bluuebunny/crossref_metadata_2025 huggingface dataset:
Myelin‐specific proteolipid protein is expressed in myelinating schwann cells but is not incorporated into myelin sheaths
Authors: Puckett C. , Hundson L. , Ono K. , Friedrich V. , Benecke J. , Dubois‐Dalcq M. , Lazzarini R. A.
Published: January 2005

Contrary to widely held beliefs, the gene encoding proteolipid protein (PLP), the major structural protein of central nervous system myelin, is ecpressed in Schwann cells and their tumors. Proteolipid mRNA was identified in human acoustic neuromas and in rat and rabbit sciatic nerves using a human PLP cDNA as probe. Proteolipid protein itself was shown to be present in human and rat sciatic nerve Schwann cells by immunofluorescence microscopy and by Western blot analysis using antisera raised to a sythetic PLP polypeptide. Although easily detected in the Schwann cell body, PLP was not detected in the peripheral myelin itself, suggesting that PLP is preferentially excluded from this protion of the Schwann cell membrane.
Migration of cultured fetal spinal cord astrocytes into adult host cervical cord and medulla following transplantation into thoracie spinal cord
Authors: Goldberg W. J. , Bernstein J. J.
Published: January 2005

Cell suspensions from 14‐days‐gestation rat spinal cord, which had previously been soaked for 1 hr in a 2 μg/ml solution of Phaseolus vulgaris leucoagglutinin (PHAL), were cultured on collagen gels containing laminin for 2 weeks. Pieces of the gel and attached cells were then transplanted into the dorsal column of adult host thoracic spinal cord. At 1, 2, and 3 months postimplantation (MPI), animals were sacrificed, and the spinal cords were removed, embaedded in paraffin, and sectioned at 8 μm for immunohistochemistry at the light microscopic level. Sections were double labeled for PHAL and utilized as a marker for transplant‐derived cells and glial fibrillar acidic protein (GFAP), a specific marker for astrocytes. Trnasplant‐derived astorcytes (PHAL‐GFAP positive cells) migrated from the transplantation site in both rostral and caudal directions and were observed within the host dorsal column ipsilateral to the transplantation site. At 2 months, latera migration into the contralateral dorsal coumn and ipsilateral dorsal horn was observed. At 3 MPI transplant‐derived astrocytes were observed in host medulla (nucleus gracilis). Transplant‐derived astrocytes were also observed on the glial limitans as far as nucleus gracilis. A migration rate of 0.72 mm/day was calculated, assuming a 14‐day delay in the initiation of migration. The ramifications of such extensive migration are discussed with regard to return of function and amelioration of lesion‐induced deficits.
Immunocytochemical demonstration of fibroblast growth factor in cultured chick and rat neurons
Authors: Janet T. , Grothe C. , Pettmann B. , Unsicker K. , Sensenbrenner M.
Published: January 2005

The presence of fibroblast growth factor (FGF) was investigated by immunocytochemistry in cultured neuronal cells derived from the peripheral (PNS) and the central nervous system (CNS) of chick and rat embryos. Polyclonal antimouse FGF antibodies, which cross‐react with basic and acidic FGF, were used in the peroxidase immunocytochemical staining method. FGF immunoreactivity was found in neurons. Staining intensity in chick and rat brain neuronal cells increased during the culture period, reached a maximum after 6–8 days, and subsequently declined. Embryonic chik ciliary and dorsal root ganglionic (DRG) neurons as well as DRG neurons from newborn rat displayed intense FGF immunoreactivity at 12 and 48 hr in culture. Nonneuronal cells were not stained. The data demonstrate for the first time that cultured neurons from the CNS and PNS contain FGF.
Brain reactions to antigen
Authors: Korneva E. A. , Grigoriev V. A. , Stoljarov I. D.
Published: January 2005

The nervous and immune system receive and process information, can develop a specific response and possess memory properties. Here we report on electrophysiological responses of brain to antigen presentation. In particular, antigenic presentation to brain affects dynamic changes in the DC potential.
Demonstration of glucocorticoid receptor‐like immunoreactivity in glucocorticoid‐sensitive vasopressin and corticotropin‐releasing factor neurons in the hypothalamic paraventricular nucleus
Authors: Uth R. M. , McKelvy J. F. , Harrison R. W. , Bohn M. C.
Published: January 2005

Many parvocelular neurons in the paraventricular nucleus of the hypothalamus express high levles of conticotropin releasing factor (CRF) or vasopressin following adrenalectomy. To determine whether gulcocorticoid receptor was used in cobination with polyclonal antisera directed against either vasopressin or CRF to permit simultanrous visulization of either peptide with gulcocorticiod receptor‐like immunoreactivity (IR). Rats were adrenlectomized (ADX) for 2 weeks to optimize numbers of vasopresin‐and CRF‐IR neurons. Six hours prior to sacrifice, a seprate group of adrenalectomized rats was treated with conticosterone (40 mg/kg). This short‐term re‐placement resulted in nuclear localization of gulcocorticoid receptor‐like‐IR but did not attenuate the increased numbers of CRF‐ and vasopressin‐Ir neurons observed after adrenlectomy. It was therfore possible to visualize vasopressin‐ or CRF‐IR and nuclear glucocorticoid receptor‐like‐Ir simultancously. Cell counts of deouble‐labeled neurons studied, which respond to adrenlectomy by increased peptide expression. Thse data suggest that a major feedback effect of gulcocorticoids on the hypothalamic‐pituitary‐adrenal axis is exerted directly within nuclei of CRF and vasopressin neurons.
Taurine and nifedipine protect retinal rod outer segment structure altered by removal of divalent cations
Authors: López‐Escalera R. , Morán J. , Pasantes‐Morales H.
Published: January 2005

