bluuebunny/crossref_metadata_2025 huggingface dataset:
Sensory neurite growth cone guidance by substrate adsorbed nerve growth factor
Authors: Gundersen R.W.
Published: January 2005

The response of growth cones from embryonic chick dorsal root ganglia to a patterned substrate of adsorbed nerve growth factor (NGF) was studied. The patterned substrate presented growth cones with an adsorbed NGF pattern and NGF‐free substrate. NGF‐responsive growth cones from 7 and 9 day ganglia could not proceed onto NGF‐free substrate, reproducing the adsorbed NGF pattern. NGF‐unresponsive growth cones from 17 day ganglia did not display any preference for adsorbed NGF or NGF‐free substrata, which resulted in neurites not reproducing the adsorbed NGF pattern. Neurite outgrowth from NGF responsive 7‐day ganglia onto a patterned NGF substrate, in NGF‐containing medium, was radially symmetrical, exhibiting no growth cone response to the patterned NGF substrate. The lack of NGF‐responsive growth cone extension onto NGF‐free substrate indicates that NGF is a requirement for neurite elongation. If NGF is withdrawn from growth cones by microperfusion, neurite elongation ceases. Thus, an adsorbed pattern of NGF may be duplicated because growth cones are not able to extend onto NGF‐free substrate, since NGF is a requirement for neurite elongation. These results indicate that substrate adsorbed NGF can support neurite formation and elongation as well as guide the direction of neurite elongation.
Electrical and ionic controls of tissue cell locomotion in DC electric fields
Authors: Cooper M.S. , Schliwa M.
Published: January 2005

The motility of fish epidermal cells (keratocytes) was examined in the presence and absence of DC electric fields. In fields of 0.5–15 V/cm, single epidermal cells, cell clusters, and cell sheets migrate toward the cathode. Cell clusters and sheets break apart into single migratory cells in the upper range of these field strengths. During locomotion, keratocytes extend broad lamellipodia, which contain a pervasive actomyosin network. The lamellipodial extension and locomotion of keratocytes are reversiby inhibited by a variety of calcium channel antagonists, whereas thir motility is unaffected by hyperpolarizing and depolarizing (low and high K+) media. Microtubule disassembly has no effect on cell morphology, motility or the ability of the cells to be guided by a DC electric field. Using these results, the role that membrane‐regulated Ca2+ influx may play in generating cytoskeletal and protrusive activity in keratocytes and other cells is discussed in some detail. Mechanisms by which an external electric field may bias transmembrane ion fluxes and thereby control cell locomotion are also examined.
Binding and internalization of lysosomal enzymes by primary cultures of rat glia
Authors: Hill D. F. , Bullock P. N. , Chiappelli F. , Rome L. H.
Published: January 2005

Highly purified cultures of rat astrocytes and oligodendrocytes were examined for their ability to bind and internalize lysosomal enzymes. Astrocytes displayed a saturable uptake to β‐glucosidase and β‐galactosidase. The uptake was specifically inhibited by mannose‐6‐phosphate but not by several other sugars or sugar phosphates, indicating that the process was mediated by mannose‐6‐phosphate receptors. When cells were allowed to take up 125I‐β‐glucosidase for 1 hr at 37°C and subcellular organelles were isolated, the enzyme was shown to comigrate with a lysosomal organelle marker enzyme, suggesting that the enzyme was targeted to lysosomal. Astrocyte receptors were probed directly by binding of 125I labeled β‐glucosidase to astrocyte membranes at 4 °C. Binding was saturable and competitively inhibited by mannose‐6‐phosphate. In contrase to the astrocytes, cultured oligodendrocytes showed no specific binding or uptake of the lysosomal enzymes. Immunocytochemical staining of mixed glial cultures supported the biochemical data; only the astrocytes stained positive with anti‐mannose‐6‐phosphate receptor antibodies.
Response to gallamine: An indicator of diminished neuromuscular function in experimental autoimmune myasthenia gravis
Authors: Hinman C. L. , Dambach G. E. , Hudson R. A. , Rauch H. C.
Published: January 2005

A test for diminished neuromuscular function in animals with experimental autoimmune myasthenia gravis is described. Within minutes following an injection of gallamine triethiodide, mice exhibit a dramatic yet transient response which is dose‐dependent. Mice previously inoculated with acetylcholine receptor are approximately twice as sensitive to gallamine as normal mice. Positive results have been found in over 80% of receptor‐inoculated BALB/c mice and in 94% of C57B1/6 mice.
Alteration of seizures resulting from pinealectomy in the rat after neonatal treatment with 6‐hydroxydopamine
Authors: Stockmeier C. A. , Larsen B. R. , Blask D. E.
Published: January 2005

Convulsions from combined pinealectomy and parathyroidectomy were examined in rats treated as neonates with 6‐hydroxydopamine (6‐OHDA). Injection of 6‐OHDA caused a significant reduction in the average latency to onset of clonictonic convulsions. Moreover, of the 6‐OHDA‐treated rats which convulsed, nearly half of them died during a seizure. In sham‐pinealectornized rats, neonatal treatment with 6‐OHDA reduced norepinephrine (NE) and increased dopamine levels in the forebrain while brainstem levels of NE were increased. Pretreatment with desmethylimipramine reversed the effects of 6‐OHDA on convulsions and catecholamine levels. The results indicate that catecholamine lesions with 6‐OHDA exacerbate convulsions from pinealectomy.
Oligodendroglial structures and distribution shown by carbonic anhydrase immunostaining in the spinal cords of developing normal and shiverer mice
Authors: Cammer W. , Sacchi R. , Kahn S. , Sapirstein V.
Published: January 2005

