strategy, we have identified a 217 bp silencer region and a 142 bp positive regulatory region. In addition, we have localized the 5′ flanking sequences in the promoter that are responsive to cAMP induction and to the transcription factor CCAAT/enhancer binding protein (C/EBP). © 1993 Wiley‐Liss, Inc.
Expression of the receptor tyrosine kinase c‐kit in oligodendrocyte progenitor cells
Authors: Ida J. A. , Dubois‐Dalcq M. , McKinnon R. D.
Published: January 2005

The growth and differentiation of neural precursor cells in the central nervous system (CNS) are regulated by their response to polypeptide growth factors which interact with specific transmembrane receptor tyrosine kinases (RTKs). We demonstrate that rat oligodendrocyte‐type 2 astrocyte (O‐2A) glial progenitor cells, precursors of the myelin‐forming cells in the CNS, express the transmembrane RTK c‐kit, the gene product of the murine dominant white spotting (W) locus and receptor for stem cell factor. Expression of c‐kit transcripts and immunoreactive protein is lost when O‐2A progenitors differentiate into postmitotic oligodendrocytes. Analysis of developing rat brain revealed an increase in the expression of c‐kit transcripts between postnatal days 10 and 12, a window of time preceding the emergence of oligodendrocytes and the onset of myelination in vivo. Expression of c‐kit in vitro and in vivo suggests a role for this receptor and its ligand during oligodendrocyte development. © 1993 Wiley‐Liss, Inc.
Bactenecin, a leukocytic antimicrobial peptide, is cytotoxic to neuronal and glial cells
Authors: Radermacher S. W. , Schoop V. M. , Schluesener H. J.
Published: January 2005

Small antimicrobial peptides are abundantly produced by leukocytes. These peptides are active against a broad range of pathogens, notably bacteria, fungi, and enveloped viruses, but hardly anything is known about their physiological and pathophysiological relevance.

We observed that bactenecin, a dodecapeptide, is strongly cytotoxic ro rat embryonic neurons, fetal rat astrocytes and human gliblastoma cells. This neurotoxicity is unique to bactenecin, as a panel of antibacterial peptides from vertebrates and invertebrates, like defensins, corticostatin, indolicidin, ceropin P1, tachyplesin I, the magainings, or apidaecins did not impair neuronal viability. © 1993 Wiley‐Liss, Inc.
HSP72 induction by heat stress is not universal in mammalian neural cell lines
Authors: Satoh J. , Tabira T. , Yamamura T. , Kim S. U.
Published: January 2005

Heat‐induced expression of 72‐kDa heat shock protein (HSP72) was investigated in a panel of neuronal and non‐neuronal cell lines by immunoblotting and immunocytochemistry using monoclonal antibodies directed to HSP72. By immunoblotting, HSP72 expression was observed in most cell lines of mouse (SN6.1b, CL8c4.7, NSC34.6, B2A, C2C12), RAT (PC12, C‐6, L3), and human (NB‐1, GOTO, IMR‐32, Hela) origin under the heat‐stressed condition. The mouse neuroblastoma cell line N18TG2, however, did not express HSP72 under the heat‐stressed condition. By immunocytochemistry, HSP72 was undetectable in the heat‐stressed N18TG2 cells, while it was identified in the heat‐stressed SN6.1b cells, a clonal hybrid neuron between N18TG2 and mouse septal cholinergic neuron. By exposure to a priming sublethal heat shock, SN6.1b cells but not N18TG2 cells acquired a significant level of tolerance to a subsequent lethal heat shock. These results suggest that heat‐induced expression of HSP72 may contribute to acquisition of the thermotolerant state in SN6.1b cells. © 1994 Wiley‐Liss, Inc.
Neuroprotective activity of acetyl‐L‐carnitine: Studies in vitro
Authors: Forloni G. , Angeretti N. , Smiroldo S.
Published: January 2005