A protective effect of taurine on morphological alterations induced in isolated ROS by remotion of divalent cations is described. ROS damage is not observed when sodium or chloride is replaced by impermeant ions or in the presence of nifedipine (10μM). An increase acccumulation of22Na but not of36CI occurs in retinas incubated in dicalent cation free medium. Taurine (5–25mM) provides a concentration‐dependent protection to ROS structure. Taurine is also able to reduce the increased22Na influx to control values. These results suggest an interaction with calcium sites on ROS membranes which regulate sodium premeability.
Localization of glial cell antigens in the brains of young normal mice and the dysmyelinating mutant mice, Jimpy and Shiverer
Authors: Cammer W. , Tansey F. A.
Published: January 2005

Tissue sections from the brains of normal, jimpy, and shiverer mice were immunostained by the peroxidase antiperoxidase method for carbonic anhydrase (CA) and the putative astrocytic “markers” glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP). The cells in normal gray matter that immunostained with anti‐CA and anti‐GS were similar to one another in size and process elaboration. In the normal gray matter there were relatively few GFAP‐positive astrocytes. When present, these cells resembled the CA‐and GS‐positive cells; however, the GFAP appeared to be concentrated in the astroglial processes, as distinguished from the cell bodies. Glial cell processes, immunostained for CA or GS, surrounded blood vessels and unstained neurons in the normal gray matter. The glial cells in shiverer gray matter were similar to those in the normal gray matter. When stained for GS or GFAP, the glial cells in the jimpy gray matter appeared to be somewhat hypertrophied, and when the glial cells in this mutant were stained for CA, the nuclei appeared to be swollen. It was concluded that some of the CA‐positive cells inthe gray matter of the normal and of each mutant mouse brain could be astrocytes. The patterns of immunostaining in the white matter emphasized the different complements of glial cells in the mutants. In the normal and shiverer mouse corpus callosum, CA, in particular, was detected only in the oligodendrocytes, their processes, and myelin. However, the data concerning the jimpy mouse suggested that the few CA‐positive cells in the corpus callosum of that mutant could be astrocytes.
Modulation of tyrosine hydroxylase gene expression in rat brain and adrenals by exposure to cold
Authors: Richard F. , Faucon‐Biguet N. , Labatut R. , Rollet D. , Mallet J. , Buda M.
Published: January 2005

The long‐term changes in tyrosine hydroxylase (TH) activity induced by chronic exposure to cold in brain noradrenergic neurons of the locus coeruleus (LC) were analyzed and compared to those measured in a peripheral tissue such as adrenals. This analysis was made possible at the level of one single tissue corresponding to one animal by the use of sensitive methods that allow assay of TH activity, protein, and mRNA levels in parallel from the same homogenate. The three parameters were measured in brain structures and adrenals of rats maintained at 4°C during 4 days and were compared to those of control animals kept at normal housing temperature (22°C). LC of rats exposed to cold contained 200% more TH mRNA than controls. The amount of TH protein in this area rose to as much as 164% that of controls. Similarly, the activity of the enzyme increased to 140% of the normal value. Thus, these observations show that (1) the increase in TH mRNA was much higher than the increase in protein levels, and that (2) the newly synthesized molecules have about the same activity as that present under normal conditions. In contrast to the LC, no variation of these parameters was observed in the substantia nigra. In the adrenals, the variations in the different parameters were qualitatively similar to that observed in the LC, although they were quantitatively higher: TH mRNA, TH protein, and TH activity levels were respectively 330%, 182%, and 167% that of control adrenals. Altogether, these results demonstrate that exposure to cold induces an alteration in TH synthesis in brain noradrenergic neurons as well as in adrenals.
Effect of epidermal growth factor on the labeling of the various RNA species and of nuclear proteins in primary rat astroglial cell cultures
Authors: Avola R. , Condorelli D. F. , Turpeenoja L. , Ingrao F. , Reale S. , Ragusa N. , Stella A. M. Giuffrida
Published: January 2005

This study investigated the effects of epidermal growth factor (EGF) on the labeling of various RNA species and of nuclear proteins in primary rat astroglial cell cultures. After 12 hours of EGF treatment in serumfree medium or chemically defined medium, significant increase in RNA labeling, and also in acid‐soluble radioactivity and RNA content, was observed. The ratio RNA/DNA was significantly higher in EGF‐treated cultures compared with controls. Ribosomal RNAs (28S and 18S), polyadenylated, and nonpolyadenylated RNAs showed a higher specific radioactivity in EGF‐treated cultures. Among the nuclear proteins, the labeling of basic proteins was enhanced by EGF treatment, whereas that of total nuclear acidic protein (NHPs) was less modified, except for some NHPs separated by gel electrophoresis with a molecular weight (MW) approximately 95‐83 and 44 kd, which were significantly more labeled in EGF‐treated cultures.
Differential modulation of glutamate metabolizing enzymes in mouse and chick cultured glial cells by insulin
Authors: Sena A. , Ferret‐Sena V. , Sarliéve L. L. , Tholey G.
Published: January 2005

The effect of physiological concentrations of insulin (2 and 20 ng/ml) on glutamine synthetase (GS) and glutamate dehydrogenase (GDH) activities were compared in mouse and chick glial cells in culture. Addition of insulin to serum‐containing medium increased the level of GS and GDH activities in glial cells prepared from 14–15‐day‐old embryonic mice. A similar but less pronounced effect was observed with glia derived from newborn mouse brain. In absence of serum, addition of insulin had no effect on the tested enzymes.