The spinal cords of young and adult normal and dysmyelinating mutant (shiverer) mice were immunostained with anticarbonic anhydrase to investigate (1) the distribution of oligodendroglial populations into the gray‐ and white‐matter regions in the developing normal and mutant animals; (2) the morphology of oligodendrocytes and their processes at the light microscopic level in gray matter and white matter; and (3) the apparent gliosis in the gray matter, as well as the white matter, of the mutants. Immunocytochemistry and enzyme assays revealed consistent increases in carbonic antigenicity and specific activity in controls and mutants between the ages of ˜ 15 days and ˜ 60 days. As shown previously in adult animals, oligodendroglia in larger than normal proportions were situated at the periphery of the “white‐matter” columns, as compared to gray matter, in the shiverers, with, however, significant numbers of oligodendroglia also occurring in gray matter. In normal white matter, at all ages, the oligodendroglia were heterogeneous with respect to shapes, configuration of processes, and intensity of carbonic anhydrase immunostaining. In the shiverer “white matter” the oligodendrocytes were smaller than normal, and their shapes and arrangement were relatively irregular. In the normal gray matter short oligodendroglial processes appeared to be associated with neuronal perikarya, and those processes were more pronounced at ˜ 90 days than at ˜ 20 days of age. Background staining in normal gray matter suggested that oligodendroglial processes were, in addition, tightly wound around many axons. In shiverer gray matter the oligodendrocytes were smaller, and their processes appeared to be wrapped more loosely around smaller numbers of conspicuous axons and to be associated less frequently with neuronal perikarya. This finding suggests that the deficiency in the myelin basic protein in the mutant may affect interaction betwen oligodendrocytes and neurons in the gray matter as well as in the white matter. The astrocytic “marker,” glial fibrillary acidic protein, was detected in gray and white matter of shiverers as young as 16 days, and the differences from carbonic anhydrase localization supported the conclusion that the processes enwrapping axons in the shiverer mouse CNS are derived from oligodendrocytes, not astrocytes.
Laminin supports short‐term survival of rat septal neurons in low‐density, serum‐free cultures
Authors: Pixley S. K. R. , Cotman C. W.
Published: January 2005

Septal neurons from embryonic rats do not survive for 24 hr when dissociated and cultured at low density in a serum‐free medium. Lamunin at 5–40 μg/ml acts as a survival‐promoting agent in the presence of 1 mM pyruvate. Laminin is effective in promoting survival only if it is added before or during cell plating; it does not support survival if added after cell attachment. Variations in laminin concentration do not effect the total number of cells attaching to the culture plate. The percentage of septal cells with neurites is increased in a dose‐dependent manner by laminin as early as 45 min after cell plating, suggesting an effect of laminin on the rate of neurite initiation. Neurite extension at 24 hr is also dependent on laminin concentration. This study suggests that some interaction between cells and the substrate, independent of cell attachment, has a profound effect on cell physiology, increasing both cell survival and the rate of neurite extension. This study also defines the survival requirements of septal neurons in a chemically defined environment in a low‐density situation where cellular interactions are at a minimum.
Characterization of antibodies against major fish CNS myelin protein: Immunoblot analysis and immunohistochemical localization of 36K and IP2 proteins in trout nerve tissue
Authors: Jeserich G. , Waehneldt T. V.
Published: January 2005

Antisera against the trout CNS myelin proteins 36K and IP2 were prepared in rabbits and characterized by immunoblot analysis and immunohistochemistry. The anti‐36K antiserum exclusively stained its corresponding antigen from trout CNS myelin but failed to recognize any myelin polypeptide from either trout PNS or mammalian CNS and PNS. Antibodies against the IP2 glycoprotein specifically cross‐reacted with related intermediate proteins (IP) of both CNS and PNS myelin from trout but only faintly labeled the P0 protein of mouse peripheral nerve. Immunohistochemical localization of both antigens in the CNS of young trout was confined to the myelin sheath, except that anti‐36K antiserum also stained oligodendrocytes. Nodes of Ranvier, neuronal cell bodies, and dendrites, as well as other glial elements, were negative. Specificity of the immunofluorescent reaction was established by crossed immunoadsorption experiments. Whereas on adjacent sections through trout brain both antigens exhibited a nearly identical distribution pattern, immunostaining in peripheral nerves was seen only with anti‐IP2 antibodies.
Gangliosides inhibit phospholipid‐sensitive Ca2+‐dependent kinase phosphorylation of rat myelin basic proteins
Authors: Kim J. Y. H. , Goldenring J. R. , DeLorenzo R. J. , Yu R. K.
Published: January 2005

Gangliosides inhibit the phosphorylation of both small and large rat myelin basic proteins (SMBP, LMBP) by an endogenous phospholipid‐sensitive Ca2+‐dependent protein kinase (C‐Kinase). Using a rat brain myelin preparation in an in vitro phosphorylation assay system, we determined the inhibition constants (IC50's) of the gangliosides GM1, GD1a, GD1b, and GT1b to be approximately 160 μM, 65 μM, 65 μM, and 40 μM, respectively. Asialoganglioside GA1, ceramide, and Nacetylneuraminic acid (NANA, sialic acid) failed to produce similar inhibition, suggesting that both the lipid and the sialic acid moieties are necessary, but neither alone is sufficient to produce inhibition. The results indicate that gangliosides may regulate protein kinase C activities in the nervous system.
Primary retinal and cortical glial cell cultures: Effects of medium and serum on attachment and growth
Authors: Trachtenberg M. C. , Hokanson K. M.
Published: January 2005

Glial cells of the retina are anatomically distinctive and are thought to contribute importantly to retinal electrophysiology. However, no adequate preparation exists for studing them in isolation, in vitro. This report provides guidelines for primary retinal glial cultures (RET) and compares basal tissue culture features with those for neocortical glia (CX) and the well‐studied rat glial line, C6.