The neuroprotective properties of acetyl‐L‐carnitine (ALCAR) were investigated in primary cell cultures from rat hippocampal formation and cerebral cortex of 17‐day‐old rat embryos. Chronic exposure to ALCAR (10–50 μM), reduced the cell mortality induced by 24 hr fetal calf serum deprivation. Protection was partial when the neuronal cells, chronically treated with ALCAR (50 μM,) were exposed to glutamate (0.25‐1 mM)and kainic acid (250–500 μM) for 24 hr. The neurotoxity induced by N‐methyl‐D‐aspartate (NMDA, 250 μm) was attenuated by the acute co‐exposure with ALCAR (1mM), the chronic treatment with ALCAR (50 μM)significantly reduced the neuronal death induced by NMDA (0.25–1mM) Cell mortality was also investigated in ALCAR‐treated hippocampal cultures chronically treated with β‐amyloid fragment 25–35. ALCAR appeared to have neuroprotective activity. This suggests an explanation of the positive results obtained with ALCAR in the treatment of alzheimer's disease. © 1994 Wiley‐Liss, Inc.
Thrombin receptor peptides induce shape change in neonatal murine astrocytes in culture
Authors: Beecher K. L. , Andersen T. T. , Fenton J. W. , Festoff B. W.
Published: January 2005

Astrocytes appear star‐shaped in the brain, increasingly so after injury. When astroglia are cultures in serum‐containing medium, they exhibit a flat, fibroblast‐like morphology. In serum‐free medium, astrocytes become stellate, with many long processes. The serine protease α‐thrombin mimics the effects of serum at subnanomolar concentractions, whereas the thrombin‐inhibiting serpin, protease nexin I (PNI), reverses the thrombin effect. In our current experiements, murine neonatal spinal cord astrocytes became stellate after 4 hr in serum‐free medium, while cortical astrocytes requires 12 hr in serum‐free medium for stellation. Astrocytes from either region flattened after 60 min in medium containing 3.0 to 300 pM proteoloytically active human α‐thrombin. After 12 hr in thrombin‐containing medium, 98% of the astrocytes had a flattened morphology. No flattening occured if α‐thrombin was replaced by γ‐thrombin, which has its fibrinogen‐recognition exosite disrupted. PNI added at 1 nM to serum‐containing medium caused stellation after 3 hr, and astroglia were 50% stellate by 12 hr. The effect of thrombin was mimicked by a 7‐amino acid peptide (TRP‐7) action on astrocytes. These results indicate that astrocytes possess a cell surface receptor for thrombin, similar to that described for platelets, endothelial cells, and neurons. © 1994 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Neurotrophic‐4/5 is a survival factor for embryonic midbrain dopaminergic neurons in enriched cultures
Authors: Hynes M. A. , Poulsen K. , Armanini M. , Berkemeier L. , Phillips H. , Rosenthal A.
Published: January 2005

Parkinson's disease is a prevalent neurological disease characterized by profound and incapacitating movement disorders. A common pathology in Parkinson's patients is degeneration of substantia nigra dopaminergic neurons that innervate the striatum and a corresponding decrease in striatal dopamine content. We now report that NT‐4/5 can prevent the death of rat embryonic substantia nigra dopaminergic neurons in low density, enriched, primary cultures. Furthermore, these neurons express messenger RNA encoding the trkB receptor for NT‐4/5 and transcripts for NT‐4/5 are present in their environment. In addition, we demonstrate that NT‐4/5 protects embryonic dopaminergic neurons from the toxic effects of the neurotoxin MPP+ THUAS, nt‐4/5 could be a physiological survival factor for midbrain dopaminergic neurons and may be useful as a therapeutic agent for parkinson's disease. © 1994 Wiley‐Liss, Inc.
Re‐expression of glia‐derived nexin/protease nexin 1 depends on mode of lesion‐induction or terminal degeneration: Observations after excitotoxin or 6‐hydroxydopamine lesions of rat substantia nigra
Authors: Scotti A. L. , Monard D. , Nitsch C.
Published: January 2005

The serine protease inhibitor and neurite outgrowth promoter glia derived nexin (GDN) is expressed in the rat CNS during embryogenesis and persists in the olfactory system of the adult where receptor neurons are replaced throughout life. We investigated whether GDN‐immunoreactivity also appears in the adult at sites of synaptic rearrangement following nerve cell death and anterograde terminal degeneration in experimental models for Parkinson's disease. Rat substantia nigra was unilaterally lesioned by stereotaxic application of different toxins: 6‐hydroxydopamine, which selectively destroys dopaminergic neurons, the excitotoxic glutamate analog ibotenic acid, or the glutamate receptor agonists N‐methyl‐D‐aspartate and quisqualate, which cause circumscript lesions of the whole substantia nigra. Nerve cell death and astroglial reactivity were monitored by parallel cresyl staining and immunocytochemistry for glial fibrillary acidic protein, at survival times ranging from 2 to 100 days.