The effects of insulin on enzymatic activities of glial cells from 14–15‐day‐old embryonic chick brain hemispheres were, in contrast, quite different. A significant decrease of GS activity was induced by the hormone, only in the absence of serum. Conversely, the presence of serum enhanced an inhibitory effect of insulin toward chick GDH. The different effects of insulin and the different serum dependence observed for the mammalian and the avian model could reflect fundamental chemical differences between both species as indicated by immunoelectrophoretic analysis. However, it can be concluded that insulin may be a physiological factor regulating glial maturation and amino acid neurotransmitter metabolism in the central nervous system.
Major rat brain membrane‐associated and cytosolic enkephalin‐degrading aminopeptidases: Comparison studies
Authors: Hui Koon‐Sea , Hui M. P.‐P. , Lajtha A.
Published: January 2005

The major cytosolic and membrane‐associated enkephalin‐degrading aminopeptidases were purified in parallel by column chromatography successively on DEAE‐cellulose, AH‐Sepharose, hydroxylapatite, Sephadex G‐200, Affigel Blue, AH‐Sepharose, and hydroxylapatite. With the final hydroxylapatite column, the cytosol (S) and the membrane (M) enzymes could each be resolved into two peaks, one eluted with 0.05 M phosphate (SI, MI) and the other with 0.25 M phosphate (SII, MII). The overall purification, with Arg BNA as substrate, for the SI and MI was about 450‐fold; for SII and MII, 1,200‐fold. The yield for each enzyme was about 2%, the major protein integral units of the four enzymes are similar; they are single polypeptide chains with a molecular weight of 100,000 daltons. Their pH optimum, substrate specificity, and sensitivity to puromycin show that they are similar to lysosomal aminopeptidase (EC 3.4.11.‐). The two forms of the cytosol and the membrane enzymes have slightly different kinetic constants. With the inhibitors, SII is more sensitive to proctolin, whereas MII is more sensitive to bestatin and Arg‐Phe‐Ala. Mn2+ activates SI on Met‐enkephalin degradation, but inhibits SII, MI, and MII.
Components of neurite outgrowth that determine neuronal cytoarchitecture: Influence of calcium and the growth substrate
Authors: Mattson Mark P. , Guthrie P. B. , Kater S. B.
Published: January 2005

The present study examined the cellular mechanisms underlying the generation of neuroarchitecture. Identified Helisoma neurons in isolated cell culture normally require factors present in brain‐conditioned medium (CM) in order to display the different components of neurite outgrowth (sprouting, elongation, branching, and growth cone motility), which ultimately determine their overall architecture. We report here that cell calcium and cell‐substrate interactions can play quite specific roles in the regulation of these different components of neuronal outgrowth. CM‐induced neurite outgrowth was inhibited by calcium ionophore A23187. In the absence of CM the calcium channel blocker La3+ (10μM) reduced intracellular calcium levels and induced neurite sprouting and elongation; growth cone motility and branching were greatly reduced in the La3+ ‐induced neurites. Neurons plated into an environment containing La3+ and a fibronectin substrate exhibited all of the components of neuronal outgrowth normally seen in response to CM. Fibronectin alone had little outgrowthpromoting activity. Neurite elongation rates and branching were increased by exposure to La3+ in neurons on either a CM or fibronectin substrate. The neurons growing on CM or fibronectin whose outgrowth was accelerated by La3+ elaborated neuritic arbors that differed from those of neurons grown in resposne to CM; differences were seen in neurite length, area of outgrowth, branching frequency, and varicosity numbers. Taken together, these results indicate that (1) calcium and the growth substrate can exert specific effects on neurite sprouting, elongation, growth cone motility, and branching; (2) appropriate levels of activation of these two sytems can elicit neurite outgrowth that closely resembles that induced by endogenous growth factors; (3) both the differential expression of the separate components of outgrowth and the kinetics of outgrowth determine a neuron' morphology.
Extraction of major acidic Ca2+ dependent phosphoproteins from synaptic membranes
Authors: Perrone‐Bizzozero N. I. , Weiner D. , Hauser G. , Benowitz L. I.
Published: January 2005

The association of several phosphoproteins with the synaptosomal plasma membrane (SPM) was investigated by phosphorylating SPM fractions from neonatal rat brain in the presence of Ca2+ and then exposing these to a variety of agents. Extraction of the major acidic phosphoproteins, GAP‐43, pp40, and pp80, was assessed by two‐dimensional gel electrophoresis and fluorography. All three proteins were best extracted from the membrane by high pH and by guanidine hydrochloride. GAP‐43 was not extracted in the presence of either low‐or high‐ionic‐strength buffers, reducing agents, or chelating agents; pp80 and pp40, however, showed a significant extraction even under low‐ionic‐strength conditions. Partition experiments with Triton X‐114 revealed an amphiphilic behavior for GAP‐43 and a strong affinity for hydrophobic environments for pp80 and pp40. None of the phosphoproteins was released from the membrane by the use of a phosphatidylinositol‐specific phospholipase C. The extraction properties of GAP‐43, pp80, and pp40 are similar to those of known extrinsic membrane proteins and therefore suggest that these phosphoproteins are peripheral rather than integral to the membrane compartment.
Calcium‐activated neutral proteinase in rat brain myelin and subcellular fractions
Authors: Chakrabarti A. K. , Yoshida Y. , Powers J. M. , Singh I. , Hogan E. L. , Banik N. L.
Published: January 2005