Cell attachment and growth of RET, CX, and C6 are unique. These differences are explored by the use of specific media and sera. RET attachment, unlike that for CX or C6, was far more sensitive to medium than serum. RET cells attached least quickly, CX most quickly; 4 hr after plating 20% of RET remained unattached. RET growth was poor and relatively insensitive to medium.

In contrast, growth of CX or C6 was medium dependent. Serum had substantial effects on the growth of all three glial lines. Pig, goat, horse, and dog sera were particularly effective, often comparing favorably to fetal calf serum. Medium or serum optimal for cell attachment, typically, was not optimal for growth and serum effects were more dramatic than those of medium. By all measures, CX and C6, both derived from brain, were more alike than were the two rabbit primaries, CX and RET. The data reveal substantial differences between presumably similar cells and indicate a need for an empirically based choice of both basal‐salt media and serum to optimize specific aspects of cell development in culture.
Neurite‐promoting activity from fetal skeletal muscle: Partial purification of a high‐molecular‐weight form
Authors: Steele J. G. , Hoffman H.
Published: January 2005

Neurite extension from sensory neuroblasts dissociated from chick embryo dorsal root ganglia can be stimulated by precoating the polylysine culture surface with extracts of skeletal muscle from bovine fetuses. The active factor(s) may be partially purified from cytosolic extracts of muscle by chromatography on Sepharose 6B and affinity chromatography on wheat germ agglutinin or Helix pomatia agglutinin columns. Extract concentrations of 10–50 μg protein per 1 ml were active in promoting neurite extention when the neurons were cultured without serum or nerve growth factor (βNGF). However, levels of 1–10 μ/m1 produced dramatic neurite extension when 10% (v/v) fetal or newborn calf serum or 0.5 ng/ml βNGF was added to the medium. The biological activity was not blocked by antiserum that was raised against purified mouse laminin and that abolished the neurite‐promoting activity of mouse laminin. The activity of the muscle extract was destroyed by trypsin or heparitinase, in contrast to the biological activity of purified mouse laminin, which was not abolished by heparitinase treatment. The activity could be resolved into two broad peaks on a Sepharose 2B column (apparent Mr between 2 × 106 and in 10 × 106 in native form). Treatment with dithiothreitol was necessary to dissociate the factor for electrophoresis in 4.25% polyacrylamide‐SDS gels, revealing three major polypeptide bands at Mr = 160,000, 195,000 and 200,000. This preliminary charcterization indicates that the neurite‐promoting activity from bovine skeletal muscle tissue consists of a high‐molecular‐weight complex, one essential component of which is a heparan sulfate.
An immunochemical analysis of a myelin basic protein serum factor: Cross reactivity with residues 69–71 of the rabbit encephalitogenic sequence 65–74 of myelin basic protein
Authors: Potter N. T. , Hashim G. A. , Day E. D.
Published: January 2005

A partially purified myelin basic protein serum factor (MBP‐SF), cross‐reactive with residues 65–74 (TTHYGSLPQK) of myelin basic protein, has been employed in an immunochemical study to identify the nature of the cross‐reacting determinant, more precisely. To probe the structural requirements of this determinant, Scatchard inhibition analyses and competitive peptide inhibition radioimmunoassays were employed with a series of peptide analogs of the 65‐74 region and with three different reagent antisera: a rabbit anti‐rat myelin antiserum (#My05) and two antisera, one rabbit (#162) and one chicken (#66), raised against synthetic peptide S24 (TTHYGSLPQKG). Scatchard inhibition analyses with MBP‐SF revealed specific inhibition of binding of 125I‐S24 to #162 and #My05, but not to #66. Further delineation of the structural requirements of the cross‐reactive determinant, employing a liquid‐phase radioimmunoassay, revealed a unique reactivity pattern for the chicken anti‐S24 antiserum which, unlike #162 and #My05, did not cross‐react under high‐affinity conditions with synthetic peptide S20 (GSLPQK, representing the C‐terminal half of S24). This, in concert with the Scatchard data, is suggestive of the presence of a cross‐reactive determinant centered around residues 69–71 of MBP.
Acetylcholinesterase of Schistosoma mansoni: Purification and characterization
Authors: Goldlust A. , Arnon R. , Silman I. , Tarrab‐Hazdai R.
Published: January 2005

Larval acetylcholinesterase (acetylcholine acetylhydrolase) EC 3.1.3.7 of the trematode Schistosoma mansoni was characterized and purified by affinity chromatography. The enzyme was solubilized from sonicated cercarial tissue and showed a Km value of 1.83 mM and a Vmax value of 102 U/mg protein. It was characterized as a true AChE since it hydrolyses acetylthiocholine more than seven times faster than butyrylthiocholine, and since it was inhibited by high concentrations of substrate. The enzyme was purified by affinity chromatography on a Sepharose column of the inhibitor [N‐(6‐aminocaproyl‐6‐aminocaproyl)‐maminophenyl] trimethyl ammonium. The purified enzyme eluted from the column by decamethonium bromide migrated as a single band of 500 kD on nondenaturing polyacrylamide gel electrophoresis (PAGE), whether stained for proteins or for enzymatic activity. Analysis by SDS‐PAGE revealed two major polypeptide bands of 76 kD and 30 kD. By labeling the enzyme with 3H‐DFP (di‐isopropyl‐fluorophosphate), the 30‐kD polypeptide was shown to contain the active site of the enzyme, with an additional labeled band of 110 kD also being detected.