Sustained de novo synthesis of GDN occurred in the dopamine depleted caudate putamen following excitotoxin or 6‐hydroxydopamine induced degeneration of the substantia nigra and of the nigrostriatal pathway provided that the lesions were nearly complete. This is consistent with compensatory changes occurring in deafferented caudate putamen and suggests a permissive role of GDN in neuronal plasticity.

In the substantia nigra astroglia exhibited GDN‐immunoreactivity following excitotoxin injection but not after application of 6‐hydroxydopamine. Thus differences in action mechanisms of neurotoxins may have distinct consequences on the astrocyte mediated response of the same affected brain region. © 1994 Wiley‐Liss, Inc.
Distribution and differentiation of A2B5+ glial precursors in the developing rat spinal cord
Authors: Fok‐Seang J. , Miller R. H.
Published: January 2005

In many regions of the rat central nervous system, oligodendrocytes develop from migratory A2B5+ precursor cells. In the rat spinal cord, during early embryonic development the capacity for oligodendrogenesis appears to be restricted to ventral regions of the spinal cord, while cultures of postnatal rat spinal cord contain a distinct population of A2B5+ astrocyte precursors. To determine if, as in other regions of the CNS, spinal cord A2B5+ cells give rise directly to oligodendrocytes and astrocytes, the initial distribution, and subsequent dispersion, proliferation, and differentiation of spinal cord A2B5+ cells have been examined in both explant and dissociated cell cultures. Spinal cord oligodendrocytes develop from A2B5+ cells. At E14, A2B5+ cells are restricted to ventral regions of the spinal cord and as development proceeds they become more uniformly distributed throughout the spinal cord. In explant cultures, greater than 95% of the explants that contain oligodendrocytes also contain A2B5+ cells and a proportion of mature oligodendrocytes retain detectable A2B5 immunoreactivity briefly on their surface. The maturation of spinal cord oligodendrocyte precursors occurs in a number of distinct stages characterized by the expression of O4 immunoreactivity, which first appears at E16, and GC immunoreactivity, which first appears at E18. As spinal cord oligodendrocyte precursors acquire O4 immunoreactivity they appear to lose the ability to proliferate in response to PDGF but retain the ability to proliferate in response to bFGF, suggesting that the control of proliferation of oligodendrocyte precursors is, in part, dependent on their maturational state. In the presence of high serum, spinal cord A2B5+ cells fail to develop in isolated E14 dorsal spinal cord cultures, while in ventral cultures they subsequently differentiate into A2B5+ astrocytes suggesting that A2B5+ astrocyte precursors are also initially ventrally located. Unlike oligodendrocyte differentiation, however, the differentiation of spinal cord A2B5+ cells into astrocytes is delayed in early embryonic‐derived cultures compared to those from older animals. These observations suggest that local influences may regulate the timing of spinal cord A2B5+ astrocyte development, but not spinal cord oligodendrocyte development. © 1994 Wiley‐Liss, Inc.
Transient expression of 3,5,3′‐triiodothyronine nuclear receptors in rat oligodendrocytes: In vivo and in vitro immunocytochemical studies
Authors: Besnard F. , Luo M. , Miehe M. , Dusssault J. H. , Puymirat J. , Sarliève L. L.
Published: January 2005

It is generally accepted that the action of thyroid hormones is mediated through specific nuclear receptors. Recent studies have demonstrated the homology of the thyroid receptor with the cellular product of the oncogen v‐erbA. So far, two genes have been identified and classified as α and β subtypes. In this study, the expression of nuclear triiodothyronine (T3) receptors (NT3RS) was examined in secondary cultures containing 85–90% oligodendrocytes (OL) prepared from newborn rat brain primary cultures enriched in OL. These cultures, which are able to produce myelin membranes, were examined by double immunolabelling with a monoclonal antibody (2B3) raised against purified rat liver NT3Rs and with antibodies against two maturation markers of OL: an early marker, galactocerebroside (GC), and myelin basic protein (MBP), which is expressed later than GC. 2B3 recognized three nuclear proteins with the same molecular weights as β, α1, and α2 sybtypes with different capacities for binding T3. In 5‐day ‐old OL secondary cultures (25 days, total time in culture), 2B3‐NT3R immunoreactivity was located in 77% of morphologically immature OL (GC)+ cells, whereas only 44% of morphologically mature OL were immunoreactive. Only 35% of the MBP+ cells co‐expressed NT3Rs. In the corpus callosum of devloping rat brain, at all ages studied from 7–60 days postnatal, the total absence of NT3Rs in dark OL (morphologically mature), confirmed by ultrastructural immunocytochemistry, indicates an even more dramatic decrease during maturation. Furthermore, the percentage of medium OL (less mature) stained by 2B3 is reduced by approximately half in 60‐ compared to 20‐day‐old rat brain. It is of interest to note that the in vitro observation with maturation markers mirrors the in vivo decrease of NT3R expression during development. It is intersting that NT3Rs are absent in vivo before the critical period of active myelination. These data indicate the presence of a nuclear T3 binding protein in the nuclei of OL at the time of myelination both in vitro and in vivo. The transient expression of these NT3Rs during active myelination argues in favour of a direct effect of thyroid hormones on OL. © 1994 Wiley‐Liss, Inc.
Amyloid precursor protein is enriched in axolemma and periaxolemmal‐myelin and associated clathrin‐coated vesicles
Authors: Sapirstein V. S. , Durrie R. , Berg M. J. , Marks N.
Published: January 2005