The activity of calcium‐activated neutral proteinase (mMCANP) was determined in subcellular fractions of rat brain. The CANP activity in whole homogenate and its membrane fractions including myelin was increased tenfold following treatment with Triton X‐100. The majority of the activity (60%) was in the primary particulate fractions P1 (nuclear), P2 (mitochondrial), and P3 (microsomal). Following subfractionation of each particulate fraction, most of the activity (50%) was found in the myelin‐enriched fractions (P1A, P2A, and P3A) and separated at the interface of 0.32–0.85 M sucrose. Only 20–30% of the total homogenate activity was in cytosol. The enrichment in the myelin fractions resembled that for 2′, 3′‐cyclic nucleotide 3′‐phosphohydrolase (CNPase) activity.

Immunoblotting revealed that the CANP was mainly in myelin and cytosol. In addition to the presence of 72–76 Kd and 80 Kd bands, there were faint highmolecular‐weight CANP bands ranging from 110–150 Kd and lower‐molecular‐weight forms in the region of 30–50 Kd in both purified myelin and cytosol. These studies suggested that CANP is present in myelin and cytosol and that it exists in the brain in membranebound and soluble forms.
Rat β‐nerve growth factor sequence and site of synthesis in the adult hippocampus
Authors: Whittemore S. R. , Friedman P. L. , Larhammar D. , Persson H. , Gonzalez‐Carvajal M. , Holets V. R.
Published: January 2005

A rat b̃‐nerve growth factor (NGF) genomic sequence encoding the entire 3′ exon of preproNGF was cloned, and its nucleotide sequence was determined. Rat NGF shows very high homology with other known NGFs in both the prepropeptide and the 3′ untranslated regions. The presumptive signal sequence, the cysteine residues important for tertiary structure, possible glycosylation sites, and dibasic amino acids required for proteolytic cleavage to mature NGF are conserved across species. Comparison of the hydrophobicity plots and amino acid sequences revealed an evolutionary divergent domain on the external surface of NGF, which may account for the poor immunologic crossreactivities of the various NGFs. In situ hydridization with a rat‐specific oligodeoxynucleotide indicated high levels of NGF mRNA synthesis in both hippocampal granule and pyramidal cell layers. These results are consistent with one role for NGF in the CNS as a neuronally released, retrogradely transported neurotrophic factor for basal forebrain cholinergic neurons.
GM1 ganglioside protects nucleus basalis from excitotoxin damage: Reduced cortical cholinergic losses and animal mortality
Authors: Mahadik S. P. , Vilim F. , Korenovsky A. , Karpiak S. E.
Published: January 2005

Ten days after bilateral injections of ibotenic acid into the nucleus basalis, rats injected daily (i.m.) with ganglioside GM1 were protected from anterograde degeneration of cholinergic projections to the frontolateral cortex. This protection was reflected by reduced losses (associated with ibotenic acid lesions) of cortical acetylcholinesterase, choline acetyltransferase, and lowered animal mortality.
Volume regulation in astrocytes: A role for taurine as an osmoeffector
Authors: Morales H. Pasantes , Schousboe A.
Published: January 2005

Astrocytes in culture regulate their volume under anisosmotic conditions by as yet unclear mechanisms. In a number of other cells this process involves a loss of intracellular osmotically active solutes, including taurine. The possibility that taurine participates as an osmoeffector in astrocytes was examined in cultured astrocytes exposed to hyposmolar conditions.

Astrocytes responded to decreases in osmolarity by rapid swelling followed by a volume regulatory phase. Hyposmotic conditions induced a dramatic increase of 3H‐taurine efflux, with a time course corresponding to the cell volume regulatory phase. Decreasing osmolarity from 310 to 254, 198 or 150 m osmoles resulted in the release of 8.2%, 17%, and 54%, respectively, of 3H‐taurine previously accumulated by astrocytes. Endogenous taurine concentration decreased 64%. The efflux of 3‐H GABA, 3H‐glycine, or 3H‐D‐aspartate was much less affected under similar conditions. These results suggest a role for taurine as an osmoeffector in astrocytes.
Ganglioside metabolism in the hippocampus after septal lesion in rats
Authors: Domańska‐Janik K.
Published: January 2005

The changes in ganglioside compositon and metabolism of deafferentated rat hippocampus were estimated after septal lesion. A significant decrease in total ganglioside concentration was found 7 days after the lesion. The reduced level of total gangliosides persisted at 17 and 25 days. Relative increase in the proportion of GD1b and Gx (O‐acetylated GT1b) and decrease in GM1 were found in hippocampus only at 25 days post‐lesion.