On the basis of our data we suggest that the principal species of S. mansoni AChE is a tetramer of four subunit polypeptides each of MW ca. 110 kD which are not linked by disulfide bonds, and which are further cleaved into two fragments, one of MW 76,000 and one of MW 30,000, the latter bears the active site.
An efficient approach for the selective isolation of specific transcripts from complex brain mRNA populations
Authors: Rhyner T.A. , Biguet N. Faucon , Berrard S. , Borbély A.A. , Mallet J.
Published: January 2005

As a consequence of the complexity of the nervous system, many characteristic proteins are only expressed at low levels, and their detection and purification often represents a formidable task. A strategy based on liquid hybridizations, which greatly facilitates the identification of the mRNA transcript for this kind of protein, is presented here. A ten‐fold‐enriched recombinant library was generated from cDNA transcribed from forebrain mRNA after subtraction of cerebellum sequences. Clones specific to forebrain could then be revealed with cerebellum subtracted probes, by colony hybridization. The use of selected cDNA populations greatly enhanced the sensitivity of the screening procedure; clones corresponding to transcripts present at an abundance as low as 0.0005% could still be detected. About 5% of specific clones were recognized with an enriched forebrain probe. Additional clones were revealed with subtracted probes from restricted areas such as cerebral cortex, brainstem, and hippocampus. The important features and potential applications of this approach are discussed.
Developmental expression of myelin basic protein mRNA in a hypermyelinating mouse
Authors: Miskimins R. , Yu R.K.
Published: January 2005

Polysomal poly (A)+ RNA and total cellular RNA were isolated from the brains of C57BL/6J (B6), DBA/2J (D2), and F1 hybrid mice. Both types of RNA were isolated at 9, 12, 16, 21, and 30 days of age. By using a 32P‐labelled cDNA probe for myelin basic protein (MBP; pMBP‐1) the amounts of RNA coding for MBP present in the three strains at each age were determined. For both the polysomal poly(A)+ and total cellular RNA the peak of MBP RNA accumulation was at about 16–18 days for all three strains. These results imply that the developmental pattern of MBP RNA synthesis for F1 mice is not shifted with respect to the parental strains. For the polysomal poly(A)+ RNA, F1 had the greatest amount of MBP mRNA, B6 the least, and D2 an intermediate amount. F1 again had the greatest amount of MBP RNA from the total cellular pool at all ages. However, B6 had a greater amount of MBP RNA in the total cellular RNA pool than did D2. This demonstrates elevated transcription of the MBP gene in the F1 or an increase in message stability in this strain. It also suggests some form of translational control for MBP RNA in the B6 strain. In addition a second species of MBP RNA was found in the total cellular RNA pool. This species is larger than the major polysomal RNA band and may be a nuclear precursor.
The hormonal regulation of gene expression of glial markers: Glutamine synthetase and glycerol phosphate dehydrogenase in primary cultures of rat brain and in C6 cell line
Authors: Kumar S. , Holmes E. , Scully S. , Birren B.W. , Wilson R.H. , de Vellis J.
Published: January 2005

Increases in the mRNA levels of two neuroglial markers, glutamine synthetase (EC 6.3.1.2; GS) and glycerolphosphate dehydrogenase (EC 1.1.1.8; GPDH), were observed in hydrocortisone‐treated cultures of astrocytes and oligodendrocytes, respectively, and in C6 cells by Northern blot analysis and in situ hybridization. In vitro transcription assays demonstrated increased GS transcription in isolated nuclei from hydrocortisone (HC)‐treated primary cultures of astrocytes and C6 cells, relative to untreated cells. This increased transcription is reflected in increased GS mRNA levels in the cytoplasm and increased levels of GS protein synthesis. Sodium butyrate (NaB) blocked the glucocorticoid‐mediated increase in GS transcription in the primary cultures of astrocytes but not in C6 cells. From our earlier observations (Kumar et al: J Neurochem 43:1455–1463, 1984) we found NaB in combination with HC to increase the levels of GS mRNA and GS protein synthesis (Weingarten et al: FEBS Lett 126:289–291, 1981). We now report that NaB, alone or in combination with HC, does not increase the rate of transcription, suggesting that NaB plays a role in post‐transcriptional regulation of GS in C6. In addition, we report the presence of two distinct sizes of GS mRNA, 2.9 and 1.8 kb, in the primary cultures of astrocytes and C6 cells.
Myelin basic protein‐specific protein methylase I activity in shiverer mutant mouse brain
Authors: Kim S. , Tuck M. , Ho L.‐L. , Campagnoni A.T. , Barbarese E. , Knobler R.L. , Lublin F.D. , Chanderkar L.P. , Paik W.K.
Published: January 2005