The amyloid precursor protein (APP) is widely distributed within the CNS, where it is expressed in both neurons and glia. We have isolated axolemma and periaxolemmal‐myelin from rat brain and have determined by Western blot that APPs, Mr 100–110 kDa, are major constituents of these membrane. Isolation of axolemma, periaxolemmal‐myelin, and compact myelin show that while APP represents 1 and 0.6% of the proteins of these respective membranes, it is absent from compact myelin. These results indicate that APP transported down the axon is deposited at sites in the axolemma as well as the synapse, and that within the myelin complex, APP is targeted to the periaxolemmal domain. Both axolemma and periaxolemmal‐myelin contained a 10.5 kDa APP peptide which, based on reactivity with anti‐C‐terminal APP antibodies but not with anti‐N‐terminal antibody, appears to be a membrane‐associated C‐terminal fragment. Western blots with antibodies to Alzheimer precursor‐like proteins (APLP) indicate that APP immune reactivity is not a result of cross reactivity with APLPs. Isolation of axolemma from human autopsy material showed nearly identical results with a clear enrichment, relative to homogenate, of APP Mr 100–100 and the 10.5 kDa C‐terminal peptide. The demonstration of APP in axolemma and periaxolemmal‐myelin was replicated in membrane isolated from bovine brain. Bovine studies were extended to analysis of white matter clathrin‐coated vesicles; these data show that coated vesicles isolated from white matter, under conditions that previous studies indicate are largely endocytic vesicles, contain levels of APP comparable to that found in axolemma and periaxolemmal‐myelin. In addition, these vesicles contain cysteinly and aspartyl proteases. Incubation of axolemma with cathepsin B at pH 6.0 caused a rapid loss in the immune reactivity of APP Mr 100–110 and Mr 10.5 when analyzed with antibodies to APP672–695. This appears to be the result of hydrolysis within the epitope and not proteolysis of APP or the C‐terminal peptide, since no loss of reactivity was observed when analyzed with antibodies to sites more distal to the C‐terminus. Thus, cathepsin B hydrolyses membrane bound APP close to the C‐terminus and may be a useful tool for altering C‐terminal APP function. © 1994 Wiley‐Liss, Inc.
Differential accumulation of gangliosides by the brains of MPTP‐lesioned mice
Authors: Saulino M. F. , Schengrund C.‐L.
Published: January 2005

Although gangliosides have been reported to enhance recovery from 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced lesions of the substantia nigra, evidence as to whether the administered gangliosides actually reach this or any other site of lesion in the central nervous system (CNS) at which they putatively enhance recovery is lacking. Therefore, studies were carried out to determine the amount of 3H‐labeled ganglioside that was accumulated by the brains of MPTP‐treated mice as well as by brains of control mice. No significant difference in the accumulation of 3/‐bovine brain gangliosides or 3H‐GD1a was seen between lesioned and control brains up to 240 min after injection of the labeled lipids. However, significantly more label was associated with the brains of MPTP‐treated mice compared to controls 120 and 240 min after the injection of 3H‐GM1. Analysis of the lipids extracted from the brain of a 3H‐GM1‐treated mouse revealed that the majority of label was still associated with 3‐GM1, 240 min after its administration. Autoradiography of tissue sections from the brains of MPTP‐treated mice injected with 3H‐GM1 showed that label was present in the ventricular spaces of the brain. This observation suggests that the administered gangliosides are present in the cerebrospinal fluid, which indicates that they have the potential to reach the lesioned CNS site at which they putatively enhance recovery. © 1994 Wiley‐Liss, Inc.