The incorporation of 3H‐N‐acetylamannoseamine into gangliosides was examined in rats whose hippocampi were lesioned 25 days prior to radioprecursor injection. Differences in the labeling pattern of total and individual gangliosides were found. Increases in the label in GM1, GD3, and GD1a and decreases in GT1b and GQ1b were found 10 hr after isotope injection. However, decreases in the specific activity of all gangliosides except GT1b and GQ1b were observed 24 hr after 3H‐N‐acetylomannosamine injection, suggesting the activated turnover of gangliosides in postlesioned hippocampus. The significance of these changes has been discussed in terms of cellular damage and repair in the hippocampal tissue.
Secretion of newly synthesized proteins into the extracellular fluid of the rabbit hippocampus
Authors: Nyström B. , Karlsson J.‐O. , Hamberger A.
Published: January 2005

Extracellular proteins were collected continuously from the hippocampus of the rabbit by slow perfusion of a protein‐permeable thin tubing. After pulse labelling with radioactive amino acids via the tubing a rapid appearance of labelled proteins was seen in the extracellular fluid. The half‐life of the proteins in the extracellular space varied between 15 min and 2 h. Protein fractions were separated with gel electrophoresis or HPLC. The extracellular fraction differed distinctly from intracellular proteins with respect to the labelling pattern.
Changes in protein kinase C activities are correlated with the metaplastic transformation of schwann cell precursors of avian embryos into melanocytes
Authors: Hess L. , Chamberlin T. , Ciment Gary
Published: January 2005

In previous work, we found that the phorbol ester drug 12‐0‐tetradecanoyl phorbol acetate (TPA) reverses the developmental restriction of melanogenesis that occurs early in neural crest development, causing Schwann cell precursors to undergo a metaplastic transformation into melanocytes. In this study, we examine whether these effects of TPA may be mediated by changes in endogenous levels of protein kinase C (PKC) activities. We report that low levels of PKC activity are correlated with this adventitious pigmentation in the crest‐derived cells of dorsal root ganglia both during normal development and following TPA treatment in culture. These results suggest that regulation of endogenous levels of PKC plays a role in developmental decisions that neural crest cells make during early embryogenesis.
Distribution and function of tenascin during cranial neural crest development in the chick
Authors: Bronner‐Fraser M.
Published: January 2005

Tenascin is a glycoprotein associated with the extra‐cellular matrix and the surface of some cell types. Here, the distribution and possible function of tenascin have been examined along the pathways followed by cranial neural crest cells. During early stages of neural crest migration, tenascin was observed in a dense matrix surrounding premigratory cranial neural crest cells. Along the neural tube, tenascin immunoreactivity was observed in a dorsoventral gradient and was also noted under the ectoderm and around the notochord. During advanced neural crest migration, tenascin immunoreactivity colocalized with and appeared to be on the surface of migrating neural crest cells. At later stages, tenascin was present around the otic vesicles, retina, lens, and in an interstitial matrix in the region of the branchial arches. At the level of the occipital somites, tenascin immunoreactivity was observed around the neural tube, notochord, dermamyotome, and on the basal surface of the ectoderm. Tenascin was also observed in an interstitial matrix within the sclerotome. At early stages of vagal neural crest migration, immunoreactivity was uniform within the sclerotome, whereas at later stages tenascin colocalized with vagal neural crest cells within the rostral half of each sclerotome. The possible function of tenascin was tested by injecting antitenascin antibodies lateral to the mesencephalic neural tube. Two predominant defects were noted in injected embryos: (1) ectopic aggregates of cranial neural crest cells external to the neural tube and sometimes located on the apical side of the ectoderm; and (2) open and deformed neural tubes. Both the distribution and results of the perturbation experiment suggest that tenascin is required for proper cranial neural crest migration.
Regional expression of myelin protein genes in the developing mouse brain: In situ hybridization studies
Authors: Verity A. N. , Campagnoni A. T.
Published: January 2005

Expression of mRNAs for the two major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), was examined in a number of regions of the developing mouse brain using in situ hybridization. In general, MBP and PLP mRNAs were observed to be coexpressed during ontogeny, prior to the histological appearance of myelin. Expression of both mRNAs was detected as early as 6 hours postpartum in the medulla oblongata and, with development, expression of these mRNAs progressed in a caudal to rostral direction. Peak expression occurred at approximately postnatal day 20 in most regions examined, regardless of time of onset of expression. As myelination proceeded, two different labeling patterns were observed with the PLP and MBP 35S‐labeled cDNA probes. In the earliest stages of myelinogenesis MBP mRNA labeling was restricted to oligodendrocyte cell bodies, but shortly after the gene began to be expressed the labeling became more diffuse. In contrast, PLP mRNA labeling remained over or surrounding oligodendrocyte cell bodies at all stages of myelinogenesis. These two distinctly different patterns of labeling are consistent with alternative intracellular trafficking of MBP and PLP mRNAs, in which PLP mRNAs remain associated with ribosomes within the cell soma and MBP mRNAs move from the cell soma to the oligodendrocyte processes at a specific stage early in myelinogenesis. However, there appeared to be a clear time lag between the onset of MBP mRNA expression and the movement of ribosomes carrying MBP mRNAs into the oligodendrocyte processes. Additionally, the in situ hybridization studies revealed a population of unidentified cells residing in cortical molecular layers that express PLP mRNA in the absence of MBP mRNA.
Stimulation of oligodendrocyte differentiation in culture by growth in the presence of a monoclonal antibody to sulfated glycolipid
Authors: Bansal R. , Gard A. L. , Pfeiffer S. E.
Published: January 2005