Myelin basic protein (MBP)‐specific protein‐arginine N‐methyltransferase (protein methylase I) activity in homozygous shiverer (shi/shi) mutant mouse brain is significantly higher than in than normal littermate brain at the onset of myelination. While the enzyme activity (expressed as pmol os S‐adenosyl‐L‐[methyl‐14C] methelination used /min/mg enzyme protein) increase coincidently during the period of myelination in the normal brain (15–18 days of age), it decreases significantly in the mutant brain during this period of time. These results are in contrast to those found with another dysmyelinating mutant, jimpy (jp/Y) mice, in which the enzyme activity in the mutant brain is similar to that in the normal animals but remains unchanged during the myelination process. There is no difference in the weight and protein concentration of the normal and shiverer mutant brains with corresponding ages, and the histone‐specific portein methylase I activity is also unaffected in the shiverer brain.
Correlation between inhibition of myelin basic protein (arginine) methyltransferase by sinefungoin and lack of compact myelin formation in cultures of cerebral cells from embryonic mice
Authors: Amur S.G. , Shanker G. , Cochran J.M. , Ved H.S. , Pieringer R.A.
Published: January 2005

Sinefungin, a known inhibitore of protein methylation, inhibited the myelin basic protein (arginine) methyltransferase activity in homogenantes of cultured cererbral cells from embryonic mice. Fifty percent inhibition was achieved with 25μ M sinefungin. Electron microscopie examination of the myelin fraction, isolated by gradient density centifugation and obtained from untreated cells, revealed numerous ringlike multilamellaar membranous substructures that had a major dense line periodicity, compactness, and the general apperance expected of myelin obtained by the same technique from whole brain. Cells treated with 30 μM sinefungin, which inhibits myelin basic protein methyltrnasferase in broken cell preprations about 60%, produced ringlike structures that were devoid of multiameller periodicity and compactness reminiscent of the vacuolated myelin observed in subacute combined degeneration and in nitrous‐oxide‐ or cycloleucine‐treated animals in which methyltransferase activity is also inhibited. The sinefungin‐induced change in multiamellar periodicity cannot be attributed to a lack of myelin basic protein, since the ratio of myelin basic protein to total protein did not decrease in sinefungin‐treated cells. This primary culture system should be useful for further evaluting the hypothesis that the methylation of mylein basic protein is related to the formation of compact myelin.
Glial culture on artificial capillaries: Electron microscopic comparisons of C6 rat glioma cells and rat astroglia
Authors: Tiffany‐Castiglioni E. , Neck K.F. , Caceci T.
Published: January 2005

The functional association of astroglial footplates with blood vessels is important because astrocytes may provide a channel between the blood and neurons deeper in the brain parenchyma for the passage of ions and metabolites. This hypothesized function is very difficulty to study in vivo or in monolayer cultures. We have produced a three‐dimensional cell culture model of perivascular astroglia by means of an artificial capillary system. Conventional primary cultures of astroglia were first prerpard from neonatal rat cererbral hemispherers in 75‐cm2 tissue culture flasks. After 25 days, the cells were seeded in Amicon Vitafiber hollow fiber culture vessels. Direct seeding of brain cell suspensions was not successful. A culture unit consists of a bundle of hollow, semi‐permeable polysulfone fibers encased in a plastic shell. The fibers were coated with fibronectin and bovine serum albumin, and astroglia were seeded on their outer surfaces. Warmed medium was pumped through the lumina of the fibers. After 13 days the cells were fixed with paraformaldehyde and examined. Scanning electron microscopy reevealed the tubes to be uniformly covered with astroglia with short processes that contacted nearby cells. Transmission electron microscopy showed glial filaments glioma cells in hollow fiber culture. The astrocytes formed a monolayer, whereas C6 cells formed a stratified culture. Furthermore, C6 cells did not form gap junctions. Astrocytes have been hypothesized to take up K+ discharged to the extracellular space by deploarizing neurons and move it to areas of low concentration, i.e., to act as a K+ spital buffer. Our culture system should permit direct testing of this hypothesis.
A reinterpretation of tyramine sympathomimetic effect and tachyphylaxis
Authors: Raiteri M. , Levi G.
Published: January 2005

In this commentary, the indirect sympathomimetic effect of tyramine and the phenomenon of tyramine tachiphylaxis are reinterpreted in terms of carriermediated excahange processes. Extracellular tyramine would exchange with intraterminal noradrenaline and, upon repeated tyramine administration, with a mixture of noradrenaline and tyramine progressively more enriched in the pharmacologically inactive amine.
Central versus peripheral effects of bombesin on the release of gastrointestinal hormones in dogs
Authors: Lu Q.‐H. , Swierczek J.S. , Zhu X.‐G. , Greeley G.H. , Thompson J.C.
Published: January 2005

This study compared the effects of bombesin given intracerebroventicularly and intravenously on circulating levels of gastrin, pancreatic polypeptide (PP), and cholecystokinin (CCK‐33). Bilateral cannulas were implanted permanently into the lateral cerebral ventricles of five dogs. An intravenous (IV) bolus injection of bombesin (0.25 μg/kg) significantly elevated circulating levels of gastrin, PP, and CCK‐33. Vagotomy inhibited the release of PP that was induced by IV bombesin, but vagotomy did not affect gastrin and CCK‐33 responses. Intracerebroventricular injection of bombesin (5.0 μg) significantly elevated circulating gastrin levels but did not affect circulating levels of CCK‐33 and PP. Vagotomy did not alter gastrin release induced by intracerebroventricular injection of bombesin.
Assignment of genes for human monoamine oxidases A and B to the X chromosome
Authors: Kochersperger L.M. , Parker E.L. , Siciliano M. , Darlington G.J. , Denney R.M.
Published: January 2005