Perturbation of myelinogenesis by monoclonal antibodies against galactolipids is being used to study the role of these lipids in oligodendrocyte differentiation. We report here a marked stimulatory effect on oligodendrocyte differentiation when mixed primary cultures initiated from 19–21 day fetal rat telencephala are grown in the presence of a monoclonal antibody against sulfogalactolipids. When such cultures were grown in the presence of the IgM antibody 04 [Sommer and Schachner, Dev Biol 83:311–327 1981], the oligodendrocytes formed aggregates connected by fasciculated processes. Immunofluorescence microscopy and biochemical analyses of treated cultures demonstrated 2–3 fold increases in the fraction of 04‐positive cells expressing myelin basic protein, and in the levels of myelin basic protein RNA, myelin basic protein, 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase activity, and 35SO4 incorporation into sulfatide. Greater than 90% of the cells positive for myelin basic protein in treated cultures were in aggregates. The specific activities of oligodendrocyte markers were unaffected in control cultures grown with nonspecific myeloma IgM. Since there was no increase in the total number of 04‐positive cells in treated cultures, the increases in the specific activities of the myelin protein markers appears to be due to an increase in the fraction of cells expressing these markers. Time course studies demonstrated that both the rate and extent of oligodendrocyte differentiation were enhanced in treated cultures. These data are discussed with regard to possible mechanisms of the stimulation, considering not only potential direct effects of the antibody on the cell physiology, but also possible indirect effects due to antibody‐induced aggregation.
Interference reflection microscopic study of dorsal root growth cones on different substrates: Assessment of growth cone–substrate contacts
Authors: Gundersen R. W.
Published: January 2005

The contact between dorsal root growth cones and substrates of type IV collagen, fibronectin, poly‐L‐lysine, and laminin were studied using interference reflection microscopy (IRM) coupled with detaching growth cones from these substrates and observing substrate‐associated membrane (SAM). IRM images of growth cones on collagen and fibronectin indicate substantial dark areas of close association between the substrate and basal region of the growth cone. The thin membranous veils and lamellapodia appear bright, indicating a greatly decreased association with the substrate. Filopodia can appear either entirely dark or light but usually are dark interspersed with brighter areas indicating varying degrees of contact. IRM images of growth cones on polylysine and laminin suggest a decreased association between the basal region of the growth cone and the substrate. The appearance of veils and lamellapodia was similar to that observed on collagen and fibronectin. Observations of SAM indicate an increased degree of contact between growth cones and substrates of collagen and fibronectin compared to laminin and polylysine. Collectively these data indicate that simple increases in adhesion are not adequate to explain substrate preference and enhancement of neurite elongation. Overall decreases in adhesion may accomplish, the same phenomena, suggesting that it is perhaps more important to consider the regional distribution of adhesive contacts in relation to growth cone movement.
Intracellular messengers in the generation and degeneration of hippocampal neuroarchitecture
Authors: Mattson M. P. , Guthrie P. B. , Kater S. B.
Published: January 2005

The actions and interactions of the neurotransmitter glutamate and the intracellular messengers calcium, cyclic AMP, and protein kinase C (PKC) in the regulation of neurite outgrowth and cell survival were examined in hippocampal pyramidal‐like neurons in isolated cell culture. Low, subtoxic levels of glutamate (10–100 μM) caused the regression of dendrites but not axons; millimolar levels caused cell death. Calcium ionophore A23187 (50–100 nM) and the PKC activator phorbol‐12‐myristate‐13‐acetate (PMA; 10–50 nM) caused the regression of both axons and dendrites, whereas the adenylate cyclase activator forskolin enhanced outgrowth rates in both axons and dendrites. The effects of glutamate, A23187, PMA, and forskolin on outgrowth were mediated locally at the growth cones; dendrites were more sensitive than axons to each of these agents. High levels of A23187 (1 μM) or PMA (100 nM) significantly reduced cell survival. Co2+ and trifluoperazine each significantly reduced glutamate‐induced dendritic regression and neurotoxicity suggesting that calcium influx and/or PKC activation mediated glutamate's actions. Fura‐2 measurements showed that glutamate caused a rapid rise in intracellular calcium levels; this rise was prevented by Co2+. PMA and forskolin did not alter intracellular calcium levels, nor did these agents affect glutamate‐induced calcium rises. Taken together, the results indicate that parallel intracellular messenger pathways that influence neurite outgrowth and cell survival are operative in hippocampal neurons; these messengers may play roles in the formation and modification of neuronal circuitry.
Secreted peptides as regulators of neuron–glia and glia–glia interactions in the developing nervous system
Authors: Giulian D. , Vaca K. , Johnson B.
Published: January 2005

Secreted peptides of the nervous system help to regulate neuron–glia and glia–glia interactions during development. These regulatory factors, referred to as glia‐promoting factors (GPFs), act on specific classes of glia and include oligodendroglia‐stimulating peptides, interleukin‐1 (IL‐1), colony‐stimulating factors (CSF), and fibroblast growth factor (FGF). The maturity of secretory and target cells determines, in part, the ability of a factor to influence glial proliferation, activation, or differentiation. During neural development, GPFs help to control such fundamentally important events as cell movement, neurite outgrowth, and myelination.
Basic fibroblast growth factor and nerve growth factor administered in gel foam rescue medial septal neurons after fimbria fornix transection
Authors: Otto D. , Frotscher M. , Unsicker K.
Published: January 2005

Basic fibroblast growth factor (bFGF) recently has been established as a survival‐ and transmitter‐promoting neurotrophic agent for embryonic neurons in vitro. Its local application to lesioned adult optic and sciatic nerves has been shown to rescue axotomized retinal and sensory neurons that otherwise die. Following transection of the fimbria fornix pathway connecting the medial septum (MS) to the hippocampus, MS neurons undergo severe cell death, which can be prevented partially by infusion of nerve growth factor (NGF). In the same lesion paradigm, we find that 87% of these neurons visualized by cresyl‐violet staining have disappeared by 4 weeks after unilateral fimbria fornix transection in adult rats. Implantation of gel foam soaked with 8 μg bFGF reduced neuron death to 68%. A similar rescue effect was seen with 0.3 μg NGF. NGF administered at 20 μg reduced cell losses to 54%. Thus, bFGF rescued 22% and NGF at 20 μg 38% of the neurons that otherwise would have died. Choline acetyltransferase immunocytochemistry revealed dramatic losses of cholinergic neurons on the lesioned, compared with the unlesioned, side. Cholinergic neuron death was clearly reduced by the bFGF and NGF treatments. Basic FGF, in contrast to NGF, did not prevent a reduction in size of surviving neuronal cell bodies. Considered in the context of FGF being present in brain and hippocampal neurons, our results suggest a possible role for FGF as a neurotrophic factor for CNS neurons in vivo.
Interaction of nerve growth factor with murine α‐macroglobulin
Authors: Koo P. H. , Stach R. W.
Published: January 2005

The murine nerve growth factor, when injected i.v. or, combined in vitro with plasma, was found largely associated with the mouse α‐macroglobulin (a homologue of human α2‐macroglobulin). The nerve growth factor α‐macroglobulin complex produced is sufficiently stable to resist separation by gel filtration in 1.0 M sodium chloride, polyacrylamide gel electrophoresis, and immunoprecipitation by antibodies against α‐macroglobulin. As determined by equilibrium binding studies and computer generated Scatchard analysis, α‐macroglobulin apparently possesses two types of binding sites with the apparent dissociation constants of 1.2 × 10−6 and 2.9 × 10−9 M, respectively, saturable by 3.7 and 0.03 moles of nerve growth factor. Hence, about one mole of nerve growth factor is bound to each of the four subunits of α‐macroglobulin. Nerve growth factor can be readily dissociated from α‐macroglobulin in sodium dodecyl sulfate gel electrophoresis in the absence of a reductant. Procedures that affect the proteinase‐binding or methylamine‐activities of α‐macroglobulin do not affect the binding of nerve growth factor, and the binding is unaffected by the presence of zinc ions or EDTA. Hence, nerve growth factor is noncovalently associated with α‐macroglobulin at a site separate from that of the proteinase‐, methylamine‐, and zinc‐binding sites of α‐macroglobulin. Mouse α‐macroglobulin can protect the nerve growth factor from inactivation by trypsin. Even in the presence of trypsin, α‐macroglobulin‐nerve growth factor complexes still can stimulate the neurite outgrowth by dorsal root ganglia of 9‐day‐old chicken embryos. Since α‐macroglobulin can specifically and noncovalently carry nerve growth factor, one important role of this α‐macroglobulin in the circulation and extracellular spaces may be to protect the nerve growth factor from proteinase inactivation.
Aged adrenal medullary tissue survives intraocular grafting, forms nerve fibers and responds to nerve growth factor
Authors: Strömberg I. , Ebendal T.
Published: January 2005

Adrenal medullary tissue from aged (24 months old) and young adult (2 months old) rats was grafted to the anterior chamber of the eye of previously sympathectomized animals. Nerve growth factor (NGF) was administered by weekly bilateral intraocular injections. Five weeks postgrafting, irides were prepared as whole mounts and processed for Falck‐Hillarp histochemistry for visualization of catecholamines. NGF appeared to partially prevent the reduction in volume that both old and young grafts underwent. In the presence of NGF, an extensive, dense fiber network, closely resembling the normal adrenergic innervation, was formed in the host irides by grafts from aged donors. The area of outgrowth from aged transplants without NGF treatment was as large as with NGF treatment but less dense. The reinnervation of irides by NGF‐treated young adult grafts occupied a similar area as that seen with aged grafts, but the pattern of innervation was irregular, particularly close to the transplants. Transplants from young adult donors without NGF treatment generated a sparse, limited network of nerves in the irides. All grafts were tyrosine hydroxylase‐, adrenaline‐, and dopamine‐β‐hydroxylase‐immunoreactive in about the same proportion of cells, but the grafts from the young donors were smaller in size. We concluded that the ability of chromaffin cells to transform toward a neuronal phenotype, produce nerve fibers, and respond to exogenous NGF is maintained in aged adrenals.
Spontaneous and beta‐adrenergic receptor‐mediated taurine release from astroglial cells do not require extracellular calcium
Authors: Martin D. L. , Madelian V. , Shain W.
Published: January 2005

Astroglial cells release taurine when stimulated by beta‐adrenergic agonists and other neuroactive agents. The Ca2+ ‐dependency of taurine release by an LRM55 astroglial cell line was investigated by removing Ca2+ from the perfusion medium and by using three inorganic and three organic Ca2+ ‐channel blockers (Mn2+, Co2+, Cd2+, verapamil, nifedipine, and diltiazem). Spontaneous release and release stimulated by the beta‐adrenergic agonist isoproterenol were not inhibited when cells were perfused with medium containing no added Ca2+ and 10 μM EGTA. Isoproterenol‐stimulated taurine release was not blocked when extracellular Ca2+ was completely replaced by Mn2+, Co2+, or Cd2+, nor was it blocked by verapamil, nifedipine, or diltiazem. In fact isoproterenol‐stimulated taurine release was increased by 50 μM diltiazem and when Ca2+ was replaced by Co2+. The rate of spontaneous release increased slowly and continually when Co2+ was substituted for Ca2+ or but was almost unaffected by substitution of Mn2+ or Cd2+. Application of diltiazem increased spontaneous release significantly, while verapamil and nifedipine appeared to cause small increases. These results indicate that entry of Ca2+ from the extracellular medium is not required for either receptor‐mediated or spontaneous taurine release from astroglial cells. Some other changes in the medium did strongly affect release. Both spontaneous and isoproterenol‐stimulated release were inhibited by elevated osmotic pressure, and spontaneous release was greatly increased when Ca2+ was completely removed without substituting another divalent cation. Spontaneous release increased when antagonistie metal ions were replaced with Ca2+ and when organic channel blockers were removed.
Sensory ganglia from tadpoles but not adult bullfrogs synthesize heat shock‐like proteins in vitro at non‐heat shock temperature
Authors: Hammerschlag R. , Maines S. , Ando M.
Published: January 2005