Monoclonal antibodies that immunoprecipitate human monoamine oxidase (MAO) A or human MAO B, but not the corresponding mouse enzymes, were used to assay for the presence of immunoprecipitable MAO A or MAO B (presumably coded by the respective human genes) in mouse‐human hybrid somatic cell lines containing small numbers of human chromosomes. The results were as follow: (1) Extracts of a human lymphoblastoid x mouse hepatoma hybrid line that retained the human X chromosome contained immunoprecipitable MAO B, while a similar hybrid line that contained the same human chromosomes, except for the human X, did not. (2) Extracts of a human fibroblast x mouse neuroblastoma hybrid cell line, whose human chromosomal material consisted solely of the X, contained both immunoprecipitable MAO A and MAO B. Extracts of a related hybrid line, whose human chromosomal material consisted solely of an autonomous fragment and a fragment translocated to a mouse chromosome, contained immunoprecipitable MAO A. However, the level of immunoprecipitable MAO B activity in extracts of this hybrid was low or undetectable. (3) Among extracts of 33 human fibroblast x mouse hepatoma hybrids that had been selected for expression of the X‐linked human enzyme HPRT, 60% contained immunoprecipitable MAO B. This figure was comparable to the 58% that expressed the X‐linked human isozyme for glucose‐6‐phosphate dehydrogenase (G6PD). When 11 of these hybrid lines, which contained immunoprecipitable MAO B and human HPRT, were selected for loss of HPRT, all lost immunoprecipitable MAO B in addition to HPRT.

These data demonstrate that genes controlling the expression of MAO A and MAO B, which can be immunoprecipited with the human‐specific monoclonal antibodies, are located on the human X chromosome. Properties of the immunological epitopes recognized by the monoclonal antibodies suggest that the X‐linked genes detected in this study are probably structural genes for the enzymes.
Substance P neurons sprout in the cervical spinal cord of the wobbler mouse: A model for motoneuron disease
Authors: Vacca‐Galloway L.L. , Steinberger C.C.
Published: January 2005

The mutant mouse, wobbler, possesses a recessively inherited degeneration of motoneurons and other ventral horn cells in the cervical spinal cord, and therefore it has been proposed as an animal model of human motoneuron disease. Affected mice have been identified by behavioral tests that also determined the extent of the motor deficit. The results from these tests were combined and used to define distinct stages of the disease process that could then be correlated histochemically with the amount of acetylcholinesterase (AChE) staining in the cervical spinal cord. AChE is used as a marker for cholinergic neurons and is known to hydrolyze the neuropeptide modulator substance P (SP). SP, a peptide neuromodulator of primary afferent transmission in the dorsal horn, excites motoneurons in the ventral horn, and may posses secondary frnctions in neuronal maintenance. Therefore, the levels of immunoreactive (IR) SP and AChE were examined in an attempt to determine the possible interaction between these factors in motoneuron degeneration.

By enzyme histochemistry, the cervical spinal cord, taken from wobbler mice at behaviorally identified stages of the motor deficit, exhibited decreased levels of AChE throughout the ventral horn. The decrease detected in the AChE staining intensity was linear and correlated with the decrease in the number of AChE‐positive cells in the ventral cervical spinal cord, as the motor deficit progressed. Presumably, the continual decrease in AChE staining represents the degeneration of cholinergic perikarya and neuronal processes in the ventral horn as the motoneuron disease proceeds.

At two well‐established stages of the motor deficit, the amount of immunoreactive SP increased in the ventral horn compared with the control mice. The elevated levels of immunoreactive SP suggest sprouting may have occurred preceding, or in response to, the motoneuron degeneration. Several additional hypotheses are discussed in respect to phenomena that might contribute to the increase of immunoreactive SP in the degnerating ventral horn of the wobbler mouse.
Dissimilarities between benzodiazepine‐binding sites and adenosine uptake sites in astrocytes and neurons in primary cultures
Authors: Bender A. S. , Hertz L.
Published: January 2005

The question whether the benzodiazepine receptor site in astrocytes or in neurons might be identical to the adenosine uptake site was studied by determining pharmacological profiles, inhibition types, and the effects of benzodiazepine antagonsts in primary cultures of either astrocytes or neurons. Fourteen different benzodiazepines and five different adenosine uptake inhibitors displaced [3H] diazepam and inhibited adenosine uptake in both astrocytes and neurons. However, the rank orders (determined as IC50 values) with which these two parameters were affected were profoundly different, indicating dissimilarities between these two sites. For several of the compounds a difference in inhibition type (competitive vs. noncompetitive) was observed between the benzodiazepine‐binding site and the adenosine uptake site in astrocytes and/or neurons, which further corroborated the conclusion of a difference between the benzodiazepine‐binding site and the adenosine uptake site. Finally, the neuronal benzodiazepine antagonists RO 15‐1788 and CGS‐8216 and the astrocytic benzodiazepine antagonist PK 11195, which reverse the action of benzodiazepines, were not able to reverse inhibition of adenosine uptake by diazepam but exerted an inhibitory effect of their own.
Denervation of serotonergic fibers in the hippocampus induced a trophic factor which enhances the maturation of transplanted serotonergic neurons but not norepinephrine neurons
Authors: Zhou F. C. , Auerbach S. , Azmitia E.
Published: January 2005