Lumbar dorsal root ganglia (DRG) from premetamorphic bullfrog tadpoles showed a selectively enhanced accumulation of two radiolabeled proteins when incubated in vitro at 22°C for longer than 1 hr. During the first hour in vitro or after in vivo labeling, these polypeptides–of 25 and 68 kD molecular mass and slightly acidic isoelectric point–were detected in the DRG only at relatively low levels. Accumulation of the 25 kD species increased in lumbar DRG from tadpoles of all metamorphic stages but waned at stage XXV (completion of metamorphosis) and was absent from DRG of adult bullfrogs. In contrast, increased levels of the 68 kD species were detected only at premetamorphic stages. Tail DRG from premetamorphic tadpoles did not increase their synthesis of either polypeptide in vitro, which is consistent with the above findings because sensory neurons in tail DRG, but not in lumbar DRG, are mature at this developmental stage. Although the molecular sizes of the two polypeptides led us initially to consider them as heat shock‐like proteins, only the 68 kD species was further increased (by approximately 100‐fold) when premetamorphic lumbar DRG were incubated at 37°C. The 25 kD species, which was the predominant protein induced at 22°C, showed no further increase in response to hyperthermia. The enhanced in vitro synthesis of these polypeptides at control temperature (22°C) is an example of cellular stress responses that may occur not as a consequence of elevated temperature or exogenous chemical agents, but as a result of transplantation‐associated trauma. This phenomenon also represents an example of a cellular stress response that is “outgrown” during development.
Muscarinic receptor subclasses in retinal cultures: Effect of corticosterone
Authors: Torelli S. , Sogos V. , Gremo F.
Published: January 2005

We have previously shown that exogenously administered corticosterone affects muscarinic receptor binding in the chick embryo retina. Analysis with the selective antagonist pirenzepine has shown that both muscarinic receptor subclasses M1 and M2 are present in treated retinas. On the contrary, only M2 is detectable in controls. Moreover, receptor affinity for agonists is modified by hormone treatment. Since these studies did not show whether or not the hormone directly influences retinal cells, a similar study was performed on retinal tissue cultures. Cells were treated at day 5 in vitro for 24 hr with 1.10−8 M corticosterone. Schatchard analysis of results obtained with 3H‐quinuclidinyl benzilate (3H‐QNB) binding showed no difference between treated and control cultures. However, displacement experiments demonstrated that both M1 and M2 were present in treated cultures, whereas controls had only M2. Also, receptor affinity for the agonist carbachol was modified, as already observed with in vivo studies. In addition, a new phenomenon was observed: treated cultures had a significantly higher number of cells. The possibility that the hormone can prevent cell death or stimulate cell mitosis is considered.
Carrier‐mediated Cl− transport in cultured mouse oligodendrocytes
Authors: Hoppe D. , Kettenmann H.
Published: January 2005

We studied the steady state and the regulation of intracellular Cl− activity (aCl−i) and the mechanisms of KCl uptake in cultured oligodendrocytes from mouse spinal cord using Cl−‐selective microelectrodes. The majority of oligodendrocytes actively accumulated Cl− above passive distribution (2–3 mM), few cells showed a passive Cl− distribution. To identify the carriers mediating Cl− uptake, oligodendrocytes were maintained in a solution with low extracellular Cl− concentration ([Cl−]0) which resulted in a rapid decrease in aCl−i. The recovery of aCl−i above its passive distribution in normal [Cl−]0 was blocked in the absence of Na+ or in the presence of furosemide and of bumetanide, which has been reported to inhibit Na+/K+/Cl− cotransport. We therefore conclude that Cl− uptake is primarily due to the activity of a Na+/K+/Cl− transport system. Cl− uptake above passive distribution was not affected in HCO3−‐free solution or in the presence of SITS and DIDS, indicating that Cl−/HCO3− exchange is not involved in Cl− uptake by oligodendrocytes. Elevation of [K+]0 induced an increase in aCl−i and, as shown earlier, intracellular K+ activity. This K+‐induced Cl− uptake was not blocked by bumetanide, furosemide, SITS, or DIDS, suggesting that under conditions of raised [K+]0 the combined uptake of K+ and Cl− is not mediated by a carrier, but can be explained by the entry through channels driven by Donnan forces.
Suppression of MYC by high expression of NMYC in human neuroblastoma cells
Authors: Breit S. , Schwab M.
Published: January 2005

Members of the MYC gene family, including MYC, NMYC, and LYMC, have been found amplified and expressed at high level in various human cancers. We have analysed the expression of two members of the MYC gene family, NMYC and MYC, in human and murine neuroblastoma cells. Whenever NMYC and MYC are co‐expressed, MYC expression predominates. Cells carrying high expression of NMYC as result of amplification lack MYC expression. The same is true for neuroblastoma cells in which expression of a single‐copy NMYC is upregulated or into which a vector forcing high expression of an exogenous NMYC had been introduced by transfection. Our studies indicate a regulatory interaction between MYC and NMYC in neuroblastoma cells.