The trophic effects of specific denervation on the growth and survival of fetal serotonergic (5‐HT) or norepinephrinergic (NE) neurons grafted into the hippocampus were assessed by means of two transplantation paradigms. In the first, fetal raphe cells (containing 5‐HT neurons) were transplanted into the control hippocampus. In the second, the transplantation was performed 2 weeks after the specific removal of 5‐HT afferents to the hippocampus with 5,7‐dihy‐droxytryptamine (5,7‐DHT). We found that a month after transplantation, the number of 5‐HT immunoreactive neurons was not significantly different between the two experimental paradigms. However, transplanted raphe neurons had 400% more 5‐HT synaptosomal high‐affinity uptake and 380% higher content of 5‐HT in the hippocampus with prior 5,7‐DHT lesion than in control hippocampus. Furthermore, immunocytochemistry showed that the transplanted 5‐HT neurons had denser processes and varicosities in the hippocampus with lesion than in control hippocampus. The somatic area of the neurons with these denser processes and varicosities was 42% larger than that of control group. A greater 5‐HT level could be achieved if transplanted neurons in the control hippocampus were treated with the supernatant extracted from the hippocampus with 5,7‐DHT lesion.

In contrast, the NE level of the implanted fetal locus ceruleus (containing NE neurons) was not significantly higher in the 5‐HT denervated hippocampus than in control hippocampus a month after transplantation. These results suggest that 5‐HT denervation in the hippocampus induces a trophic substance which promotes the maturation rather than survival of 5‐HT neurons but not NE neurons.
Alteration of calmodulin distribution does not accompany dopaminergic supersensitization of the mouse striatum
Authors: May P. C. , Osterburg H. H. , Mandel R. J. , Morgan D. G. , Randall P. K. , Finch C. E.
Published: January 2005

Membrane‐bound calmodulin increases following dopaminergic supersensitization of the rat striatum. To assess the generality of this relationship, mice were treated with two different supersensitization paradigms. Calmodulin levels and subcellular distribution were determined by radioimmunoassay. Chronic haloperidol treatment increased striatal D2 dopamine receptor density by 25% but had no effect on membrane‐bound calmodulin levels. Similarly, 6‐hydroxy‐dopamine (6‐OHDA) lesions depleted striatal dopamine content > 95% without affecting membrane‐bound calmodulin. In contrast, soluble calmodulin levels decreased by 15% in the 6‐OHDA‐lesioned striatum, suggesting that soluble calmodulin is enriched in presynaptic dopaminergic terminals. We conclude that dopaminergic supersensitization can occur in the mouse striatum in the absence of any change in calmodulin distribution.
Comparisons of proteins and glycoproteins in neuronal plasma membranes, axolemma, Synaptic Membranes, and oligodendroglial plasma membranes
Authors: Hampson D. R. , Poduslo S. E.
Published: January 2005

Neuronal membranes are unique in that they consist of several functionally disinct segments: the perikaryal plasma membrane, the axolemma, the synaptic membrane, and the dendritic membrane. Methods are now available to isolate the first three types of membranes as well as to isolate oligodendroglial plasma membranes. The protein and glycoprotein compositions for each set of membranes were analyzed by silver staining after separation by SDS polyacrylamide gradient gel electrophoresis and by radiolabeled lectin binding to glycoproteins transferred to nitrocellulose. Analysis of the composition of each set of membranes reveals that they are all complex structures consisting of heterogeneous mixtures of proteins and glycoproteins, ranging in molecular weights from < 200,000 to 15,000. Each membrane fraction presents a unique pattern of staining and of lectin binding. As there were proteins and glycoproteins in common among the membranes, there were also differences. Synaptic membranes and axolemma appeared to have more proteins of higher molecular weight than the other membranes. Neuronal plasma membranes had a major concanavalin A binding glycoprotein at 79 kDa, which was not found in the other membranes. The three neuronal membrane fractions had a common wheat germ agglutinin binding glycoprotein at 82 kDa. The most interesting finding was the intense binding of neuronal plasma membrane glycoproteins to Ulex europaeus, suggesting high levels of fucose‐containing glycoproteins.
Effects of coculture on the morphology of identified raphe and substantia nigra neurons from the embryonic rat brain
Authors: König N. , Han V. , Lieth E. , Lauder J.
Published: January 2005

The substantia nigra (SN) is one of the earliest targets of the 5‐HT neurons of the raphe nuclei (RN). To test the hypothesis that embryonic 5‐HT and catechol‐amine neurons may influence the differentiation of their target cells or source neurons, we have produced dissociated cell cultures from embryonic day 14 (E14) rat rhombencephalon (containing the serotonergic RN) and mesencephalon (containing the dopaminergic substantia nigra, SN). These cells were grown for 6 days in vitro, either as single cultures (RN or SN) or cocultures (RN + SN). Effects of coculture on the morphological development of neurons immunoreactive (IR) for 5‐HT or tyrosine hydroxylase (TH) were studied by measuring a series of morphological parameters related to size of the cell body and dendritic field, as well as to the complexity of neurites within this field, using computer‐assisted morphometry. Increases in a number of these parameters were found in cocultures compared to single cultures for both types of monoamine neurons, but a greater number of parameters were increased for TH‐IR cells, including size of the cell body. Although this might suggest that there was a greater effect of coculture on the TH‐IR (dopaminergic) cells of the SN than on the 5‐HT‐IR cells of the RN, we must consider the fact that a significant population of TH‐IR cells were present in single RN cultures, which contributed to the total population of TH‐IR cells in cocultures. Indeed, when morphometric parameters for TH‐IR cells in RN and SN single cultures were compared, it was found that TH‐IR cells from the RN were generally larger and more complex than those from the SN. Therefore, an analysis was made of which parameters were significantly increased for TH‐IR cells in cocultures compared to single cultures from both SN and RN. This was the case for two parameters: cell body size and absolute field area, indicating that these increases were probably due to the effects of coculture itself rather than to contamination by the larger and more complex TH‐IR cells from the RN. It is impossible to ascertain, however, whether this effect was on cells from the RN, SN, or both. Coculture effects on 5‐HT‐IR cells were easier to analyze, since no such cells were found in single cultures of SN. Three parameters were consistently increased for 5‐HT‐IR cells in coculture: cumulative length of all segments, segment length density, and absolute field area. This indicates that 5‐HT‐IR cells in coculture had larger dendritic fields that contained a more complex array of neurites than their counterparts in single cultures. In sum, coculture seems to promote the growth of both 5‐HT and TH‐IR neurons, but in different ways and to different degrees. These results suggest that some factor or factors related to growth of these monoamine neurons are altered when the rhombencephalon and mesencephalon are cultured together. The exact nature of this developmental signal remains to be determined.
Development of innervation within syngeneic thymus tissue transplanted under the kidney capsule of the nude mouse: A light and ultrastructuarl microscope study
Authors: Bulloch K. , Cullen M. R. , Schwartz R. H. , Longo D. L.
Published: January 2005

The devloping thymus, prior to the onset of its functional and structural organization, is innervated by the autonomic nervous system (ANS). The present study extends these earlier findings by analyzing at the light and ultrastructural microscope level the distribution of ANS nerves within normal 18‐day‐old embryo thymus, adult thymus, and thymic tissue transplanted under the kidney capsule of syngeneic nude mice. The results of this study showed that prior to birth the murine thymus is innervated by AChE‐positive fibers that are distributed at the corticomedullary boundaries and throughout the adjacent cortex, as well as to cells beneath the thymic capsule. This pattern of distribution remains constant during adult life. The examination of the various thymic tissues by ultrastructural analysis demonstrated that myelinated and nonmyelinated fiber bundles penetrate the thymic capsule and the interface between the kidney and the thymus transplant. The myelinated fibers measured 2 m̈m or less in diameter; the nonmyelinated fibers were 1 m̈m in diameter. Myelination did not accompany the nerves into the parenchyma of the normal gland but was observed in the intralobular trabeculae of the transplanted thymus. Subcapsular nerves form a network that terminates among the thymocytes, whereas intrathymic nerves enter the parenchyma in bundles along the vasculature and interlobular septa before penetrating into the deeper layers of the thymic cortex. Some larger nerve fibers terminate in the corticomedullary boundaries and in the interlobular septa. Smaller fibers form en passant boutons near parenchymal cells. The innervation of both the normal thymus prior to the onset of thymic immune function supports a role for ANS innervation in the development of thymic competency.
Noradrenergic sympathetic innervation of the spleen: I. Nerve fibers associate with lymphocytes and macrophages in specific compartments of the splenic white pulp
Authors: Felten D. L. , Ackerman K. D. , Wiegand S. J. , Felten S. Y.
Published: January 2005

Sympathetic noradrenergic nerve fibers, stained with antiserum for tyrosine hydroxylase (TH), richly innervate the splenic white pulp. These fibers distribute with the vascular and trabecular systems, and associate mainly with the central artery and its branches, the periarteriolar lymphatic sheath (PALS), the marginal sinus, and the parafollicular zone, with occasional delicate fibers also present in the follicles. Simultaneous staining of TH‐positive nerve fibers and markers for specific lymphoid cells has shown several regions of contact between nerves and lymphocytes or macrophages. The TH‐positive nerve fibers in the plexuses around the central arterial system and in the PALS are present among T lymphocytes (OX‐19‐positive cells) including both T helper and T suppressor cells, and interdigitating cells. At the marginal sinus, TH‐positive fibers run adjacent to macrophages (ED3‐positive cells), B lymphocytes (IgM‐positive), and intensely fluorescent IgM‐positive cells. Along the parafollicular zone, TH‐positive nerve fibers run adjacent to T lymphocytes, peripheral follicular B lymphocytes, and intensely fluorescent IgM‐positive cells. Within some follicles, delicate fibers end adjacent to both T and B lymphocytes. These relationships suggest a direct interaction between norepinephrine release from the TH‐positive nerve terminals and the lymphocytes and macrophages closely associated with them, and focuses attention on the potential neural modulation of related functions such as T and B lymphocyte entry into the spleen and antigen capture (marginal zone), antigen presentation and T cell activation (PALS), B cell activation (parafollicular zone and marginal zone), and lymphocyte egress (outer marginal zone).
Modulation of T‐Suppressor cell activity by central nervous system catecholamine depletion
Authors: Cross R. J. , Brooks W. H. , Roszman T. L.
Published: January 2005

This study extends our previous findings, which indicate that depletion of CNS catecholamines has a marked inhibitory effect on humoral immune responsiveness. These data show that depletion of CNS catecholamines by injection of 6‐hydroxydopamine (6‐OHDA) into the cisterna magna in conjunction with immunization enhances activity of a population of splenic T‐suppressor cells as evidenced by the transfer of these cells to normal recipients. Increased suppressor cell activity does not results solely from 6‐OHDA treatment, but rather requires concomitant immunization. Further characterization shows that these suppressor cells are not antigen specific. Hypophysectomy abrogates the effect of 6‐OHDA injection suggesting that catecholamine depletion modulates immune function via the release of pituitary hormones. Thus, depletion of CNS catecholamines impairs immune responsiveness by inducing enhanced T‐suppressor cell activity, providing additional evidence of the involvement of the CNS in gegulation of immune responsiveness.